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ICREA International Symposium

BioNanoVision of cellular

architecture: from the nucleus

to the cell membrane

25-27 May 2016 | Barcelona, Spain

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FOREWORD

We are most proud to welcome you in Barcelona for attending the ICREA International Symposium BioNanoVision of molecular architecture: from the nucleus to the cell membrane. The aim of the Symposium is to bring together a multidisciplinary group of world-leading scientists to further our understanding on the fundamental molecular mechanisms that regulate cellular architecture, from nuclear organization to the cell membrane. The Symposium will cover topics related to the organization of the plasma membrane at the nanoscale, their role on initiating cell signaling, intracellular and nuclear organization and architecture. Moreover, the entire event places a special emphasis on novel technologies that are advancing biological understanding, including super-resolution microscopy, correlative techniques and single molecule approaches.

We are very grateful to all of you by actively participating in this event. We sincerely hope that you will enjoy this meeting and that through the many lively discussions we expect to have, you will get further stimulated in your research.

Barcelona, May 2016.

We Welcome you to BarcelonaThe Organizing Committee

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Sponsors

Local organizing committee

Important information

ProgramWednesday 25 may

Thursday 26 mayFriday 27 may

Conference dinner information

Abstracts and oral comunications

Posters

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Summary

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Sponsors

The event is sponsored by the Institució Catalana de Recerca i Estudis Avançats (ICREA).

Additional support comes from:ICFO-Institute of Photonic SciencesFundación Privada CellexExcelencia Severo OchoaEMBOThe Company of Biologists

ponsors

ció Catalana de

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Prof. Maria Garcia-Parajo (ICFO & ICREA, Barcelona)

Prof. Melike Lakadamyali (ICFO, Barcelona)

Prof. Pia Cosma(CRG & ICREA, Barcelona)

Local Organizing Committee

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Important informationDear colleague,

We are looking forward to welcome you to the ICREA symposium “BioNanoVision of cellular architecture: from the nucleus to the cell membrane” that will take place during 25-27 May 2016 at ICFO. With the aim of making your stay as comfortable as possible, we would like to remind you of some information that may be of interest:

PROGRAMYou will fi nd the updated program of the event on the offi cial website. Notice that we will not provide a printed booklet with the contributed abstracts. You can download the full program including the abstracts at the con-ference website (http://icrea-bionanovision2016.com). You may pick up your documentation, including a printed copy of the three-day program, your identifi cation badge and an USB containing the full program and abstracts on May 25th from 08:00 to 09:00.To avoid any inconveniences, we remind you of wearing your identifi cation badge at all times during the event.

LOCATIONThe event will take place on the grounds of ICFO, which is situated in Castelldefels, just a short ride from Bar-celona. If you would like more information on how to get there you can refer to the following links:• From the Barcelona Airport (BCN) to Castelldefels (http://www.castelldefelsturisme.com/ca/cl-

aeroport?idioma=3&id_pagina=ca/cl-aeroport)• Trains from Barcelona to Castelldefels (http://www.castelldefels.com/ingl/trenin.htm)• Location of ICFO (http://goo.gl/maps/x8c9)

INFORMATION FOR THE SPEAKERSShort talks should last 20 min in total, including questions. Since the program is quite tight and we would like to have lively discussions, we recommend you to carefully prepare your talk for a total duration of 15 min max, leaving at least 5 min for discussion. Remember that there will be a prize for the best short talk. The best talk award will be given on the last day of the conference, Friday 27th May. To avoid last minute problems we highly recommend you to bring your talk in a pen-drive so that it can be rapidly uploaded on the local computer. Please upload your presentation and check that it is correct well before your session starts.

PRESENTATION POSTERSThe most suitable format for preparing your poster is A0 (vertical format). You may place your poster on May 25th in the morning. Panels for placing your poster will be identifi ed by a number. Please check the number of your poster at the website: http://icrea-bionanovision2016.com.Posters will be displayed during the entire event and should be removed on Friday after lunch. Remember that there will be a poster prize to be awarded on the last day of the Conference, Friday 27 May.

WI-FIPlease be informed that the ICFO campus has WI-FI.

CONFERENCE DINNERThe conference dinner is scheduled for Thursday 26 May. There will be buses at the entrance of ICFO to bring all participants to the Restaurant Fosbury. Buses will depart at 20:00 sharp and will return to the Hotels at 22:30h (fi rst bus) and 23:00h (second bus).

LAB TOURSWe will organize Lab tours on Friday from 16:00-17:30 for those interested. There will be lists available for you to sign in case you are interested on visiting our Labs.

FINAL REMARKS: Our team is at your disposal for anything you may need. Please do not hesitate to contact us and we will be happy to help you.24/7 Phone: Oriol Galgo (+34) 600 93 86 86

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programme

Scientifi c

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08:00-09:00Registration

09:00-09:15Welcome

09:15-09:55Jeorg Bewersdorf (Yale University, USA): Live-cell Optical Microscopy Beyond the Diffraction Limit

09:55-10:35Philip Tinnefeld (TU Braunschweig, Germany): Getting more photons for super-resolution and single mol-ecule biophysics

10:35-10:55Laura Zanetti (Lasers For Science Facility, CLF, STFC, UK): Determining the architecture of inactive EGF receptor oligomers on cells with 5 nm resolution

10:55-11:30Coffee break

11:30-12:10Keith Lidke (University of New Mexico, USA): Single Objective Light-Sheet Microscopy for High-Speed Whole-Cell 3D Super-Resolution Imaging

12:10-12:30Erik Garbacik (ICFO-Institute of Photonic Sciences, Barcelona, Spain): Excitation-multiplexed multispec-tral microscopy with a single monolithic color-blind detector

12:30-12:50Florian Baugmart (TU Vienna, Austria): Label density variation to probe membrane protein nanoclusters in dSTORM and PALM

12:50-13:10Samuel Ojosnegros (CMRB, Barcelona, Spain): Enhanced Number and Brightness analysis reveals a com-peting dynamics of Eph receptor activation and large-scale clustering

13:10-14:30Lunch

14:30-16:30Poster Session (includes coffee & drinks)

16:30-17:10Aleksandra Radenovic (EPFL, Switzerland): The power of correlative super-resolution imaging

17:10-17:30Ione Verdeny-Vilanova (ICFO-Institute of Photonic Sciences, Barcelona, Spain): Unravelling 3D cargo transport dynamics at the microtubule network

17:30-17:50 Anika Raulf (Goethe-University Frankfurt, Germany): Small Labeling Pair for Single-Molecule Localization Microscopy

17:50-18:30Jacob Hoogenboom (Technical University Delft, Netherlands): Super-resolution in the cell structural con-text using focused electron beams

Scientifi c Programme

Wednesday, May 25BioNanoVision: New technological advances in super-resolution & single molecule imaging

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09:00-09:40Christian Eggeling (Weatherall Institute of Molecular Medicine, Oxford, UK): Advances in super-resolution STED microscopy: Prospects for studying membrane bioactivity

09:40-10:00Oriol Gallego (IRB-Institute for Research in Biomedicine, Barcelona, Spain): The molecular organization of the exocyst determined by live cell imaging

10:00-10:20Jonas Ries (EMBL, Heidelberg, Germany): Superresolution imaging of clathrin-mediated endocytosis in yeast

10:20-11:00Diego Krapf (Colorado State University, USA): Anomalous diffusion and compartmentalization on the sur-face of mammalian cells

11:00-11:30Coffee break

11:30-12:10Diane Lidke (University of New Mexico, USA): Imaging the early events in membrane receptor signalling

12:10-12:30Stefan Balint (Manchester Collaborative Centre for Inflammation Research, UK): Ligation of activating FcγRI nanodomains at macrophage surfaces causes them to segregate from nanodomains of inhibitory SIRPα and re-organize into concentric rings

12:30-13:10Grégory Giannone (CNRS, Bordeaux, France): Deciphering the spatiotemporal regulation of integrin and actin regulators at the nanoscale

13:10-14:30Lunch

14:30-16:30Poster Session (includes coffee & drinks)

16:30-17:10Alessandra Cambi (Institute for Molecular Life Sciences, Radboud umc, Nijmegen, Netherlands): Integrat-ing advanced microscopy techniques reveal actin nanoscale architectures and mesoscale dynamics of mechanosensory podosomes.

17:10-17:30Amanda Remorino (Institut Curie, Paris, France): Spatio-temporal regulation of Rac1 signalling

17:30-17:50Helge Ewers (Free University Berlin, Germany): Nanoscopic compartmentalization of membrane protein motion at the axon initial segment

17:50-18:30Lukas Kapitein (University Utrecht, Netherlands): Navigating the cytoskeleton: novel tools to dissect and direct intracellular transport

20:00Conference dinner at the Fosbury Restaurant-Lounge (www.fosburycafe.es). Bus shuttle provided. Meet-ing point: ICFO 19.45h.

Scientifi c Programme

Thursday, May 26BioNanoVision of cell membrane architecture and membrane traffi cking

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09:00-09:40Xavier Darzacq (University California, Berkeley, USA): Single molecule imaging reveals nuclear domains modulating transcription regulation

09:40-10:00Diego Cattoni (Centre de Biochimie Structurale, INSERM U1054, France): Super-resolution imaging of topological barriers reveals higher-order chromatin folding principles

10:00-10:20Kyle Douglass (EPFL, Switzerland): Quantitative imaging of human telomere structure

10:20-11:00Marcelo Nollmann (CNRS/INSERM, Montpellier, France): Chromosomes from bacteria to humans are or-ganized at the sub-megabase scale into topological domains (TDs).

11:00-11:30Coffee break

11:30-12:10Pia Cosma (CRG, Barcelona, Spain): The nanoscale structure of chromatin fi bers correlates with cellular state

12:10-12:30Alessandra Agresti (San Raffaele Scientifi c Institute, Italy): Chromatin organization in nucleosome-rich and nucleosome-poor cells

12:30-12:50Anna Oddone (ICFO-Institute of Photonic Sciences, Barcelona, Spain): Chromatin organization in Dros-ophila melanogaster’s developing neural system

12:50-14:30Lunch

14:30-15:10Bo Huang (University California San Francisco, USA): Life inside the cell: STORM, CRISPR and imagenomics

15:10-15:40Kyle Legate (Springer Nature, London, UK): Insights into Editorial policies of Nature Communications.

15:40-16:00Best short talk & Best poster Awards – end of the meeting

16:00-17:30Guided Lab tours at ICFO (optional)

Scientifi c Programme

Friday, May 27BioNanoVision of nuclear architecture

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Scientifi c Programme

WEDNESDAY 25 MAY THURSDAY 26 MAY FRIDAY 27 MAY

08:00 - 09:00 Registration

09:00 - 09:15 Wellcome speach

09:00 - 09:40 Christian Eggeling

09:00 - 09:40 XavierDarzacq

09:40 - 10_00 Oriol Gallego

09:40 - 10:00 DiegoCattoni

09:15 - 09:55 Jeorg Bewersdorf

10:00 - 10:20 JoansRies

10:00 - 10:20 KyleDouglass

09:55 - 10:35 PhilipTinnefeld

10:35 - 10:55 LauraZanetti

10:20 - 11:00 DiegoKrapf

10:20 - 11:00 Marcelo Nollmann

10:55 - 11:30 COFFEE BREAK

11:30 - 12:10 KeithLidke

11:30 - 12:10 DianeLidke

11:30 - 12:10 PiaCosma

12:10 - 12:30 ErikGarbacik

12:10 - 12:30 StefanBalint

12:10 - 12:30 Alessandra Agresti

12:30 - 12:50 Florian Baugmart

12:30 - 13:10 Grégory Giannone

12:30 - 13:10 AnnaOddone

12:50 - 13:10 Samuel Ojosnegros

13:10 - 14:30 LUNCH

14:30 - 16:30 POSTER SESSION (coffee break) 14:30 - 15:10 BoHuang

15:10 - 15:40 KyleLegate

16:30 - 17:10 Aleksandra Radenovic

16:30 - 17:10 Alessandra Cambi

15:40 - 16:00 Best short talk & Best poster Awards - end of meeting

17:10 - 17:30 Ione Verdeny-Vilanova

17:10 - 17:30 Amanda Remorino

16:00 a 17:30 Guided Lab tours at ICFO (optional)

17:30 - 17:50 AnikaRaulf

17:30 - 17:50 HelgeEwers

17:30 - 17:50

17:50 - 18:30 Jacob Hoogenboom

17:50 - 18:30 LukasKapitein

17:50 - 18:30

invited speakers

talk abstracts

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Conference dinnerAt the Fosbury Restaurant-Loungewww.fosburycafe.es

Bus shuttle provided. Meeting point: ICFO 19.45h.

LocationPasseig Maritim, 29908860 CastelldefelsT: +34 936 367 372

ICFO

RESTAURANT

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Oral ComunicationsAbstracts and

Joerg Bewersdorf. Yale University School of Medicine, USALive-cell Optical Microscopy Beyond the Diffraction Limit

Philip Tinnefeld. Technical University Braunschweig, GermanyGetting more Photons for Superresolution and Single-Molecule Biophysics

Laura Zanetti. Lasers for Science Facility - STFC, UKDetermining the architecture of inactive EGF receptor oligomers on cells with 5 nm resolution

Keith Lidke. University of New Mexico, USASingle Objective Light-Sheet Microscopy for High-Speed Whole-Cell 3D Super-Resolution Imaging

Erik Garbacik. ICFO-Institute of Photonic Sciences, SpainExcitation-multiplexed multispectral microscopy with a single monolithic color-blind detector

Florian Baugmart. Technical University Vienna - AustriaLabel density variation to probe membrane protein nanoclusters in dSTORM and PALM

Samuel Ojosnegros. Centre de Medicina Regenerativa de Barcelona (CMRB), SpainEnhanced Number and Brightness analysis reveals a competing dynamics of Eph receptor activation and large-scale clustering

Aleksandra Radenovic. Ecole polytechnique fédérale de Lausanne EPFL, SwitzerlandThe power of correlative super-resolution imaging

Ione Verdeny-Vilanova. ICFO-Institute of Photonic Sciences, SpainUnravelling 3D cargo transport dynamics at the microtubule network

Anika Raulf. Johann Wolfgang Goethe-University, GermanySmall Labeling Pair for Single-Molecule Localization Microscopy

Jacob Hoogenbom. Technical University Delft, NLSuper-resolution in the cell structural context using focused electron beams

Wednesday, May 25

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Christian Eggeling. Weatherall Institute of Molecular Medicine, University of Oxford, UKAdvances in super-resolution STED microscopy: Prospects for studying membrane bioactivity

Oriol Gallego. IRB-Institute for Research in Biomedicine, Spain The molecular organization of the exocyst determined by live cell imaging

Jonas Ries. EMBL, GermanySuperresolution imaging of clathrin-mediated endocytosis in yeast

Diego Krapf. College of Engineering, Colorado State University, USAAnomalous diffusion and compartmentalization on the surface of mammalian cells

Diane Lidke. Department of Pathology, University of New Mexico, USAImaging the early events in membrane receptor signaling

Stefan Balint. Manchester Collaborative Centre for Inflammation Research. University of Manchester - UKLigation of activating FcγRI nanodomains at macrophage surfaces causes them to segregate from na-nodomains of inhibitory SIRPα and re-organize into concentric rings

Grégory Giannone. Institut Interdisciplinaire de Neuroscience (IINS), CNRS/Université Bordeaux, FranceDeciphering the spatiotemporal regulation of integrin and actin regulators at the nanoscale

Alessandra Cambi. Institute for Molecular Life Sciences, Radboud umc, Nijmegen, NLIntegrating advanced microscopy techniques reveal actin nanoscale architectures and mesoscale dyna-mics of mechanosensory podosomes

Amanda Remorino. Institut Curie, FranceSpatio-temporal regulation of Rac1 signalling

Helge Ewers. Free University BerlinNanoscopic compartmentalization of membrane protein motion at the axon initial segment

Lukas Kapitein. Department of Cell Biology, University Utrecht, NLNavigating the cytoskeleton: novel tools to dissect and direct intracellular transport

Xavier Darzacq. University California, Berkeley, USASingle molecule imaging reveals nuclear domains modulating transcription regulation

Diego Cattoni. Centre de Biochimie Structurale, INSERM U1054Super-resolution imaging of topological barriers reveals higher-order chromatin folding principles

Kyle Douglass. EPFL, SwitzerlandQuantitative imaging of human telomere structure

Marcelo Nollmann. Center for Structural Biochemistry, CNRS/INSERM, Montpellier, FranceChromosomes from bacteria to humans are organized at the sub-megabase scale into topological do-mains (TDs).

Pia Cosma. CRG-Centre for Genomic Regulation, Spain The nanoscale structure of chromatin fi bers correlates with cellular state

Alessandra Agresti. San Raffaele Scientifi c Institute, ItalyChromatin organization in nucleosome-rich and nucleosome-poor cells

Anna Oddone. ICFO-Institute of Photonic Sciences, SpainChromatin organization in Drosophila melanogaster’s developing neural system

Bo Huang. University of California San Francisco, USALife inside the cell: STORM, CRISPR and imagenomics

Kyle Legate. Springer Nature, London, UKInsights into Editorial policies of Nature Communications.

Thursday, May 26

Friday, May 27

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Joerg Bewersdorf1

1 Department of Cell Biology and Department of Biomedical Engineering. Yale University, USA

Optical Nanoscopy (super-resolution) techniques such as STED and FPALM/PALM/STORM microscopy utilize either targeted or stochastic switching of fluorescent molecules to achieve ~25 nm spatial resolution – about 10-fold be-low the diffraction limit. However, their primary application has been focused on fi xed samples because of (i) a lack of suitable live-cell compatible labels and (ii) time resolutions often limited to minutes, especially for FPALM/PALM/STORM.

In this talk, I will present recent advances in live-cell nanoscopy using newly developed probes, labeling procedures and instrumentation that have been optimized for live-cell imaging.

I declare a fi nancial interest in Hamamatsu and Bruker.

Live-cell Optical Microscopy Beyond the Diffraction Limit

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02Guillermo Acuna1, Mario Raab1, Anastasya Puchkova1, Birka Lalkens1, Philip Tinnefeld1

1 Institute for Physical & Theoretical Chemistry – NanoBioScience, and LENA (Laboratory of Emerging Nanometrolo-gy), and BRICS (Braunschweig Integrated Center for Systems Biology) Braunschweig University of Technology 38106 Braunschweig, Germany Email: p.tinnefeld@tu-braunschweig.de

The number of photons per molecule and their brightness ultimately determine the resolution and signal-to-noise ratio in superresolution microscopy and single-molecule detection. We present recent advances to use the full photon budget from single dye molecules in the superresolution technique DNA PAINT 1,2. We also show how self-assembled nanophotonic structures assembled by the DNA origami technique can act as nanolenses to increase the bright-ness3,4 and photostability5 of fluorescent dyes and might enable biomolecular assays at elevated concentrations.

(1) Molle, J.; Raab, M.; Holzmeister, S.; Schmitt-Monreal, D.; Grohmann, D.; He, Z.; Tinnefeld, P. Curr Opin Biotechnol 2016, 39, 8. (2) Raab, M.; Schmied, J. J.; Jusuk, I.; Forthmann, C.; Tinnefeld, P. Chemphyschem 2014, 15, 2431. (3) Puchkova, A.; Vietz, C.; Pibiri, E.; Wunsch, B.; Sanz Paz, M.; Acuna, G. P.; Tinnefeld, P. Nano Lett 2015, 15, 8354.

(4) Acuna, G. P.; Moller, F. M.; Holzmeister, P.; Beater, S.; Lalkens, B.; Tinnefeld, P. Science 2012, 338, 506. (5) Pellegrotti, J. V.; Acuna, G. P.; Puchkova, A.; Holzmeister, P.; Gietl, A.; Lalkens, B.; Stefani, F. D.; Tinnefeld, P. Nano Lett 2014, 14, 2831.

Getting more Photons for Superresolution and Single-Molecule Biophysics

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03Laura C. Zanetti-Domingues1, Sarah R. Needham1, Selene K. Roberts1, Christopher J. Tynan1, Daniel J. Rolfe1, Michael Hirsch1, Dimitris Korovesis1, David T. Clarke1, Marisa Martin-Fernandez1

1 Central Laser Facility, Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Didcot, United Kingdom

The human epidermal growth factor receptor (EGFR) initiates signals for cell proliferation and transformation. This receptor has an extracellular growth factor-binding domain (ECD), a single-pass transmembrane region, and an in-tracellular domain that has tyrosine kinase activity.

The activation of the EGFR is triggered by the binding of small growth factor polypeptide ligands and involves the formation of dimers of these receptors. Oligomers are also commonly observed on cell surface, yet little is known about their structures and their functional role in EGFR signalling. This is largely attributable to the lack of methods with suffi cient resolution.

We developed a super-resolution method based on fluorophore localisation imaging with photobleaching (FLImP) to investigate the geometry and size of oligomers of the EGFR family on the cell surface with ~ 5 nm resolution. By using non-activating peptide markers and combining the FLImP super-resolution method with fluorescence resonance en-ergy transfer to determine intra-receptor conformation, and with single-particle tracking to assess receptor dynamics in real time, we are beginning to determine conformational changes and interactions in oligomers that regulate EGFR signal transduction across the plasma membrane.

Determining the architecture of inactive EGF receptor oligomers on cells with 5 nm resolution

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04Keith Lidke1 , Marjolein Meddens1 , Sheng Liu1 , Conrad James2 , Thayne Edwards2

1 University of New Mexico2 Sandia National Laboratories

Single molecule super-resolution (SM-SR) imaging in 3D throughout a whole cell is hampered by wide-fi eld activation because imaging light cannot be targeted to the in-focus image plane. This results in high background fluorescence that degrades detection and localization precision. We have developed a technique, termed Single Objective Light-Sheet Microscopy (SO-LSM) that uses a common, single objective, inverted epi-fluorescence microscope along with a reflective surface to generate illumination only in the image focal plane. We use a reflective, planar surface which is placed at an angle of 45° on a cover slip to reflect the beam. This surface forms the side wall of a microfluidic channel incorporated into a microfluidic device. A light-sheet is generated through the objective and reflected by the surface such that it illuminates only the in-focus plane of the cell (Fig.1). The light-sheet is scanned through the cell for whole-cell imaging and an astigmatic lens in the emission light path enables the 3D localization of individual emitters, creating a whole-cell 3D super-resolution image. SO-LSM provides greatly improved localization accuracy due to ~3-4 fold background reduction. By combining astigmatism to determine the Z-position and a PSF model that incorporates system abberations [1] we can achieve a signifi cant improvement in localization precision as compared to wide fi eld illumination. An additional benefi t of SO-LSM comes from reduced photobleaching of un-imaged, out of focus fluorophores. Moreover, the light-sheet confi nes the laser light in one dimension, thereby increasing the excitation intensity 7 fold, allowing inexpensive lasers to genearte high intenisty excitation. This increased intensity makes the probes photo-switch faster and reduces the aquisition time for a single whole-cell SM-SR image. The microfluidics device provides a closed environment that allows for fast and automated buffer exchange, which is uti-lized to maintain a reduced oxygen environment needed for many SM-SR probes. SO-LSM is a system that is easily implemented on a regular wide-fi eld fluorescence microscope. The development of this microscope and its imaging and analysis routines will therefore be of great value to investigators seeking a relatively straightforward, inexpensive method for whole-cell 3D super-resolution microscopy and high-speed live cell imaging.

[1] S. Liu, E.B. Kromann, W.D. Krueger, J. Bewersdorf, K.A. Lidke, “Three dimensional single molecule localization using a phase retrieved pupil function”, Optics express, 21, 24: 29462-87, 2013.

Single Objective Light-Sheet Microscopy for High-Speed Whole-Cell 3D Super-Resolution Imaging

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05Erik Garbacik1 , Maria Sanz-Paz1 , Kyra J.E. Borgman1 , Felix Campelo1 , Maria F. Garcia-Parajo1

1 ICFO-Institute of Photonic Sciences, Mediterranean Technology Park, 08860 Castelldefels, Spain

A common theme in cell biology is the direct relationship between the spatial distributions and functional inter-actions of biomolecules. To elucidate these relationships there has recently been a very determined push toward fluorescence-based imaging of as many individual species of biomolecules as possible, either simultaneously or in sequence. Unfortunately, these new methods generally require extensive sample preparation, complicated optical setups, long imaging times, or a combination of these traits. We have approached the problem of multicolor imag-ing from a different direction. Recognizing that each species of fluorophore in a sample will by defi nition have its own unique absorption spectrum, we perform fluorescence microscopy by encoding all spectral information in the frequency domain during excitation of the sample. Our excitation multiplexing technique enables us to discriminate between multiple species of fluorophore solely on the basis of their absorption cross sections. As a result, we can relay all of the fluorescent signal photons to a single detector with minimal spectral fi ltering, maximizing the photon budget of the system. We have demonstrated its utility in cell biology by simultaneously imaging and determining the spatial distributions of fi ve different organelles with only four excitation sources and relying only on notch fi lters to remove the residual excitation light on the detection arm of the setup. This system is simple, low-cost, highly robust, and can be integrated into nearly any microscopy confi guration.

Excitation-multiplexed multispectral microscopy with a single monolithic color-blind detector

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06Florian Baumgart1 , Andreas Arnold1 , Gerhard Schütz1

1 TU Vienna

The spatial organization of signaling components on the T cell plasma membrane is generally believed to regulate T cell function. Indeed, local enrichment in so-called microclusters and subsequent rearrangement of key compo-nents of the T cell signaling machinery result in down-stream signaling and T cell activation1. Recently, using super-resolution microscopy, a number of reports have proposed the existence of nanoscopic clusters of T cell signaling components2-4, suggesting that nano-scale spatial organization is a fundamental regulatory mechanism of T cell signaling processes.

Here, we develop a new approach to distinguish random from clustered spatial distributions of molecules in PALM and dSTORM data. The method is based on characteristic changes in the relative area and the localization density of randomly distributed and clustered molecules, when the labeling density is varied. Most importantly, it is insensitive to common pitfalls inherent to single molecule localization-based super-resolution techniques, such as blinking ar-tifacts or residual diffusion in fi xed samples, which severely hamper the bona fi de detection of nano-clusters in cells.

Finally, we used the new method to study clustering phenomena on the T cell plasma membrane. Investigating the nanoscopic spatial distribution of Lck, a key kinase of early T cell signaling, we fi nd, contrary to a previous report2, Lck to be homogeneously distributed on the T cell plasma membrane. Our data thus suggest that Lck activity and hence T cell activation cannot depend on the formation of nanoscopic Lck clusters. Rather, our data support the view that Lck is randomly distributed on the T cell plasma membrane and that it is specifi cally recruited to signaling hotspots during T cell activation.

1. Dustin ML (2014) Cancer Immunol Res2. Owen DM et al (2012) Nat Commun3. Rossy J et al (2013) Nat Immunol4. Lillemeier BFF et al (2010) Nat Immunol

Label density variation to probe membrane protein nanoclusters in dSTORM and PALM

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07Samuel Ojosnegros1, Francesco Cutrale2, Dani Rodriguez3, Jason J Otterstrom4, Chi Li Chiu5, Veronica Hortigüela6, Carolina Tarantino1, Anna Seriola1, Stephen Mieruszynski7, Elena Martinez6, Melike Lakadamyali4, Angel Raya1, Scott E Fraser2

1 Center of Regenerative Medicine in Barcelona (CMRB)2 University of Southern California, Translational Imaging Center, Molecular and Computational Biology3 Laboratory of Theoretical & Applied Mechanics (LMTA) Dept of Mechanical Engineering, Universidade Federal Fluminense4 ICFO-The Institute of Photonic Sciences, The Barcelona Institute of Science and Technology5 Center for Applied Molecular Medicine, University of Southern California6 Biomimetic Systems for Cell Engineering group, Institute for Bioengineering of Catalonia (IBEC)7 European Molecular Biology Laboratory (EMBL) Australia, Australian Regenerative Medicine Institute, Monash University

Signal transduction of the Eph tyrosine kinase receptor is intimately linked to its oligomerization, yet the stoichiom-etry of the active species remains largely unknown. Eph receptors form large, homomeric clusters containing hun-dreds of monomers, and current methods cannot discriminate whether high-order clusters or smaller oligomers are key to receptor signaling. Through the use of an enhanced image processing based on Number and Brightness theory, we assay the oligomerization state of the receptors over time, and exploit a mathematical model to compare this to receptor activation. The analysis reveals that hexamers and octamers are the most active signaling species; large-scale clusters arise after maximum activation from the coalescence of smaller oligomers. The decoupled activation and large scale clustering revealed by our quantitative imaging, allows the Eph receptor to balance the needed signal amplifi cation with the wide dynamic range necessary to reliably sense ligand gradients.

Enhanced Number and Brightness analysis reveals a competing dynamics of Eph receptor activation and large-scale clustering

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08Aleksandra Radenovic1

1 EPFL School of Engineering, EPFL, Institute of Bioengineering, Laboratory of Nanoscale Biology1015 Lausanne, Switzerland

In this talk I will review recent progress in correlative super-resolution imaging. First part of the talk will be dedicated to our efforts to build and characterize a correlated single molecule localization microscope- with an atomic force mi-croscope (SMLM)/AFM that allows localizing specifi c, labelled proteins within high-resolution AFM images in a bio-logically relevant context. The technique has been successfully applied to the live and fi xed samples. The second part of my talk will be dedicated to the discussion on the complementarity between SMLM and fluctuation imaging (SOFI).

References High resolution correlative microscopy: Bridging the gap between Single Molecule Localization Microscopy and Atom-ic Force Microscopy Pascal D. Odermatt, Arun Shivanandan, Hendrik Deschout, Radek Jankele, Adrian, P. Nievergelt, Lely Feletti, Michael W Davidson, Aleksandra Radenovic and Georg E. Fantner, Nano Lett. 15 (8), pp 4896–4904 2015

The power of correlative super-resolution imaging

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09Ione Verdeny Vilanova1 , Fabian Wehnekamp2 , Nitin Mohan1 , Ángel Sandoval Álvarez1 , Joseph S. Borbely1 , Jason J. Otterstrom1 , Don C. Lamb2 , Melike Lakadamyali1

1 ICFO-Institut de Ciències Fotòniques, The Barcelona Institute of Science and Technology, 08860 Castelldefels (Bar-celona), Spain2) Ludwig-Maximilians-Universität München, Department Chemie, Physikalische Chemie, Butenandt-str. 11, Haus E, D-81377 München, Germany

Motor-driven cargo transport helps deliver several types of vesicles to the proper location of function. Motor proteins, such as dynein and kinesin, tether and move vesicles through the complex 3D microtubule network. Motors encounter different roadblocks on their way such as microtubule intersections, patches of microtubule associated proteins, or other vesicles and organelles. The mechanisms that allow motors to overcome these roadblocks still remain unclear.

We have developed a correlative imaging approach that combines single particle tracking with super-resolution mi-croscopy (Bálint, Verdeny, Sandoval, Lakadamyali, PNAS 2013). Using this method, we showed that vesicles pause at tight microtubule intersections, likely because the intersecting microtubule constitutes a roadblock. Our initial imag-ing approach was limited to 2D single particle tracking making it diffi cult to determine the precise mechanisms that motors use to overcome these roadblocks.

In this work, we overcame several technical challenges to extend the correlative imaging method to 3D single particle tracking, which allowed us to visualize 3D motion of vesicles in the context of the microtubule network. We show that vesicles move in two different modes along individual microtubules. Typically, they follow the microtubule long axis in a straight line although in 30% of cases they can also move in 3D around the microtubule. Importantly, this second mode of motion is correlated to events such as switching from one microtubule to another at microtubule intersec-tions and passing other vesicles on the same microtubule. In addition, vesicles perform longer runs when moving in 3D around the microtubule. Thus, this mode of motion seems to be a more effi cient way of transport and it likely plays an important role in avoiding roadblocks. Overall, these results provide new insights into the mechanisms that motor proteins may use to overcome different types of obstacles when navigating through the microtubule network.

Unravelling 3D cargo transport dynamics at the microtubule network

24.

10Anika Raulf1 , Ralph Wieneke2 , Alina Kollmannsperger2 , Robert Tampé2 , Mike Heilemann1

1 Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Germany2 Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Germany

Single-molecule localization microscopy (SMLM) allows near-molecular resolution imaging of target proteins deco-rated with fluorescent probes of different size, and therefore labeling strategies are an important factor to consid-er. Here, we introduce a small labeling pair (SLaP)1 consisting of the genetically encoded His-tag and a Ni-trisNTA coupled fluorophore. Ni-trisNTA has a high affi nity for His-tagged proteins (KD ≤ 10 nM)2 and facilitates labeling of target proteins with bright organic fluoro¬phores required for direct stochastic optical reconstruction microscopy (dSTORM)3.

The high target specifi city was determined by labeling GFP- and His10-tagged β-actin with Ni-trisNTAAlexa647 via SLaP and measuring the colocalization of both signals using confocal microscopy. The suit¬¬ability of SLaP for SMLM was demonstrated by imaging actin fi laments with widths down to 40 nm and a localization precision of 16.6 nm. Further¬more, the small size of the SLaP label (~1 nm) minimizes the localization inaccuracy in SMLM tech-niques. We demonstrated this by measuring the apparent cluster size of the ABC transporter TAP which was either immuno¬stained or SLaP labeled. We found a difference of 22 nm in cluster size, which can be largely explained by the difference in the size of the label. A combination of SLaP with microfluidic cell squeezing4 enables specifi c in-cell targeting of His-tagged proteins for live-cell microscopy with high uptake effi ciencies (< 80 %) and cell survival rates (>90 %)5.

Small Labeling Pair for Single-Molecule Localization Microscopy

25.

Jacob Hoogenboom1

1 Department of Imaging Physics, Delft University of Technology, Delft, the Netherlands

Superresolution (SR) fluorescence has revolutionized the fi eld of optical microscopy by achieving molecular locali-zation well below the diffraction limit of light[1]. However, the non-labelled structural context of the cell remains dark, while this context may be crucial in understanding how biological molecules function. Correlation of SR data with structural images obtained with electron microscopy (EM) has been demonstrated [2], but requirements for SR microscopy are often in conflict with those for EM. Moreover, registration of separate SR and EM images may be dif-fi cult and introduce additional errors on top of the optical localization accuracy. I will present alternative approaches to achieve molecular localization within the cellular context using integrated light and electron microscopy. In our system, a high numerical aperture fluorescence microscope is integrated inside a scanning EM in such a way that the electron beam can be positioned anywhere within the fi eld of view of the fluorescence microscope[3]. This allows to record fluorescence-guided EM snapshots during live-cell dynamics[4], as well as to achieve SR within a high-resolution structural EM image by monitoring electron-induced modifi cations of the fluorescence signal from labelled bio-molecules.

[1] B. Huang, M. Bates, and X. Zhuang, Annual review of biochemistry, 78, 993-1016 (2009).[2] P. de Boer, J.P. Hoogenboom, and B.N.G. Giepmans, Nature Methods 12(6.), 503–513 (2015).[3] A.C. Zonnevylle et al., Journal of Microscopy 252, 58-70 (2013).[4] N. Liv et al, ACS Nano 10, 265-273 (2016)

Super-resolution in the cell structural context using focused electron beams

11

26.

Christian Eggeling2

1 MRC Human Immunology Unit & Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford, OX3 9DS, United Kingdom, christian.eggeling@rdm.ox.ac.uk

Plasma membrane interactions such as the transient protein-protein or protein-lipid complexes, the formation of lipid nanodomains (often denoted “rafts”), or diffusional restrictions by the cortical cytoskeleton are considered to play a functional part in a whole range of membrane-associated processes. However, the direct and non-invasive observation of such structures in living cells is impeded by the resolution limit of >200nm of a conventional far-fi eld optical microscope. Here we present the use of the combination of super-resolution STED microscopy with fluores-cence correlation spectroscopy (FCS) for the disclosure of complex nanoscopic dynamical processes. By performing FCS measurements in focal spots tuned to a diameter of down to 30 nm, we have obtained new details of molecular membrane dynamics, such as of transient lipid-protein interactions and of diffusional restrictions by the cortical cytoskeleton. Further insights will be given for molecular dynamics in the plasma membrane of immune cells, specifi -cally during T-cell activation.

Advances in super-resolution STED microscopy: Prospects for studying membrane bioactivity

12

27.

Oriol Gallego1

1 IRB-Institute for Research in Biomedicine, Spain

During exocytosis the secretory vesicles are tethered to the plasma membrane and subsequently fuse with it. The exocyst is a hetero-octameric protein complex that mediates the tethering step and is essential for the viability of eu-karyotic cells. The structure of the exocyst has been extensively investigated but its molecular organization remained unknown, probably because of the large size of the complex and the flexible nature of its subunits.

To determine the molecular organization of the exocyst we designed a hybrid approach that integrates live cell im-aging with the structural information available for each of the exocyst subunits. First, we engineered yeast cells to express defi ned anchor platforms on their plasma membrane to which we recruited the complex in controlled orientation. The anchor was labeled with a fluorescent protein to act as a landmark and the different subunits were tagged one at the time with a different fluorophore at their N- or C-termini. We imaged yeast cells at their equatorial plane where each exocyst subunit and the anchor platform appeared as pairs of colocalising fluorescent spots on the plasma membrane. We measured the centroid positions of the spots in each pair and, by observing a large number of pairs, we estimated the average distance between the spots with a precision of 5 nm or better. These distances positioned the termini of each subunit in respect to the anchor platform. We then used these distances, which we integrated with the structural properties of the subunits, as constrains to reconstruct the molecular organization of the full complex.

Our results show the molecular organization of the exocyst complex for the fi rst time and directly in living cells. The exocyst subunits are rod-shaped and they use one end of the rod to form the core of the complex where all the subunits are connected. The other ends of the subunits protrude outward from the core to mediate vesicle tethering. We also reconstructed the exocyst complex with an attached vesicle. This reconstruction revealed how the exocyst complex can tether the vesicle to the plasma membrane without hindering the subsequent fusion step. Our results provide the structural basis for understanding the molecular mechanisms of exocytosis.

The molecular organization of the exocyst determined by live cell imaging

13

28.

14Markus Mund1 , Jan van der Beek1 , Andrea Picco2 , Marko Kaksonen2 , Jonas Ries1

1 European Molecular Biology Laboratory (EMBL)2) University of Geneva

Clathrin-mediated endocytosis is a highly intricate cellular process, which involves the ordered recruitment and dis-assembly of around 60 proteins. Live-cell microscopy has led to tremendous insight into composition and dynamics of the endocytic machinery, but its diffraction-limited resolution is above the size of endocytic structures. Electron microscopy on the other hand offers nanometer resolution, but lacks molecular specifi city. Thus, the structural or-ganization of endocytic proteins in situ is largely unknown.

We use single-molecule localization microscopy (PALM/STORM) to determine this crucial missing information on protein localizations within the endocytic machinery and focus on Saccharomyces cerevisiae as a model organism. Currently, live-cell superresolution microscopy is still too limited in terms of spatial and temporal resolution, the num-ber of frames that can be acquired and the number of proteins that can be studied simultaneously. Thus, our approach is to image specifi c proteins in a large number of unsynchronized fi xed endocytic sites in dual-color in combination with a reference protein. With the help of this reference protein we assign an exact time point to each site and inte-grate data for many different proteins into one common coordinate system. The aim is to acquire a time-resolved localization map of the entire endocytic machinery, which would provide us with a comprehensive structural picture of endocytosis in yeast.

In yeast endocytosis, polymerizing actin provides the force to invaginate the membrane with a high effi ciency and remarkable temporal and spatial regularity. We directly visualize the structural relation between polymerized actin and actin interacting proteins to ultimately understand how actin polymerization is regulated in situ. We discovered that several actin nucleation promoting factors and coat proteins show a striking ring-shaped organization even prior to actin polymerization [1]. This lateral pre-patterning of the endocytic site provides an elegant explanation for an ef-fi cient force generation by the actin machinery and force transfer to the membrane.

[1] Picco, A. et al. Visualizing the functional architecture of the endocytic machinery. Elife 4, (2015).

Superresolution imaging of clathrin-mediated endocytosis in yeast

29.

15Diego Krapf1

1 Department of Electrical and Computer Engineering and School of Biomedical Engineering, Colorado State University

Tracking individual proteins on the surface of live mammalian cells reveals complex dynamics involving anomalous diffusion. Theoretical models show that anomalous subdiffusion can be caused by different processes. By perform-ing time series and ensemble analysis of extensive single-molecule tracking we show that two anomalous sub-diffusion processes simultaneously coexist and only one of them is ergodic. Weak ergodicity breaking is found to be maintained by immobilization events that take place when the proteins are captured within clathrin-coated pits. Furthermore, using a combination of dynamic super-resolution imaging and single-particle tracking, we observe that the actin cytoskeleton introduces barriers leading to the compartmentalization of the plasma membrane and that proteins are transiently confi ned within actin domains. Our results show that the actin-induced compartments are scale free and that the actin cortex forms a self-similar fractal structure.

Anomalous diffusion and compartmentalization on the surface of mammalian cells

30.

16Diane Lidke1

1 University of New Mexico, Department of Pathology

Complex cellular processes are governed by signal transduction, which in turn is controlled by protein-protein inter-actions at the plasma membrane and along the signaling cascade. Fluorescence imaging technologies are making it possible to quantify the protein dynamics that regulate cell signaling in living cells. We have used single molecule imaging to visualize the early events in membrane receptor signaling, with a focus on FcεRI. FcεRI is a member of the immunoreceptor family that also includes the TCR, the BCR and several IgG receptors. Crosslinking of FcεRI by multivalent antigen initiates signaling cascades involving Lyn, Syk and LAT that ultimately lead to release of key me-diators of allergic inflammation. Single particle tracking of FcεRI using a variety of probes (quantum dots, fluorogen-activating peptides) has revealed the relationship between receptor mobility, stability and membrane interplay during signaling. We have also quantifi ed the kinetics of signaling molecule recruitment to the FcεRI complex by tracking individual Syk molecule dynamics in living cells, revealing the influence of functional Syk mutations on binding life-times and cellular outcomes. The high spatiotemporal data sets provided by single molecule imaging are fi lling the gaps in our picture of cell signal transduction.

Imaging the early events in membrane receptor signaling

31.

17

Stefan Balint1 , Filipa B. Lopes1 , Daniel M. Davis1

1 Manchester Collaborative Centre for Inflammation Research, University of Manchester

Phagocytosis by macrophages is controlled by the balance of signals from activating Fc receptors (FcRs) and inhibi-tory receptors such as signal regulatory protein alpha (SIRPα). Despite the importance of signal integration between these receptors, their spatial organization at the nanometer-scale has not been examined at the surface of human macrophages. Here, using dual-color direct stochastic optical reconstruction microscopy (dSTORM), we report that FcγRI, FcγRII and SIRPα are constitutively organized in discrete nanodomains with a radius of 76 nm, 60 nm, and 49 nm, respectively, at the surface of primary human macrophages. Nanodomains of FcγRI, but not FcγRII, are con-stitutively in close proximity to nandomains of SIRPα, with a nearest neighbor distance (NND) of 35 nm. However, on surfaces coated with human IgG, nanodomains of FcγRI and SIRPα segregate (NND, 165 nm). In addition, upon activation, FcγRs re-organize at a micrometer-scale to form periodically spaced concentric rings. The segregation of nanodomains, as well as the formation of FcγRI rings, was dependent on Src-family kinase signaling and the actin cytoskeleton. These results are consistent with the proximity of nanodomains of activating and inhibitory receptors being important for signal integration in human macrophages.

Ligation of activating FcγRI nanodomains at macrophage surfaces causes them to segregate from nanodomains of inhibitory SIRPα and re-organize into concentric rings

32.

18Grégory Giannone1

1 CNRS, Interdisciplinary Institute for Neuroscience, University of Bordeaux, UMR 5297, F-33000 Bordeaux, France

Super-resolution fluorescence microscopy techniques revolutionized biomolecular imaging in cells by delivering op-tical images with spatial resolutions below the diffraction limit of light. The direct observation of biomolecules at the single molecule level enables their localization and tracking at the scale of a few tens of nanometers and opens new opportunities to study biological structures at the scale of proteins inside living cells. We are using super-resolution microscopy techniques and single protein tracking (SPT) to study adhesive and protrusive sub-cellular structures, including integrin-dependent adhesion sites and neuronal dendritic spines.

Integrin-mediated cell adhesion to the extracellular matrix and mechano-transduction are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies such as cancer. Yet the molecular events controlling integrin biochemical and mechanical activation within adhesion sites (FAs) are still not understood. We unravel the key spatiotemporal molecular events leading to integrins activation by their main activator talin in mature FAs. We performed SPT combined with PALM (sptPALM) and super-resolution microscopy to study integrins and talin displacements and distributions outside versus inside mature FAs. We dem-onstrated that FAs are specialized platforms priming integrins immobilization. Using the same experimental strategy, in collaboration with the group of Valerie Weaver (UCSF, USA), we studied how bulky membrane glycoproteins regulate integrin diffusive behavior and activation. Our fi ndings support a model where large glycoproteins act as physical “steric” barriers impeding integrins immobilization and thus funneling integrins clustering into adhesive contacts. Thus control of membrane nano-topology by the glycocalyx could mechanically enhanced integrin activation and could foster metastatic progression. Using the same approaches we studied the nanoscale dynamic organization of F-actin regulators in neuronal dendritic spines. We show that within spines branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at membrane protrusion tips. This organization is opposite to classical lamel-lipodial protrusive structures where branched F-actin nucleation and elongation occur at protrusion tips. Overall, our studies suggest that the spatial segregation of specifi c protein interactions into molecularly distinct nano-domains could be a general mechanism to regulate locally protein activity within sub-cellular structures.

Deciphering the spatiotemporal regulation of integrin and actin regulators at the nanoscale

33.

19Koen van den Dries1 , Marjolein Meddens1 , Elvis Pandzic2 , Ben Joosten1, Johan Slotman3 , Leila Azar4 , Kees Jalink4 , Adriaan Houtsmuller3 , Paul Wiseman2 , Alessandra Cambi1

1 Radboud University Medical Center2 McGill University 3 Erasmus MC4 The Netherlands Cancer Institute & van Leeuwenhoek Centre of Advanced Microscopy

Podosomes are dynamic integrin- and actomyosin-based multi-molecular adhesion structures formed at the ventral cell membrane and involved in cell protrusion, topography sensing and extracellular matrix degradation. Formed by a variety of cells, they have a dotted shape and undergo irregular cycles of growth and shrinkage driven by actin po-lymerization and myosin contractility. With an actine core of ~0.5um, hundreds of podosomes organize in different mesoscale spatial arrangements ranging from large circular belts in bone-degrading osteoclasts to small rosettes in endothelial cells and well-defi ned large clusters in antigen-presenting cells. The molecular mechanisms regulat-ing formation and maintenance of these mesoscale arrangements are still poorly defi ned. By integrating a variety of advanced microscopy techniques, we aim to unravel the nanoscale structural and dynamic complexity of individual podosomes as well as formation, architecture and function of mesoscale podosome clusters. We previously demon-strated that, at the nanoscale, single podosomes comprise a dense actin core with radiating actin fi laments decorated by vinculin, and that integrins and talin densely cover the entire cluster area except the actin core regions. At present, by exploiting an extension of spatiotemporal image correlation spectroscopy (STICS), we measured the evolution of spatiotemporal molecular dynamics of podosome components, revealing self-organized dynamic spatial patterns of various cytoskeletal components throughout the clusters. Furthermore, by multi-color SIM and 3D STORM, we revealed the presence of membrane-subcortical actin substructures regulating the core nanoscale architecture that likely contribute to their protrusive behaviour and tensional integrity. Together, our fi ndings propose podosomes as highly organized sub-membrane cortical actin microarchitectures mediating tensional integrity at the nanoscale and providing a feedback mechanism for cellular mechanosensing at the mesoscale.

Integrating advanced microscopy techniques reveal actin nanoscale architectures and mesoscale dynamics of mechanosensory podosomes

34.

20Amanda Remorino1 , Aurelien Duboin1 , Jean-Baptiste Masson2 , Maxime Dahan1 , Mathieu Coppey1

1 Institut Curie, UMR 168, Paris, France2) Institut Pasteur, CNRS URA 2171, Unit In Silico Genetics, 75724 Paris Cedex 15, France

The small RhoGTPase Rac1 is involved in the signaling network regulating cell polarization and migration. Rac1 is activated and deactivated at the plasma membrane and its active form recruits a variety of effectors control-ling the cytoskeleton dynamics. It has been shown that Rac1 presents detailed patterns of activation/deactivation with micrometer and millisecond resolutions key to the generation and maintenance of a polarized cell state. Here, we present a methodology combining optogenetics, single molecule tracking, super-resolution imaging, and protein micro-patterning, to interrogate the molecular architecture and dynamics of Rac1 at the molecular level as a function of its activation state. Moreover, using novel computational tools, we could obtain diffusivity maps of Rac1 across the whole cell with high temporal and spatial resolution. The results show a link between activity and diffusivity of Rac1 mediated by the formation of Rac1 clusters that are localized at the cell front. These clusters behave as signaling platforms composed of Rac1, lipids and upstream and downstream molecules and could be used as mechanism to localize Rac1 activity to specifi c regions.

Spatio-temporal regulation of Rac1 signalling

35.

21Helge Ewers4 , David Albrecht2 , Christian M. Winterflood2 , Thomas Tschager3

1 Institut für Chemie und Biochemie, Freie Universität Berlin, Germany2 Randall Division of Cell and Molecular Biophysics, King’s College London, United Kingdom3 Institute of Biochemistry, ETH Zurich, Switzerland4 Free University Berlin

The axon initial segment (AIS) is enriched in specifi c cytoskeletal and adaptor molecules that anchor transmembrane proteins to the cytoskeleton. Concurrent with the establishment of this dense and complex structure during neuronal development, a barrier to the lateral exchange of membrane molecules between the axon and the somatodendritic domain is formed. Recently, a periodic pattern of actin, spectrin and ankyrin forming 190 nm distanced, ring-like structures perpendicular to the direction of axonal propagation has been discovered. However, whether this structure is related to the diffusion barrier function is not clear.

Here, we performed single-particle tracking timecourse experiments on hippocampal neurons during AIS develop-ment. We furthermore analyzed the lateral mobility of lipid-anchored molecules by high-speed single-particle track-ing. Finally, we correlated positions of membrane molecules with the nanoscopic organization of the AIS cytoskeleton.

We fi nd that membrane protein mobility becomes reduced in the AIS early during development. The lateral motion of membrane proteins in the AIS plasma membrane is confi ned to a repetitive pattern of ~190 nm spaced segments along the AIS axis as early as DIV4 and this pattern is framed by actin rings. Our data provide a new model for the mechanism of the AIS diffusion barrier.

Nanoscopic compartmentalization of membrane protein motion at the axon initial segment

36.

22Lukas Kapitein1

1 Utrecht University

The Kapitein lab studies the mechanisms by which cells establish and maintain their precise shape and intracellular organization. This is important, because form and function are closely connected and cellular disorganization often leads to cellular dysfunction and disease. By combining protein engineering, optogenetics, advanced microscopy techniques and mathematical modeling, we aim to obtain a mechanistic understanding of cellular organization in health and disease.

In my lecture, I will highlight two recent breakthroughs from the lab. First of all, we successfully engineered a sys-tem to control the transport and positioning of intracellular components with light. This allows us to directly explore the functional consequences of organelle mislocalization. In addition, we have developed a novel approach for the super-resolution imaging of microtubules, the polarized biopolymers that serve as tracks for intracellular transport. Importantly, our approach enables us to directly determine the polarity of individual microtubules, which is the key determinant of motor directionality. This method allows us to better resolve microtubule organization in different cel-lular compartments, such as the axons and dendrites.

Navigating the cytoskeleton: novel tools to dissect and direct intracellular transport

37.

23Xavier Darzcaq1

1 University California, Berkeley, USA

Formation of the large protein complexes regulating transcription such as the pre-initiation complex (PIC) is the result of an equilibrium in between the frequency at which its principal components assemble and disassemble. To date a lot of information has been collected on how the off rate of a specifi c interaction is regulated. Here I will concentrate on two mechanisms controlling the on rate of proteins assembly at the level of their ability to sample the nucleoplasm using effi cient strategies to search for their targets.

While the on rate of a particular interaction is a well known parameter defi ned by the Smoluchowski equation link-ing the diffusion coeffi cient, the concentration and the cross section of the interaction. We recently reported that the concentration of RNA polymerase II is not homogeneous in the nucleoplasm; this may play an important role in regu-lating transcription (Cisse et al, Science 2013). Moreover we showed that different transcription factors explore the nucleoplasm using different mechanisms. Notably the Positive Elongation Factor b (P-TEFb) massively oversamples space redundantly revisiting the same sites before translocating to other positions (Izeddin et al, Elife 2014 ; Woringer et al Curr. Opin Chem Biol 2014). Our observations are consistent with a model in which the exploration geometry of the P-TEFb is restrained by its interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of P-TEFb target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time. Here we report that the 7SK snRNP complex acts as a chaperone controlling its engagement with the transcription machinery and therefore tightly regulates the P-TEFb exploration mode. I will discuss these different mechanisms and their im-plications on enzymatic processivity regulation and gene expression control.

Single molecule imaging reveals nuclear domains modulating transcription regulation

38.

24Diego Cattoni1 , Andrés Cardozo Gizzi1 , Mariya Georgieva1 , Alessandro Valeri1 , Fréedéeric Bantignies2 , Delphine Chamousset1 , Pau Farré4 , Stephanie Dejardin1 , Jean-Bernard Fiche1 , Olivier Cuvier3 , Eldon Emberly4 , Giacomo Cavalli2 , Marcelo Nollmann1

1 Centre de Biochimie Structurale, CNRS UMR5048, INSERM U1054, Université de Montpellier, 29 rue de Navacelles, 34090 Montpellier, France2 Institut de Génétique Humaine, CNRS UPR 1142, 141 rue de la Cardonille, 34396 Montpellier, France3 Laboratoire de Biologie Moléculaire Eucaryote, CNRS, Université de Toulouse, Toulouse, France4 Simon Fraser University, Burnaby, Canada

Chromosomes from bacteria to humans are organized at the sub-megabase scale into topological domains (TDs). Borders between TDs are constitutive, while their internal organization and their association dynamics are cell-type specifi c. Recent studies proposed that in mammalian genomes sequential TD borders associate at sites containing convergent sites of the CTCF insulator protein. Here, we combined super-resolution and oligoPAINT technologies to investigate the roles of Beaf-32 -the insulator protein most overrepresented at TD borders in Drosophila- in the as-sociation of domain boundaries at the single-cell level. We found that sequential and nonsequential barriers in two genomic loci in chromosomes 2L and 3R followed the path expected for a self-avoiding random polymer and did not display specifi c association. Distances between barriers flanking black TDs followed exactly the model. Interestingly, barriers flanking active TDs exhibited larger distances than those expected by the model, while boundaries surround-ing Polycomb TDs were closer, consistent with distances among barriers reflecting the transcriptional activity of the intervening TD. 69 TD barriers homogeneously spread across chromosome 3R appeared in average as single clusters, consistent with constitutive association between TD borders. Finally, the size, number and composition of Beaf-32 clusters imaged at super-resolution are consistent with Beaf-32 clusters representing single TD borders. Interestingly, Beaf-32 clusters surround H3K27me3 and Polycomb territories while it overlaps to a large extent with transcriptionally active sites. Overall, our data is in support of a model by which TDs in Drosophila are in part formed and maintained by the combined roles of active transcription and self-association of chromatin elements of the same epigenetic types.

Super-resolution imaging of topological barriers reveals higher-order chromatin folding principles

39.

25Kyle M. Douglass1 , Aleksandra Vancevska2 , Verena Pfeiffer2 , Joachim Lingner2 , Suliana Manley1

1 Institute of Physics, EPFL, Lausanne, Switzerland2) Swiss Institute for Experimental Cancer Research (ISREC), EPFL, Lausanne, Switzerland

Telomeres are kilobase-scale regions that terminate eukaryotic chromosomes and protect their ends from degrada-tion, DNA rearrangements, and DNA damage signalling [1]. To achieve these functions, telomeres associate with the shelterins, proteins that are thought to confer structural stability [2]. Furthermore, telomeres exhibit an unusually tight arrangement of nucleosomes [3], which suggests that telomere architecture is important for their functioning. To date, however, methods to determine their architecture and its relationship to the telomeres\’ function have been lacking.

The reasons for this are two-fold. While much is known about the composition of telomeres through ensemble-based biochemical techniques, there are currently few ways to assess the variability between individual telomeres. Further-more, the telomeres\’ small sizes and rich nucleo-protein composition leaves tools like fluorescence imaging—which are typically well-suited for structural studies of larger organelles—unable to extract detailed information about the arrangement of the telomeric proteins and DNA.

We addressed these issues by developing a quantitative imaging and analysis platform based on the super-resolved STORM modality [4] for studying fluorescently labeled telomeres in situ. Though the individual telomeric fi bers and proteins could not be resolved, a number of features could still be well-determined from the super-resolution data.

These measurements, when combined with quantitative telomere length assessments, the known abundance of epi-genetic markers, and polymer simulations, allowed us to make the following conclusions: the packing density (the linear density of base pairs per length of the telomere fi ber) in two different but isogenic cell lines can vary signifi -cantly and correlates with the known abundance of heterochromatic marks. Furthermore, we found that the pres-ence of shelterin proteins have a weak affect on the telomeres\’ physical size, which we will discuss in the context of recent and related work [5]. Finally, we found that the telomeres are not, in fact, as compact as a canonical model for chromatin architecture would predict, forcing us to re-examine what it means for telomeres to be “compact” in quantitative terms.

[1] de Lange, T., Science 326, 5955 (2009).[2] Doksani, Y., Wu, J.Y., de Lange, T. and Zhuang, X. Cell 155, 345 (2013).[3] Lejnine, S., Makarov, V.L. & Langmore, J.P. Proc. Natl. Acad. Sci. U.S.A. 92, 2393 (1995).[4] Rust, M. J., Bates, M., and Zhuang, X. Nat. Methods 3, 793 (2006).[5] Bandaria, J. N., Qin, P., Berk, V., Chu, S. and Yildiz, A. Cell 164, 735 (2016)

Telomeres are kilobase-scale regions that terminate eukaryotic chromosomes and protect their ends from degrada-tion, DNA rearrangements, and DNA damage signalling1. To achieve these functions, telomeres associate with the shelterins, proteins that are thought to confer structural stability2. Furthermore, telomeres exhibit an unusually tight arrangement of nucleosomes3, which suggests that telomere architecture is important for their functioning. To date, however, methods to determine their overall compaction and shape have been lacking

Quantitative imaging of human telomere structure

40.

26Marcelo Nollmann1

1 CNRS/INSERM, Montpellier, France

Chromosomes from bacteria to humans are organized at the sub-megabase scale into topological domains (TDs). We will present examples of how 3D super-resolution microscopies can be combined with new DNA labeling technolo-gies and genome-wide chromosome capture methods to shed light into the mechanisms of formation and regulation of TDs. In particular, these methods allow us to understand how heterogeneity in a population may play a biological role.

Chromosomes from bacteria to humans are organized at the sub-megabase scale into topological domains (TDs).

41.

27Maria Pia Cosma1

1 Center for Genomic Regulation (CRG) and ICREA, Barcelona, Spain

One of the interests of my group is to dissect how the dynamics of the Wnt pathway controls somatic cell reprogram-ming and pluripotency in embryonic stem cells. We use experimental and modeling approaches to associate the pathway dynamics to biological functions. Recently, in collaboration with Lakadamyali and Garcia-Parajo groups by using quantitative super-resolution nanoscopy, we identifi ed a novel model of chromatin fi ber assembly and the rela-tion among the decoded structure and naïve pluripotency. We showed that nucleosomes arrange in groups of various sizes along the chromatin fi ber, which we named ‘nucleosome clutches’ (in analogy with clutches of eggs). Clutches are interspersed with nucleosome-free regions. Interestingly, we found that ground-state pluripotent stem cells have, on average, clutches that are less dense and contain fewer nucleosomes. We are now investigating the remodeling of the chromatin fi ber during the transition of cells undergoing differentiation and reprogramming upon perturbation of the Wnt pathway.

The nanoscale structure of chromatin fi bers correlates with cellular state

42.

28Davide Mazza2, Rohini Nair1, Marco E. Bianchi3, Alessandra Agresti1

1 Division of Genetics and Cell Biology, San Raffaele Scientifi c Institute, Milan, Italy2 Center for Experimental Imaging, San Raffaele Hospital, Milan, Italy3 San Raffaele University, Milan, Italy

Contrary to what written in textbook, the amount of histones – and therefore of nucleosomes – is not a fi xed param-eter but a tunable one that cells exploit to adapt or respond to the external environment.

We showed that in macrophages exposed to stress signals, nucleosome reduction facilitates the transcriptional re-sponse of inflammatory genes1. Along the same line, the chromatin of ES cells acquires nucleosomes as differentia-tion progresses, suggesting that the difference in histone content is an additional hallmark of pluripotency2, in addi-tion to and besides histone modifi cations.

Mouse Embryo Fibroblasts (MEFs) progressing toward senescence reduce their nucleosome content3. A 30 % nuc reduction is also detected when HMGB1 (High Mobility Group Box 1 protein) is genetically ablated thereby causing increased sensitivity to DNA damage and increased transcription genomewide4.

We thus wondered about chromatin structure when nucs are 30% less and used HMGB1 KO as a proxy for such nu-cleosome decrease.

Starting from the observation that nuclei of human and murine cells depleted of HMGB1 are larger, we studied chro-matin compaction by quantitative super-resolution nanoscopy in live cells (STORM). Chromatin fi bers with a reso-lution of 20 nm were visualized by tagging the core histone H2Ain G1 synchronised MEFs from wt and HMGB1 ko embryos. We found that the average size of histone clutches 5 is reduced in nuc-poor cell as compared to those in nuc-rich cells.

Single molecule tracking of chromatin-incorporated histones revealed that in nuc-poor living cells the nucleosome movements in the chromatin context are corralled within a smaller confi ned radius but have a higher diffusion coef-fi cient.

When analysed by FRAP, the histone exchange rate was also lower in nuc-poor chromatin that also showed a broader immobile fraction. Finally, by confocal time-lapse microscopy, the turnover rate of histones is faster in nuc-poor cells

All together these preliminary results in living cells are consistent with our previous in vitro observations4 and support the notion that stable nucleosomes are very stable and labile nucs are even more labile in nuc-poor cells. Overall, chromatin appears to be more decondensed and more accessible.

REFERENCES: 1) De Toma, I. D., doi:10.1111/joim.12286, 2) Karnavas, T., doi:10.3389/fphys.2014.00330, 3) O\’Sullivan, R. J., doi:10.1038/nsmb.2754, 4) Celona, B., doi:10.1371/journal.pbio.1001086, 5) Ricci M. A., doi:10.1016/j.cell.2015.01.054 (2015).

Chromatin organization in nucleosome-rich and nucleosome-poor cells

43.

29Anna Oddone1 , Petra Stockinger2 , Melike Lakadamyali1 , Jerome Solon2 , Manuel Mendoza2

1 The Institute for Photonic Sciences (ICFO)2 Centre for Genomic Regulation (CRG)

Chromosome condensation, the dramatic reorganization of sparse interphase chromatin into compact chromosome structure, is a fundamental aspect of cell division that ensures correct matching of chromosome size to spindle size. Yet, the molecular mechanisms underlying this process are still poorly understood.

Here, we investigate chromatin compaction in Drosophila melanogaster\’s developing neural system. During development, Drosophila’s neuron precursors divide asymmetrically yielding two daughter cells of different size: a large neuroblast (NB) and a small ganglion mother cell (GMC). NBs typically undergo additional cycles of asymmetric division, whereas GMCs eventually divide and differentiate into neurons. We show that chromatin compaction upon entry into mitosis is similar for NBs and GMCs, suggesting that the observed difference in mitotic chromosomes’ length is due to a difference in chromatin packaging already established in interphase. We investigate interphase chromatin architecture in NBs and GMCs using a combination of 3D STORM imaging and biophysical analysis, and show that the chromatin nanoscale organization is different between these siblings despite their identical DNA content. We now aim at understanding the role of nuclear volume and cell fate in chromatin packing in these two types of cells.

Chromatin organization in Drosophila melanogaster’s developing neural system

44.

30Bo Huang1

1 Department of Pharmaceutical Chemistry, University of California, San Francisco

Cellular processes are carried out by coordinated participation of many biomolecules in a tiny volume. Many people have been dreaming to see clear pictures of these processes in order to understand how these molecules work together. Taking on this challenge, we are developing new imaging techniques and imaging probes, including the use of super-resolution microscopy to dissect the architecture of centrosome, as well as fluorescent probes based on the CRISPR-Cas9 system to visualize the spatial organization of the genome.

Life inside the cell: STORM, CRISPR and imagenomics

45.

31Kyle Legate1

1 Kyle Legate

Nature Communications is the flagship open access journal of the Nature family. This talk will describe the hierar-chy of the Nature family of journals, and what distinguishes the journals from each other. It will also cover editorial policies within the journal, and will describe new initiatives that Nature Communications is taking to improve author services and the publishing experience.

Insights into Editorial policies of Nature Communications

46.

Abstracts

Poster

47.

P01. Rafael Porcar-Guezenec. Imagine Optic - SPAIN. Ultimate adaptive optics correction for 3D single molecule localization microscopy

P02. Pamina Winkler. ICFO-Institute of Photonic Sciences, Spain. Plasmonic nanogap antennas to reveal spatiotemporal heterogeneities of model lipid membranes

P03. Lin Wang. STFC, Research Complex at Harwell, Rutherford Appleton Laboratory, UK. Using single molecule localisation microscopy to study the three-dimensional structure of the assembly factor LhaA in the photosynthetic membrane of purple bacterium Rhodobacter sphaeroides

P04. Maria Victoria Neguembor. CRG-Centre for Genomic Regulation, Spain. Unraveling DNA looping at the nanoscale resolution

P05. Laurent Ladepeche. ICFO-Institute of Photonic Sciences, Spain. Synaptic nanoscale reorganization during anti-NMDAR autoimmune encephalitis

P06. Lara Laparra. ICFO-Institute of Photonic Sciences, Spain. Multi-color STORM in neurons reveals molecular organization of the LGI1 synaptic complex in autoimmune encephalitis

P07. Hanae Shimo. Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge Structure and dynamics of actin assembly factors during fi lopodia formation

P08. Alvaro Castells Garcia. CRG-Centre for Genomic Regulation, Spain. Cell differentiation can be investigated by imaging the nuclear organization of RNAs with STORM

P09. Nitin Mohan. ICFO-Institute of Photonic Sciences, Spain. Tubulin Post Translational Modifi cations Influence Cargo Transport in Live Cells

P10. Bassam Hajj. Institut Curie, Physico Chimie PSF engineering for single molecule localization in multifocus microscopy

P11. Laura Martinez. Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Campus de Cantoblanco, Madrid CXCR4 nanoscale organization and dynamics influence CXCL12-mediated responses

P12. Antonios Lioutas. Center for Genomic Regulation (CRG), Barcelona, Spain Steroid hormone receptor interaction with the genome studied with super-resolution microscopy

P13. Jason J. Otterstrom. ICFO-Institute of Photonic Sciences, Spain. Visualizing Chromatin Structure at the Nanometer Scale

P14. Izabela Piechocka. ICFO-Institute of Photonic Sciences, Spain. Shear flow induces changes in ICAM-1 spatial distribution that modulate leukocyte mobility across endothelium

P15. Laurent Fernandez. Centre de Biochimie Structurale, CNRS UMR 5048-INSERM UMR 1054, Montpellier, France The metastasis suppressor CD82 and gangliosides : two key players of the plasma membrane organization?

P16. Guillaume Cordier. ICFO-Institute of Photonic Sciences, Spain. Motor-cargo organisation studied with single localisation

P17. Kyra Borgman. ICFO-Institute of Photonic Sciences, Spain. Tyrosine Kinase Receptor MerTK: from the cell membrane to the nucleus

P18. Pablo Mateos. Department of Biotechnology & Biophysics, University Wuerzburg, Germany Super-resolution imaging of plasma membrane proteins with click chemistry

P19. John Danial. Max Planck Institute for Intelligent Systems Single molecule analysis of membrane-embedded BAXP20. Catalina Martinez .ICFO-Institute of Photonic Sciences, Spain. Spatial organization of the HIV-binding receptor

Siglec-1 on the membrane of human dendritic cellsP21. Anne-Marie Byrne. School of Medicine Trinity College Dublin, Irland. Golgi structure and membrane traffi cking in

patient tissue; can we translate in vitro observations to the clinic?P22. Klaus Harter. Center for Plant Molecular Biology (ZMBP), University of Tübingen Protein response modules at the

plasma membrane regulating plant cell elongation growthP23. Fabian Wehnekamp. Fablab, Department Chemie, Ludwigs-Maximilians-Universität Munich, Germany 3D Real-

Time Orbital tracking in zebrafi sh embryos: Spatiotemporal analysis of mitchondrial dynamics in rohon-beard sensory neurons

P24. Carmen Krueger. Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University, Frankfurt (Main), Germany Copy number and cluster analysis of toll-like receptor 4 upon treatment with lipopolysaccharide using single-molecule localization microscopy

P25. Cyril Favard. CPBS, CNRS, UM Mapping the energy of HIV-1 assembly in T cells using spt-PALM.P26. Franziska Fricke. Institute of Physical and Theoretical Chemistry, Goethe-University, Frankfurt, Germany

Molecular counting with single-molecule localization microscopy and its application to death receptorsP27. Marta Gironella. Small Biosystems Lab, Universitat de Barcelona Mechanical phenotyping of single cancer cellsP28. Basu Srinjan. Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA,

United Kingdom. Combining single-molecule FRET and PALM for single particle tracking and super-resolution imaging

P29. Sandrine Lévêque-Fort. ISMO-CNRS. 3D molecular architecture of podosomes revealed by Supercritical Angle Fluorescence

P30. Armando Maestro. Cavendish Laboratory, University of Cambridge, JJ Thompson Avenue, CB3 0EH Cambridge, UK. Unravelling the complex choreography of endocytosis through in-vitro experiments

P31. Laura Maddalena. ICFO-Institute of Photonic Sciences, Spain.. A multicolor single particle tracking approach to reveal molecular interactions in a dense milieu.

48.8.

P01

49.

P02Pamina M. Winkler1 , Raju Regmi2 , Valentin Flauraud3 , Hervé Rigneault2 , Jürgen Brugger3 , Jérôme Wenger2 , María F. García-Parajo1

1 ICFO-Institut de Ciences Fotoniques, The Barcelona Institute of Science and Technology2 Institut Fresnel, CNRS, Aix-Marseille Université, Centrale Marseille3 Microsystems Laboratory, Ecole Polytechnique Fédérale de Lausanne4 Institució Catalana de Recerca i Estudis Avançats (ICREA)

Resolving the various interactions of lipids and proteins in the eukaryotic plasma membrane (such as the possible formation of ‘lipid rafts’) with high temporal and spatial resolution is of the utmost interest [1]. Here we introduce an innovative design of plasmonic nanogap antennas to monitor single-molecule events at physiologically relevant concentrations by means of fluorescence correlation spectroscopy (FCS). Arrays of planar nanogap antennas embedded in nanometric-sized boxes facilitate cell culturing in close proximity to the antenna substrates while reducing unwanted membrane curvature effects [2]. Moreover, embedding the antennas in boxes minimizes the contribution of surrounding background enabling the recording of individual molecules as they diffuse on the cell membrane in real time with nanometre precision [2,3]. The antenna gaps work as effective ‘hot spots’ of illumination where local FCS is performed. The different gap sizes provide distinct effective confi nement volumes, from which the characteristic diffusion times of molecules on living cell membranes can be probed.

To demonstrate the validity of our approach, we have fi rst focused on model lipid membranes consisting of a ternary mixture of cholesterol, sphingolipids, and saturated phospholipids. Although relatively simple, this mimetic system recapitulates some of the most important features of biological membranes, i.e., spatiotemporal compartmentalization and lipid phase separation at the nanometer scale. We present results of a model membrane consisting of DOPC and SM on top of the nanogap antenna arrays labelled with the lipophilic fluorescent dye DiD. FCS measurements were performed using antenna gaps in the range of 8-45 nanometres. The binary mixture shows an increase of diffusion time as compared to dyes in solution and a linear dependence of the diffusion time vs. the gap area, indicating random diffusion of the dye within the homogenously mixed bilayer. By adding cholesterol, the behavior of the fi rst steps of phase segregation into domains smaller than 50 nanometres (if existent) is induced and studied.

References:[1] D. Lingwood, K. Simons, Science 327, 46 (2010).[2] V. Flauraud et al, in preparation.[3] D. Punj et al, Nature Nanotechnology 8, 512–516 (2013)

Plasmonic nanogap antennas to reveal spatiotemporal heterogeneities of model lipid membranes

50.

P03

Lin Wang1 , Marisa Martin-Fernandez1 , David Mothersole2 , C. Neil Hunter2

1 Science and Technology Facilities Council, Research Complex at Harwell, Rutherford Appleton Laboratory2 Department of Molecular Biology and Biotechnology, University of Sheffi eld

The purple phototroph Rhodobacter (Rba.) sphaeroides houses an extensive system of vesicular intracytoplasmic membranes (ICM), which increases the internal surface area of membranes and photosystem complexes for ab-sorbing and trapping solar energy. Energy harvested by light-harvesting LH2 complexes migrates to the reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complex, where it is ultimately converted to quinols that are oxidised by the cytochrome bc1 complex. The resulting protonmotive force is used for the production of ATP, and also for other energy-consuming processes such as motility. The mature architecture of the photosynthetic membrane of the Rba. sphaeroides has been highly characterised both by spectroscopic techniques and topographic imaging, however the early stages of its development are less well studied. Moving on from the atomic-level models of bac-terial photosystems and membranes, it is now of interest to investigate the cellular distribution of intracytoplasmic membranes. There is a need to image living cells at high resolution, in order to identify the cellular distribution of specifi cally labelled, functionally essential proteins and complexes; this need can be met by three-dimensional (3D) super-resolution (SR) single molecule localisation microscopy (SMLM). SMLM rely on the labelling of proteins with a chromophore that, when excited, becomes a point emitter that can be localised by a suitable camera/detector system with a precision that varies between 10—100 nm, a signifi cant improvement on conventional diffraction-limited opti-cal microscopy. Protein labelling with fluorophores is best achieved with dyes, if they can gain access to the interior of the cell, but for bacteria in vivo labelling with fluorescent proteins is currently the best method. In this work we have established a method to tag the targeted photosynthetic proteins with SYFP2, a variant of yellow fluorescent protein, and use SMLM to image and analyse the 3D distribution of the assembly factor LhaA, which is involved in the assembly of the LH1 complex, in living Rba. sphaeroides cells. In-frame genomic constructs were used to ensure that gene expression was driven by native promoters and that the native gene copy number was not altered. This fi rst application of 3D SR SMLM to photosynthetic bacteria has been used to optimise the experimental conditions, and to calculate the yield of signals, based on the known cellular levels of one of the tagged proteins, RC-H. The 3D re-constructions of cells grown under high and low oxygen show how the ICM proliferates within the cell, and how much lower levels of the LhaA assembly factor are present, remaining more localised to the peripheral regions of the cell membrane. LhaA is also found predominantly in the indented membrane initiation regions that arise as indentions of the cytoplasmic membrane.

Using single molecule localisation microscopy to study the three-dimensional structure of the assembly factor LhaA in the photosynthetic membrane of purple bacterium Rhodobacter sphaeroides

51.

P04Maria Victoria Neguembor 1

1 CRG

The tridimensional conformation of the genome plays a fundamental role in the control of nuclear function. Chromosomes are folded in a hierarchical manner to establish both constant interactions, conserved acrossspecies and cell types, and dynamic interactions that reflect cell type specifi city. Chromosome looping results from the interplay of specifi c DNA sequences, epigenetic modifi cations and a myriad of nuclear proteins. In particular, CTCF and the cohesin complex are the main regulators of DNA looping. Although variations in nuclear architecture have been mainly studied during cell differentiation, the impact of chromosome folding on somatic reprogramming is still largely unexplored. Yet, reprogramming leads to profound changes of the cellular transcriptome and epigenome that are likely accompanied by major variations in chromosome looping. Taking advantage of STORM super resolution microscopy, we are currently investigating how CTCF andcohesin are organized and remodeled upon somatic cell reprogramming at a nanoscale resolution. Preliminary results suggest that CTCF and cohesin form large nuclear clusters that vary in dimension and distribution between stem and differentiated cells. By extending our analysis to cells at different steps of the reprogramming process, we aim to unravel the morphological bases and dynamics of DNA looping and to assess the contribution of folding factors to reprogramming.

Unraveling DNA looping at the nanoscale resolution

52.

P05Laurent Ladépêche1 , Jesús Planagumà2 , Joe Borbely1 , Lara Laparra1 , Josep Dalmau2 , Melike Lakadamyali1

1 Advanced Fluorescence Imaging and Biophysics group (AFIB), Institut de Ciències Fotòniques (ICFO), Av. Carl Friedrich Gauss 3, 08860 Castelldefels (Barcelona), Spain2 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic c/ Villarroel 170, 08036 Barcelona, Spain

Glutamate synapses, which mediate most excitation in the brain, have the capability to adapt their strength during the maturation and plasticity of neuronal networks. These processes require the activation of the postsynaptic glutamate N-Methyl-D-aspartate receptor (NMDAR). Recently, it has become clear that the nanoscale distribution and the mobility of NMDAR are key parameters for neurotransmission. Disruptions of this delicate balance can engender severe neuropsychiatric disorders, such as the anti-NMDAR encephalitis that has been recently described. Affected patients produce antibodies that specifi cally react with surface NMDAR causing a massive internalization, associated with prominent memory and behavioral defi cits. However, the early molecular changes taking place at the synaptic level remain poorly understood. Until recently, the diffraction limit was preventing the access to details about receptor nano-organization and dynamics due to the small size of synapses (250-500 nm wide). The newly developed super-resolution microscopy techniques can now overcome these challenges. In this study we use Stochastic Optical Reconstruction Microscopy (STORM) to observe the effect of anti-NMDAR patient autoantibodies on the nanoscale organization and stoichiometry of the synaptic components. Our preliminary results show modifi cations of both the area and relative content of NMDAR clusters upon treatment with patient autoantibodies. Using multicolor STORM imaging to image NMDAR together with the postsynaptic marker PSD95, we are currently analyzing the nanoscale alterations of NMDAR distribution at the synapse after autoantibodies treatment. This study will provide a quantitative insight on the nanoscale mechanisms leading to synaptic depletion of receptors described during autoimmune encephalitis.

Synaptic nanoscale reorganization during anti-NMDAR autoimmune encephalitis

53.

P06Lara Laparra Cuervo1 , Laurent Ladépêche1 , Jesús Planagumà2 , Ángel Sandoval1 , Joseph Steven Borbely1 , Josep Dalmau2 , Melike Lakadamyali1

1 ICFO – The Institute of Photonic Sciences2 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic

LGI1 autoimmune encephalitis is a severe neuropsychiatric disorder related to epilepsy. It is an antibody-mediated pathogenesis where the patients produce autoantibodies against leucine rich glioma activated 1 (LGI1), which alter synaptic plasticity. However, the molecular mechanisms that lead to the observed problems in patients still remain largely unknown.

LGI1 is a secreted protein that is thought to form a transynaptic bridge linking pre- and post-synaptic transmembrane proteins (ADAM22 and ADAM23) and helping organize a multimeric complex at the synapse including AMPAR and voltage-gated potassium channels. Yet, the molecular architecture of the LGI 1 complex has not been directly visual-ized due to the small length scale and the crowded environment of the synapse. This molecular architecture is highly important for maintaining neuronal homeostasis and is likely disrupted by autoantibodies during LGI 1 encephalitis.

By means of multi-color STORM, we have visualized the synaptic organization of the different proteins and recep-tors directly or closely involved in LGI1 functioning at the nanoscale level. Using well-characterized synaptic mark-ers (Homer and Bassoon) as molecular standards, we have determined the positioning of LGI1 and related proteins (AMPAR, ADAMs and voltage-gated potassium channels) within the synaptic space at nanoscale resolution in 3-color STORM images. Comparing this molecular architecture in healthy versus diseased neurons will give important in-sights into how the LGI1 antibodies may alter synaptic localization and stoichiometry of the LGI1 macromolecular complex leading to loss of synaptic homeostasis.

Multi-color STORM in neurons reveals molecular organization of the LGI1 synaptic complex in autoimmune encephalitis

54.

P07Hanae Shimo1 , Jennifer L. Gallop1

1 Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge

Filopodia are fi nger-like protrusions comprised of bundled actin, which play an important role in numerous essential cellular processes such as cell migration, adhesion, and metastasis. A previously established in vitro assay for fi lopodia like structures (FLS) using Xenopus laevis egg extracts and supported lipid bilayers, has enabled us to study fi lopodia formation in a system with reduced complexity. While the composition of the membrane-localized initiation site of FLS has had preliminary investigation, the molecular architecture and dynamics of this “tip complex” remains unknown. We have combined super-resolution microscopy and photomanipulation to investigate the dynamics and organization of the FLS tip complex components at nanoscale resolution. Multi-color 3D direct stochastic optical reconstruction microscopy (dSTORM) analysis suggests the existence of axial organization in the assembly of actin regulatory factors on the membrane. Furthermore FRAP and photoconversion experiments indicated an active turnover of proteins in the tip complex during early FLS formation. Further experiments investigating the effects of perturbations and extension of imaging to in vivo fi lopodia in Xenopus retinal growth cones will yield mechanistic insight on the interactions and functionality of actin regulators during fi lopodia formation.

Structure and dynamics of actin assembly factors during fi lopodia formation

55.

P08Alvaro Castells Garcia 1

1 CRG

STORM (STochastic Optical Reconstruction Microscopy) imaging opens a new path to investigate the nuclear organization of RNA at a nanoscale level.We aim to analyze the distribution and organization of RNA, both in the entire nucleus and in a gene locus specifi c approach. We aim to investigate aspects of RNA biology such as its interplay with the chromatin and the differential distribution of RNAs during cell processes such as differentiation.

Using click (copper catalyzed azide alkyne cycloaddition) chemistry, we have been able to visualize the distribution of all RNAs in the entire nucleus with STORM microscopy. Adding Ethynyl-Uridine (a modifi ed nucleotide) we can visualize different RNA populations by using different levels of nucleotide incorporation. With this approach, we found that RNAs do not distribute in a homogeneous way but rather show specifi c distribution patterns inside the cell.

Thanks to the recent work of Ricci et al. (Cell, 2015), we now know that the nucleosomes are distributed in clutches of varying size. We aim to correlate the size and number of nucleosomes per clutch with the amount of mRNAs and thus the transcriptional level associated to specifi c clutches. For that, we are currently using a 2-Color non-sequential labeling strategy to co-image both RNAs and nucleosomes.

We are also carrying out dual color imaging of RNA with different key components of the RNA transcription machinery. Our aim is to correlate their spatial distribution and location in the nucleus with RNA distribution. We will then investigate their changes during cellular processes such as differentiation.

With the obtained data, we intend to develop a model of RNA organization and distribution in the nucleus in a previously non-achieved resolution. This model will provide us with a better understanding of the differentiation mechanisms.

Cell differentiation can be investigated by imaging the nuclear organization of RNAs with STORM

56.

P09Nitin Mohan1, Ione Verdeny1, Vilanova1, Angel Sandoval1, Joe Borbely1, Melike Lakamyali1

1 ICFO-Institute of Photonic Sciences, Mediterranean Technology Park, 08860 Castelldefels, Spain

Intracellular transport mediated by cytoskeletal network is important for cell functioning and survival. Microtubules and Actin fi laments, serves as tracks on which motor proteins can translocate large protein complexes and vesicles over long distances within the cell. Microtubules are fi lamentous structure built from heterodimers of α- and β-tubulin monomers. Motor proteins interact with tubulin and generate movement along the microtubule utilising the chemical energy of ATP hydrolysis. The Dynein and Kinesin motors transport cargo to and from the Microtubule-Organizing Center of the cell, respectively. To effectively perform diverse function there exist distinct subtypes of microtubules generated by post translational modifi cations of tubulin, such as Acetylation, Detyrosination, Polyglutamylation and Polyglycylation. The Tubulin-Code hypothesis proposes that these post translational modifi cations can medi-ate unique interactions with microtubule associated proteins including the motor proteins and thereby coordinating cargo transport. Here we investigate the regulation of cargo transport by post translational modifi cations Acetylation and Detyrosination in live BSC1 African monkey kidney cells using correlative fluorescence imaging method based on single particle tracking (SPT) and super-resolution Stochastic Optical Reconstruction Microscopy (STORM). Fluores-cently labelled vesicles such as Lysosomes and Autophagosomes are tracked with high temporal resolution of 100 milli- seconds for about a minute and the cells are then fi xed in situ and immunostained to visualise the Acetylated or Detyrosinated tubulin along with unmodifi ed tubulin with high spatial resolution achieved by STORM imaging. The ve-sicular trajectories could be mapped on to individual Microtubules with high spatio-temporal resolution to precisely understand the cargo dynamics at the modifi ed and unmodifi ed microtubules. Our results show that the lysosome Speed and Run-length for anterograde motion driven by Kinesin motors over Detyrosinated tubulin get signifi cantly reduced in comparison to that of Tyrosinated microtubules. Acetylation, on the other hand shows signifi cant decrease in Run-length of Kinesin directed motion while the speed of the motion is not signifi cantly affected in comparison to non-acetylated tubulin. In stark contrast, the retrograde motion of the lysosomes mediated by Dynein motors is not affected by Detyrosination or Acetylation of the microtubules. These results suggest that the post translational modi-fi cations of microtubules are recognised by the motors in very distinctive manner and could be cue for cargo sorting or organelle interactions such as vesicular fusion.

Tubulin Post Translational Modifi cations Influence Cargo Transport in Live Cells

57.

P10Bassam Hajj1,2, Maxime Dahan1,2

1 Laboratoire Physico Chimie, Institut Curie, 11 rue Pierre et Marie Curie, 75005 Paris, FRANCE2 Howard Hughes Medical Institute, Janelia Research Campus, Transcription Imaging Consortium, 19700 Helix drive, Ashburn , VA 20147, USA

Email: bhajj@curie.fr

Keywords: 3D imaging, super-resolution microscopy, whole-cell

Single molecule localization-based microscopy techniques that overcome the optical diffraction limit have become a prominent tool for researchers. Increased interest in single molecule imaging in biological studies, however, has led to new technical challenges. Among the most pressing issues is the ability to rapidly image and localize molecules in thick 3D volumes. Our recently developed multifocus microscopy technique (MFM) enables instantaneous acquisition of volumetric images by tiling fluorescence images of nine axial sections on the same detector [1].We have also shown that MFM can be combined with super-resolution imaging techniques such as PALM or STORM [2,3]. When imaged using the MFM, single molecules can be localized axially within ~4 μm, a volume mainly limited by the sampling requirement of each PSF. To avoid this limitation we have combined MFM imaging with astigmatism-based PSF engineering [4]. As such, molecules can be localized axially compared to a single plane. Combining localizations between the different planes further improves the localization precision in all three dimensions. We illustrate the capacity of this approach by single molecule localization of nucleopore complex proteins over 5μm depth. Furthermore, when imaging bright emitters, it is possible to increase the spacing between planes up to 800nm. In this confi guration single bright emitters can be localized over 8μm depth when tracked in solution. Additionally, we discuss the different parameters and grating designs dictating image quality, and system performance over the imaging depth.

References:[1] S. Abrahamsson, J. Chen, B. Hajj, S. Stallinga, A. Y. Katsov, J. Wisniewski, G. Mizuguchi, P. Soule, F. Mueller, C. D. Darzacq, X. Darzacq, C. Wu, C. I. Bargmann, D. A. Agard, M. Dahan and M. G. L. Gustafsson, “Fast and sensitive multi-color 3D imaging using multi-focus microscopy,” Nature Methods 10 (1), 60–63 (2013).[2] B. Hajj, J. Wisniewski, M. El Beheiry, J. Chen, A. Revyakin, C. Wu, M. Dahan, Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy, PNAS 111 (49) 17480-17485 (2014)[3] B. Hajj, M. El Beheiry, I. Izeddin, X. Darzacq, M. Dahan, Accessing the third dimension in Localization-based Super-Resolution Microscopy, PCCP journal 16, 16340-16348 (2014)[4] B. Hajj, M. El Beheiry, M. Dahan, PSF engineering in multifocus microscopy for increased depth volumetric imaging, Biomedical Optics express (2016)

PSF engineering for single molecule localization in multifocus microscopy

58.

P11Laura Martínez-Muñoz1 , Juan A. Torreno-Pina4 , Rubén Barroso1 , Carlos Oscar Sorzano3 , Pilar Lucas1 , José Miguel Rodríguez-Frade1 , Francisco Sánchez-Madrid2 , María García-Parajo4 , Mario Mellado1

1 Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Campus de Cantoblanco, Madrid2 Servicio de Inmunología, Hospital Universitario de la Princesa, UAM, IIS-IP and Department of Vascular Biology and Inflammation, Fundación Centro Nacional de Investigaciones Cardiovasculares-Carlos III, Madrid3 Biocomputing Unit, Centro Nacional de Biotecnología/CSIC,Madrid4 Department of Pharmacology, CFO-Institut de Ciencies Fotoniques, Barcelona Institute of Science and Technology, Barcelona, Spain

Although some G protein-coupled receptors (GPCR) are able to function in monomeric form, many of them including the chemokine receptors are found as dimers and/or higher-order oligomers. Resonance energy transfer techniques (BRET, FRET) have provided evidence that chemokine receptors form homo- and heterodimers during their synthesis and maturation. The use of such techniques and those based on total internal reflection fluorescence microscopy (TIRF-M) show that, like other receptors, GPCR form dynamic complexes at the cell membrane. These dynamics are regulated by their oligomeric assembly, cell membrane composition (lipids, proteins) and by receptor expression and ligand activation. To analyze receptor dynamics at the cell surface, we used single-particle tracking (SPT) of the chemokine receptor CXCR4. This receptor and its ligand CXCL12 are central for mammalian development and hemat-opoiesis, and also have a key role in cancer, pulmonary fi brosis, HIV-1 infection and autoimmune diseases.

CXCR4 formed motile nanoclusters at the cell membrane, which were spatially structured by the cortical cytoskeleton. Movement of most of these nanoclusters was confi ned. CXCL12 binding promoted formation of larger CXCR4 na-noclusters, which led to changes in CXCR4 dynamics. We also found that the underlying actin cytoskeletal network, as well as other membrane proteins such as CD4, modulated CXCR4 cluster size and dynamics. Using transmem-brane peptide-based screening, we identifi ed the CXCR4 amino acid residues involved in receptor oligomerization, and generated a CXCR4 point mutant. This mutant receptor is expressed normally in T cells, where it formed dimeric complexes at the cell surface. CXCL12 binding to the mutant receptor did not trigger cluster coalescence, however, leading to response defects. Whereas CXCL12-mediated calcium flux was similar in wild-type and mutant CXCR4-expressing cells, the latter did not migrate in response to ligand binding.

These fi ndings indicate that receptor compartmentalization and cluster size are essential for regulating chemokine-mediated functions.

CXCR4 nanoscale organization and dynamics influence CXCL12-mediated responses

59.

P12Antonios Lioutas1, Miguel Beato1

1 Center for Genomic Regulation (CRG), Barcelona, Spain

The organization of the eukaryotic genome in the cell nucleus is not random and follows hierarchical architectural principles going from nucleosomes, to loops, topological associating domains (TADs), chromatin compartments and chromosome territories. Various factors are involved in maintaining this organization, some aspects of which are conserved and others are cell type specifi c or even change in response to external cues. Using Hi-C we found that in breast cancer cells TADs represent distinct epigenetic domains that show homogeneous changes in response to progesterone or estrogens (Le Dilly et al., 2014). In this work we focus on analyzing the dynamics of the interaction of Progesterone Receptor (PR) with genome at the single cell level as a validation of the observations at the cell population level. We aim at understanding how PR affects the organization of the genome when breast cancer cells T47D are exposed to progesterone.

To this end we use two different Super-Resolution imaging methods, DNA-PAINT (Jungmann et al., 2014) and 3D STED (Donnert et al., 2007) to accurately localize PR in individual T47D breast cancer cells. A combination of PR localization with the visualization of specifi c genomic regions will allow to perform single molecule tracking for a better understanding on how activated PR fi nds its target sequences to initiate chromatin re-arrangements. Our data suggest that the changes of PR localization might be involved in the genome re-arrangements observed in Hi-C experiments at cell population level.

Steroid hormone receptor interaction with the genome studied with super-resolution microscopy

60.

P13Jason J Otterstrom1 , Alvaro Castells García2 , Joseph Borbely1 , Maria Aurelia Ricci2 , Pia Cosma2 , Melike Lakadamyali1

1 The Institute of Photonic Sciences (ICFO)2 The Center for Genomic Regulation (CRG)

Chromatin is an ordered complex between DNA and histone proteins, which together form nucleosomes (~11 nm in diameter) and are the basic structural unit for genome organization. Chromatin’s high-order structuring is mediated by interactions between nucleosomes and is commonly described using only the qualitative classifi cations of open or closed. The open-state euchromatin is transcriptionally active and highly susceptible to DNA cleavage by endonu-cleases, whereas the closed-state heterochromatin is inactive and less susceptible to cleavage. Current ensemble, biochemical methods to study chromatin structure are unable to probe structures at the level of nucleosome-nucle-osome interactions and are blind to transient chromatin remodeling that is likely involved with active transcription. Conventional fluorescence microscopy (CFM) and electron microscopy (EM) are also limited to qualitative descrip-tions. The diffraction limit of ~250 nm impedes further structural characterization by CFM, and advances using EM are thwarted by low image contrast and poor structure labelling specifi city.

Here we apply STORM (stochastic optical reconstruction microscopy) super-resolution imaging to visualize and quantitatively describe fi ne chromatin structure at the level of a few to dozens of nucleosomes. STORM breaks the diffraction barrier by sequentially imaging and localizing individual organic dye molecules densely bound along chro-matin as they cycle between ‘on’ and ‘off’ fluorescence states. STORM benefi ts from the labeling specifi city and high contrast of CFM and localizes each single molecule with ~20 nm lateral and ~50 nm axial precision.

Previously, we used STORM to demonstrate that H2B forms clusters, which we called clutches, and found that they reorganize when fi broblasts are treated with Trichostatin A (TSA) –a histone deacetylase inhibitor that induces an open chromatin state with consequent transcriptional activation. Expanding on these observations using two spec-trally separated dyes we obtain 3D images of both H2B histones and the surrounding DNA in individual, intact hu-man fi broblast nuclei. This study represent the fi rst super-resolved 3D in situ visualization of both DNA and histone proteins and our analysis provides a fi rst stepping stone towards quantitatively describing previously invisible local chromatin states.

Visualizing Chromatin Structure at the Nanometer Scale

61.

P14Izabela Piechocka1 , Alberto Sosa-Costa1 , Carlo Manzo1 , Nitin Mohan1 , Melike Lakadamyali1 , Maria Garcia-Parajo1

1 ICFO- Institut de Ciencies Fotoniques, Castelldefels (Barcelona), Spain

In vivo leukocyte arrest on endothelial cells (ECs) occurs in the context of blood flow which exerts shear forces on both types of cells. Despite the evidence that leukocyte arrest generates in vitro a specifi c distribution of the ECs Intracellular Adhesion Molecule-1 (ICAM-1), the sole effect of the shear flow on the spatial organization of ICAM-1 have not been investigated so far. Using a microfluidic device we show that pre-exposure of ECs to the shear flow introduces a spatial heterogeneity within the apical membrane of ECs as consequence of translocation of ICAM-1 to the up-stream to the direction of flow (Fig. 1 A). This is accompanies by an increase in ICAM-1 nanoclustering and re-organization of the actin cytoskeleton. As a result, T-cells adhered to such pre-sheared ECs migrate faster and show shorter periods of interactions with ECs compared to their counterparts adhered to static ECs (Fig. 1 B). Our results indicate thus that continuous shear flow can directly modulate the organization of ICAM-1 molecules, influencing in turn leukocyte migratory behaviour. To explain these results we propose a stochastic 1-D model based on motor-clutch hypothesis whereby an increase in the strength of ligand-integrin bond leads to reduction of the actin retrograde flow, increasing in turn the velocity of T-cell.

Figure 1. The effect of shear flow on distribution of ICAM-1 molecules at the plasma membrane of ECs. (A) Single plain confocal microscopy images (top row) and 3D orthogonal views (bottom row) of ICAM-1 molecules under shear-free and after 4 hours of continuous shear flow pre-stimulation. White arrows indicate the direction of shear flow. Yellow arrows point at the up-stream accumulation of apical membrane ICAM-1 molecules. (B) Example of trajectories (grey and blue lines) of leukocytes migrating across 4 hours shear flow pre-stimulated ECs. Segmentation algorithm divides the curves in small straight traces (red lines superimposed on the blue trajectory curve) from which the velocity and time of interaction of Jurkat cell with ECs are estimated. (Inset) Example of the super-imposed trajectory (blue line) of a migrating Jurkat cell. Arrow indicates the direction of shear flow.

Shear flow induces changes in ICAM-1 spatial distribution that modulate leukocyte mobility across endothelium

62.

P15Laurent Fernandez1 , Christine Bénistant1 , Morgane Malrieu1 , Patrice Dosset1 , Eric Rubinstein3 , Fedor Berditchevski2 , Pierre-Emmanuel Milhiet1

1 Centre de Biochimie Structurale, CNRS UMR 5048-INSERM UMR 1054, Montpellier, France2 School of Cancer Sciences of the University of Birmingham, Birmingham, UK3) Inserm, U935 - Université Paris-Sud, Institut André Lwoff, 94807 Villejuif, France

CD82, a transmembrane protein belonging to the family of tetraspanins, is one of the rare metastasis suppressors identifi ed so far. However the mechanism of CD82-induced metastasis suppression remains not well understood. The most relevant hypothesis relates CD82 function to its ability to inhibit cell motility through its interaction with key motility relevant proteins including integrins and EGFR.

Tetraspanins, including CD82, have the unique property to create a network of protein-protein interactions within the plasma membrane and can be organized in microdomains known as tetraspanin-enriched microdomains. Within this network CD82 is known to interact with tetraspanins including CD9, CD81 and CD151 in addition to non-tetraspanin proteins such as, integrins, growth factor receptors. Additionally, it has been shown that the interaction of CD82 with EGFR, alpha3 and integrin within TEMs depends on the expression of gangliosides at the plasma membrane. Specifi -cally, CD82 negatively regulates EGFR function by influencing its partitioning and ligand-induced dimerization. This effect is correlated with an increase in expression levels of the ganglioside GD1a in CD82-expressing HB2 breast cells. Moreover ganglioside depletion specifi cally decreases CD82 interaction with both EGFR and alpha3 integrin. Taken together, these studies strongly suggest that gangliosides could tune the partition of CD82 and its interaction with alpha3, resulting in the regulation of the molecular landscape of integrins.

To date, studies in this fi eld have employed ensemble-averaging techniques, which are unable to analyze in details dynamics of membrane although it is now well established that the spatio-temporal organization of its components is crucial for cellular functions. It is thus essential to understand the regulation of CD82-induced metastasis suppres-sion by gangliosides at the single molecule level. We aim to study both the dynamics and partitioning of CD82 and its partners. To do so, a TIRF-based Single Molecule Tracking approach is employed to provide direct nanoscale insights of individual proteins in living cells.

Our preliminary results indicates that:

(1) At low expression levels of gangliosides, a specifi c decrease in the number of confi ned CD82 molecules and an increase in the apparent diffusion coeffi cient of CD82 at the plasma membrane are detected, strongly suggesting that gangliosides favor both CD82 partnership and the formation of CD82-enriched domains.(2) Downregulation of GD1a expression demonstrates that the ganglioside GD1a is specifi cally involved in the dy-namics of CD82 but also in its localization.(3) CD82 down regulation surprisingly decreases CD81 dynamics, specifi cally increasing its confi nement in TEMs. In addition, CD82 down regulation increases signifi cantly the dynamics of the alpha3 integrin. These results clearly indicate that CD82 can modify both CD81 and alpha3 dynamics and partitioning by modifying the tetraspanin-based interaction network.

Taken together our results clearly indicate that gangliosides modify CD82 dynamics, which also influence its mem-brane partners in terms of dynamics and partition.

The metastasis suppressor CD82 and gangliosides: two key players of the plasma membrane organization?

63.

P16Guillaume Cordier1 , Joe Borberly1, Angel Sandoval1, Melike Lakadamyali1

1 ICFO-Institute of Photonic Sciences, Mediterranean Technology Park, 08860 Castelldefels, Spain

Intracellular transport is essential for healthy functioning of cells. Organelles and proteins among other materials need to be moved with a precise timing to keep the cell homeostasis. To achieve this essential role, a family of proteins called motor proteins, are in charge of dragging large cellular organelles and vesicles via the cytoskeleton of the cell. Therefore, the organization of motors on the cargo membrane can have important implications for intracellular traffi c. However, thus far, visualizing the organization of motors on the cargo membrane has been challenging due to the low spatial resolution of light microscopy and low molecular specifi city and low throughput of electron microscopy. Using super-resolution microscopy combined with a new versatile method to implement multicolor imaging , we are surpassing the limitations of previous methods to study at the nanoscale level how the motor protein dynein is organized on the cargo membrane in 3D. Our preliminary results show that we are able to detect small nanoclusters of dynein motors on the lysosome membrane. We are using quantitative analysis to study if these dynein nanoclusters are distributed uniformly across the lysosome membrane or they are enriched in specifi c places, whether the number of nanoclusters depends on the size or the shape of the lysosome and the stoichiometry of the dynein motors within the nanocluster.

Motor-cargo organisation studied with single localisation

64.

P17Kyra J E Borgman1 , Daniel Benítez-Ribas2 , Maria Garcia-Parajo3

1 ICFO-The institute of Photonic Sciences, The Barcelona Institute of Science and Technology, 08860 Castelldefels (Barcelona) Spain.2 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) and Centre Esther Koplowitz, Barcelona, Spain 3 ICREA-Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Spain

The tyrosine kinase receptor MerTK is a transmembrane receptor involved in phagocytosis of apoptotic cells, without generating an immune response. However, recent results have shown that in vitro generated tolerogenic dendritic cells (tolDCs), used to clinically temper the immune response of T cells in cases of autoimmunity, signifi cantly up-regulate MerTK gene expression. Highly increased levels of MerTK were subsequently found on the cell membrane of tolDCs, as well as intracellularly. When MerTK on the membrane was blocked, tolDCs regained their ability to generate a strong immune response by stimulating T cell growth and activity, further indicating a new function for the mem-brane receptor in inducing tolerance.[1] However, the role and precise localization of the intracellular pool of MerTK has not been investigated yet. Our study aims to elucidate the role of the surprisingly large, around 50% of the total fraction, intracellular pool of MerTK found in tolerogenic DCs.

Using confocal microscopy we show that the intracellular pool of MerTK almost entirely resides inside the nucleus (Fig. 1A). A signifi cant increase in the levels of nuclear MerTK is observed upon tolerogenic treatment of DCs (Fig. 1B), suggesting a function in immune tolerance for not only membranal but also nuclear MerTK.

We further ow how the abundance of soluble ligand affects the nuclear localization of MerTK, and how membranal MerTK accumulates into the nucleus over the time course of 1 hour. This suggests that MerTK not only has a dual function and localization, but that the same molecule can shuttle between the membrane and the nucleus in less than an hour. This indicates that the process of traffi cking must have an enormous relevance, considering the cellular energy spent on it. Finally, we show for the fi rst time that a receptor tyrosine kinase resides in the nucleus of healthy primary immune cells directly isolated from human donors, contributing to the hypothesis that these membrane re-ceptors can play a physiological role inside the nucleus

Taken together our results clearly indicate that gangliosides modify CD82 dynamics, which also influence its mem-brane partners in terms of dynamics and partition.

Tyrosine Kinase Receptor MerTK: from the cell membrane to the nucleus

65.

P18Pablo Mateos-Gil1 , Sebastian Letschert1 , Anne Burgert1 , Sören Doose1 , Markus Sauer1

1 Department of Biotechnology & Biophysics, University Wuerzburg, Germany

Besides its function as a passive cell wall, plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have potential implications in several biological fi elds such as cell development, neurobiology, or immunology. Fluorescence imaging was limited in the past to resolve the nanoscale protein organization in cells due to the resolution barrier imposed by the diffraction of light. This problem has been recently overcome by super-resolution fluorescence microscopy methods, such as direct Stochastic Optical Reconstruction Microscopy (dSTORM), which have revealed the presence of nanoclusters of specifi c proteins in the cell membrane and rather homogeneous distributions of other proteins and glycans. However, probing the existence of universal mechanisms underlying mesoscale spatial distribution of all PM membrane proteins remains challenging. To test this hypothesis, more global approaches aimed to image simultaneously a large population of membrane proteins is required. Here we present a bioorthogonal chemical strategy, based on click chemistry and metabolic labeling, combined with dSTORM to visualize plasma membrane proteins containing co-translational incorporated non-canonical amino acids, and post-translational modifi cations such as clickable sugars.

Super-resolution imaging of plasma membrane proteins with click chemistry

66.

P19John Danial1 , Ana-Jesus Garcia-Saez1

1 Max Planck Institute for Intelligent Systems

BAX, a pore-forming protein and key player in the intrinsic apoptotic pathway, accumulates at the mitochondrial outer membrane, assembles into oligomers and punches holes to cause cell death. We have recently observed the cluster-ing of BAX monomers into oligomeric arcs and rings on apoptotic mitochondria using super-resolution microscopy and confi rmed their ability to form size-tunable pores. Despite the specifi city of its function, BAX forms structurally- and dimensionally- different oligomers which, surprisingly, do not affect its role as a pore former. To address this paradox, we fi rst reported the structure of membrane-embedded BAX and identifi ed two domains: a rigid dimerization domain that controls the extent of BAX assembly, and a flexible piercing domain that mediates the formation of size-tunable pores. To investigate the obscure role of those domains in inducing structurally-different, functionally-similar, BAX oligomers at the molecular level, we measure intra- and inter- monomeric and dimeric distances in fluorescent-ly-labeled membrane-embedded BAX mutants using single molecule Forster Resonance Energy Transfer (smFRET).

Single molecule analysis of membrane-embedded BAX

67.

P20Catalina Martínez-Guillamon1 , Kyra J.E. Borgman1 , Susana Benet2 , Juan A. Torreno-Pina1 , Núria Izquierdo-Useros2 , Javier Martínez-Picado3 , María Garcia-Parajo3

1 ICFO-The institute of Photonic Sciences, The Barcelona Institute of Science and Technology, 08860 Castelldefels (Barcelona) Spain2 AIDS Research Institute IrsiCaixa, Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain.3 ICREA-Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Spain.

HIV, the virus that causes AIDS, has an extensive repertoire of tricks to scape immune surveillance and to profi t from the immune system to spread the infection. For instance, the virus highjacks mature dendritic cells (mDCs) avoiding degradation, and takes advantage of the interaction between mDCs and T cells to effi ciently spread the infection1. Recent studies have shown that the transmembrane receptor Siglec-1, which is highly upregulated upon DCs maturation, is involved in HIV capture by mDCs and subsequent T-cell infection by selectively binding to viral envelope gangliosides2. However, very little is known regarding the mechanisms by which Siglec-1 captures HIV, and whether the spatiotemporal organization of Siglec-1 and potential interactions with different co-receptors influence its binding capabilities to the virus.

The aim of our study is to elucidate how Siglec-1 captures and further processes viral particles by imaging the spatial organization of the receptor on the membrane of human mDCs using super-resolution microscopy.

By imaging Siglec-1 on immature (iDCs) and mDCs using STED nanoscopy (Fig 1A), we show that Siglec-1 is organized in small nanoclusters in both cell types. Nevertheless, the number of molecules per cluster is signifi cantly different, with larger clusters in mDCs compared to iDCs (Fig 1B). Moreover, the degree of clustering in both cell types deviates signifi cantly from random organization suggesting that the organization of Siglec-1 on the cell membrane is not only a consequence of its molecular density, but the result of an active mechanism forming nanoclusters.

To further study the influence of Siglec-1 nanoclustering on HIV capture dual color fluorescence microscopy is currently being performed. First, iDCs and mDCs are pulsed with HIV for 30 minutes. Second, images of Siglec-1 labeled with antibodies and images of the viral particles containing the protein mCherry on their envelope are taken by STED and confocal microscopy, respectively. Images are superimposed with each other and the nanoclustering degree of Siglec-1 with respect to HIV binding is analyzed using a custom based algorithm.

Preliminary results show co-localization of Siglec-1 with HIV particles and increased Siglec-1 clustering associated with viral localization. Studying and quantifying different time points of HIV binding would allow us to determine the degree of HIV-dependent Siglec-1 nanoclustering. Furthermore, studying earlier time points of virus binding before rearrangement of Siglec-1 would tell us whether clustering is also essential for the HIV capture or whether increased clustering is only a consequence of binding, necessary for processing.

References 1. Wu, L. & KewalRamani, V. N. Dendritic-cell interactions with HIV: infection and viral dissemination. Nat. Rev. Immunol. 6, 859–868 (2006).2. Izquierdo-Useros, N. et al. Siglec-1 Is a Novel Dendritic Cell Receptor That Mediates HIV-1 Trans-Infection Through Recognition of Viral Membrane Gangliosides. PLoS Biol. 10, e1001448 (2012).

Spatial organization of the HIV-binding receptor Siglec-1 on the membrane of human dendritic cells

68.

P21Anne-Marie Byrne1 , Aideen Long1 , Graham Pidgeon1

1 School of Medicine Trinity College Dublin, Irland

Membrane traffi cking is crucial for interaction between the tumour cell and its microenvironment. As part of the transformation process, tumour cells undergo a “secretory switch” to provide the cell with increased secretory prop-erties required for cell growth and survival. Altered Golgi associated processes such as protein glycosylation are also characteristics of cancer contributing to pro-survival and metastatic mechanisms.

In this study we demonstrate that the structure of the Golgi apparatus is fragmented in both colorectal and oesopha-geal cancer tissue. One of the key environmental factors underlying the development of these diseases is the expo-sure of the cells to bile acids, in particular Deoxycholic acid (DCA). We investigated the effect of bile acid exposure in terms of the structure and functions of the Golgi.

We demonstrate that DCA caused Golgi structure disassembly in colorectal and oesophageal cell lines (HCT116, HET1A, QH-tert,GO-tert, SKGT4) resulting in impaired post translational glycosylation and membrane traffi cking of E-Cadherin. DCA exposure also causes ER stress activating the PERK arm of the Unfolded Protein response. Increased traffi cking and secretion of a Golgi-associated protein, GOLPH2, was observed in SKGT4 oesophageal cancer cells in vitro compared to a non-malignant oesophageal cell line (HET1A). In addition GOLPH2 is cleaved and secreted in response to DCA exposure. The cleaved and secreted form of GOLPH2 is required for cell migration and tumour cell invasion. Translating to the clinical setting we show this protein acts as a tissue biomarker capable of distinguishing between pre-metaplastic, dysplastic and adenocarcinoma of the oesophagus.

Both Golgi architecture and associated proteins are highlighted as having integral roles in cancer progression which can be exploited as novel biomarkers and therapeutic strategies for these diseases.

Golgi structure and membrane traffi cking in patient tissue; can we translate in vitro observations to the clinic?

69.

P22Klaus Harter1 , Sebastian Wolf2 , Margret Sauter3

1 Center for Plant Molecular Biology (ZMBP), University of Tübingen2 Centre for Organismal Studies (COS), University of Heidelberg3 Developmental Biology and Plant Physiology, University of Kiel

Several growth modulating factors initiate very early phases of cell elongation growth in plants. This phase is independent of gene expression and includes re-arrangements of the cortical cytoskeleton and re-orientation of the cellulose microfi bers in the wall. By applying - amongst other methods - different spectro-microscopic techniques such as in vivo one chromophr lifetime spectroscopy and high resoltuion FRET-FLIM, we study plant growth factor-controlled dynamic responses at the cell wall - plasma membrane -cytoplasm interface. Our analyses suggest that different response modules exist in the plasma membrane, which consist not only of receptors and co-receptors for specifi c growth-modulating factors but also of other proteins such as proton pumps and ion channels. These functional units appear to directly translate signal reception into defi ned subcellular processes. On the basis of selected examples, our fi ndings and concepts of early events in plant cell elongation growth will be discussed.

Protein response modules at the plasma membrane regulating plant cell elongation growth

70.

P23Fabian Wehnekamp1 , Gabriela Plucinska2 , Rachel Thong2 , Thomas Misgeld2 , Don C. Lamb1

1 Fablab, Department Chemie, Ludwigs-Maximilians-Universität Munich, Germany2 Neuronal Cell Biology, Technical University Munich, Germany

The main function of mitochondria is to provide cells with adenosintriphosphate (ATP) in regions with high-energy demand. A complex machinery of motor proteins (kinesin, dynein, myosin, etc.) and signaling molecules are respon-sible for the distribution and recycling of mitochondria in cells. A malfunction in the dynamics of mitochondrial trans-port is one possible reason for neurodegenerative diseases (tauopathies).

To follow the trajectory of individual mitochondria in rohon-beard sensory neurons, we developed a home-build Or-bital Tracking microscope with simultaneous widefi eld observation capabilities with which we are able to follow a single particle in real-time with nm precision in a complex 3D environment. By using photoactivation, we are able to track single mitochondria over distances of more than 100 μm. Due to our high spatial and temporal resolution, we can identify several different dynamic populations involved in mitochondrial transport and classify the relationship between these populations. In addition, the environmental information gives insight into the interactions between stationary and moving mitochondria and the propensity to change the movement behaviour in the vicinity of such an obstacle. Combining the results from the fast and precise tracking microscope with the widefi eld data we obtain an in vivo overview of the dynamic processes in rohon-beard sensory neurons which can be used to study the effects of neurodegenerative diseases.

3D Real-Time Orbital tracking in zebrafi sh embryos: Spatiotemporal analysis of mitchondrial dynamics in rohon-beard sensory neurons

71.

P24

Carmen Krueger1 , Marie Theres Zeuner2 , Darius Widera2 , Mike Heilemann1

1 Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University, Frankfurt (Main), Germany2 School of Pharmacy, University of Reading, Reading, United Kingdom

The human toll-like receptors (TLRs) are known to play an important role in innate immunity. The interaction of TLR4 and its ligands of the family of lipopolysaccharides (LPS) [1] is the most studied system in this fi eld. TLR4 is a transmembrane receptor initially located on the plasma membrane of mammalian cells, and upon activation triggers the signaling and the initiation of immune response through different signaling pathways. The level of response is dependent on the respective LPS. LPS itself is a component of the outer membrane of gram-negative bacteria, e.g. Escherichia coli. The structure of LPS varies depending on the bacterial species and leads to a different immune response [2]; LPS derived from E.coli is known to induce a strong inflammatory response whereas the inflammation caused by Salmonella LPS is generally weaker. Recently, it was discovered that TLR4 is also present on adult neural stem cells and plays a role in their neurogenesis [3].

Single-molecule fluorescence techniques are a useful toolbox to investigate assembly, activation, organization and interaction of membrane receptors and their ligands at the single-cell level [4, 5]. Here, we investigate the activation of TLR4 in neural cells by LPS derived from E.coli and Salmonella using single-molecule imaging. The aim of this study is to characterize the behavior of the receptor on neural cells in the presence of different LPS species over time, which is still unknown. Here, we quantify the number of receptor sites with respect to ligand treatment over different time points. Furthermore, we study the size of receptor clusters using coordinate-based and image-based analysis. Analysis of the Ripley’s H- function shows a clear maximum for the characteristic cluster size at 60 nm for all applied conditions. For the number of particles on the cell, we count 7 TLR4 particles per μm² in the uninduced case. Interestingly, this number only varies slightly after treating the cells with different LPS species.

[1] Takeda, K. & Akira, S. Toll—like receptors in innate immunity. International Immunology,17, 1-14 (2005)[2] Miller, S.I., Ernst, R.K. & Bader, M.W. LPS, TLR4 and infectious disease diversity. Nature reviews. Microbiology,3, 36-46 (2005)[3] Okun, E., Griffi oen, K.J. & Mattson, M.P. Toll—like receptor signaling in neural plasticity and disease. Trends in neurosciences, 34, 269-281, (2011)[4] Dietz, M. S. et al. Receptor–Ligand Interactions: Binding Affi nities Studied by Single—Molecule and Super—Resolution Microscopy on Intact Cells. ChemPhysChem., 15, 671-676 (2014)[5] Fricke,F. et.al. Quantitative single—molecule localization microscopy combined with rule—based modeling reveals ligand—induced TNF—R1 reorganization toward higher—order oligomers. Histochemistry and Cell Biology, 142 91-101 (2014)

Copy number and cluster analysis of toll-like receptor 4 upon treatment with lipopolysaccharide using single-molecule localization microscopy

72.

P25Floderer Charlotte1 , El Beihery Mohamed2 , Dahan Maxime2 , Masson Jean-Baptiste4 , Sibarita Jean-Baptiste3 , Favard Cyril1 , Muriaux Delphine1

1 CPBS, CNRS, Université de Montpellier, Montpellier, France2 Physico-Chilie Curie, Institut Curie, CNRS, Université Pierre et Marie Curie, Paris, France3 IINS, CNRS, Université de Bordeaux, Bordeaux, France4 Institut Pasteur, Paris, France and HHMI Janelia, Virginia, USA

The retroviral Gag protein has been shown to be the most important component for virus assembly as it is suffi cient to produce newly formed particles, called virus-like particles (VLP). The assembly of Gag proteins is mainly occurring at the inner leaflet of the infected cell plasma membrane [1]. Interestingly, Gag can be tagged internally by a fluorescent protein without impairing its capacity to assemble into a virion [2, 3]. Therefore, we are currently deciphering VLP as-sembly at the plasma membrane of human T lymphocytes, the main host cell for HIV-1 cell replication, by monitoring the dynamic organization of Gag-i(mEOS2) protein, at the nanometric level, with the help of single-protein tracking Photo-Activable Localization Microscopy (sptPALM) coupled to TIRF microscopy [4]. This method produces high resolution images where clusters of Gag-i(mEOS2) localisations can be observed. Our results showed between 100 and 250 of these clusters per cell. Simple morphological analysis shows clusters diameter to be approximately 150 nm, in the range of VLP sizes. We could also determine the persistence mean time of these clusters and we found it in between 6 and 15 minutes, in agreement with what has already been described in the adherent Hela model cell line [2][3]. We are now exploring how these clusters are built molecule after molecule. We will present how massive analyze of the single trajectories using newly developed tools [5] and big data facilities gives access to new molecular details about HIV-1 Gag assembly dynamic in its natural host cell.

1. Mariani C et al., 2014, Front Microbiol. 5:312.2. Ivanchenko S et al. 2009, PLoS Pathog 5:e1000652.3. Jouvenet N et al. 2009. Proc Natl Acad Sci USA 106:19114–19119.4. Sibarita JB, 2014 Histochem Cell Biol.141(6):587-95.5. El Beheiry M et al.,2015, Nat Methods. 12(7):594-5.

Mapping the energy of HIV-1 assembly in T cells using spt-PALM.

73.

P26Franziska Fricke1 , Joel Beaudouin2 , Sjoerd van Wijk3 , Ivan Dikic4 , Roland Eils5 , Mike Heilemann1

1 Institute of Physical and Theoretical Chemistry, Goethe-University, Frankfurt, Germany2 Institut de Biologie Structurale, Grenoble, France3 Institute of Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany4 Institute of Biochemistry II, Goethe-University Medical School, Frankfurt, Germany5 Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany

Death receptors are important mediators of inflammation and proliferation in mammalians, but can also induce programmed cell death under certain circumstances. The oligomeric state of death receptors is considered critical for receptor activation and signaling selectivity, but its nature remains largely controversial: There is structural and biochemical evidence for pre-assembled dimeric as well as trimeric receptors. Interestingly, pre-formed receptor dimers are hypothesized to mediate higher-order cluster formation upon binding of the respective trivalent ligand effectively leading to honeycomb-like signaling networks [1].

To test this hypothesis, we developed a molecular counting strategy based on single-molecule localization microscopy (SMLM) to assess the stoichiometry of death receptors tumor necrosis factor receptor 1 (TNFR1) and CD95 in intact cells. Our strategy is based on a photokinetic model that allows predicting the blinking statistics of photoactivatable/-convertable fluorescent proteins and possibly photoswitchable organic dyes [2]. Notably, the blinking statistics hold information about the number of underlying fluorophores, hence target molecules of stoichiometrically labeled samples. We validate our molecular counting strategy with plasma membrane proteins of well-known and defi ned stoichiometry [3]. Using our counting concept, we investigate whether TNFR1 and CD95 exist as pre-formed homomers in the plasma membrane of mammalian cells. We further address the open question of higher-order receptor oligomerization in response to ligand-binding to substantiate the current model of oligomerization-mediated death receptor activation.

[1] H. Wajant, Cell Death Diff. 22, 1727-1741 (2015)[2] S.-H. Lee et al., Proc. Natl. Acad. Sci. 109, 17436-17441 (2012)[3] F. Fricke et al., Sci. Rep. 5, 14072 (2015)

Molecular counting with single-molecule localization microscopy and its application to death receptors

74.

P27Marta Gironella1 , Marco Ribezzi1 , Fèlix Ritort1

1 Small Biosystems Lab, Universitat de Barcelona

Targeted therapies are one of the most promising advances in the fi ght against cancer. They require the differentation and characterization of the malignan and healthy cells with hight accuracy. Fluctuation membrane spectroscopy, using optical tweezers, is an extremely reliable technique that allows us to characterize the mechanics of the cells such as the drag coeffi cient and the membrane stiffness.

In order to have a full and precise knowledge of our results we have started studying the mechanics of red blood cells (RBC) which are one of the simplest cell types. We are able to measure the global and local membrane deformability using two different experimental procedures:

- Applying a flow to a trapped micron-sized bead attached to the RBC membrane - Measuring the force signal fluctuations of the RBC membrane using micron-sized beads coated with fi bronectin

that bind to the cellular membrane.

Once completed this study we will be able to use the gained knowledge to differentiate heterogeneous cellular populations.

Mechanical phenotyping of single cancer cells

75.

P28Srinjan Basu1 , Lisa-Maria Needham2 , Edward J. R. Taylor1 , Lawrence Bates1 , Yi Lei Tan1 , David Klenerman2 , Steven F. Lee2 , Ernest D. Laue1

1 Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom2 Department of Chemistry, University of Cambridge, Lensfi eld Road, Cambridge CB2 1EW, United Kingdom

Single particle tracking and super-resolution imaging approaches have allowed us to image proteins in living cells at close to molecular resolution, shedding light on protein function and the mechanisms of fundamental cellular pro-cesses such as gene expression. Photoactivated localisation microscopy (PALM) is one such approach that takes advantage of photoswitchable fluorophores to image a protein of interest. However, it is often diffi cult to image more than one protein at a time and so study protein complexes. Here, we report progress in developing a single-mole-culeFörster resonance energy transfer (smFRET) cassette that takes advantage of the photoswitchable properties of a donor fluorophore and the longer lifetimes of a fused acceptor fluorophore. We present the use of this smFRET cas-sette in single-particle tracking and super-resolution imaging, providing a framework for studying protein complexes in living mammalian cells.

Combining single-molecule FRET and PALM for single particle tracking and super-resolution imaging

76.

P29Sandrine Lévêque-Fort1

1 ISMO-CNRS

Super-resolution microscopy offers a unique access to reveal nanometer cellular organization. However in Single Molecule Localization Microscopy (SMLM) the axial localization precision is always worse compared to the lateral one. Rather than introducing a known deformation in the detection path of the microscope to retrieve axial information, we propose here to take advantage of the axial information given by the supercritical angle fluorescence (SAF) emission already presents in the pupil plane [1]. When a fluorophore is located in the vicinity of the coverslip interface, its near-fi eld fluorescence component becomes propagative and can be collected with a high numerical aperture objective. In the objective back focal plane, this SAF component appears in a ring beyond the critical angle qc. We recently demonstrated that by extracting SAF emission, a unique dual depth wide fi eld imaging can be retrieved in dense samples, allowing to monitor membrane and intracellular events simultaneously [2]. Furthermore, when associated to single molecule localization approach, SAF emission offers an absolute access to the fluorophore axial position. Indeed for a fluorophore at the interface, the number of photons in the SAF ring, NSAF, represents 50% of all the collected photons Ntot. Since NSAF decreases approximately exponentially with the fluorophore distance to the coverslip. The absolute axial position of each fluorescent dye is retrieved by comparing NSAF versus Ntot. In practice, only the detection path of our SMLM setup is modifi ed to insert a compact home-made dual view module, which permits to simultaneously measure NSAF and Ntot, and compute the absolute axial position of each fluorophore in real time. This 3D absolute method, called “Direct Optical Nanoscopy with Axially Localized Detection” (DONALD), gives an axial localization precision of 15 nm within an axial range of ~150 nm above the coverslip, while preserving typical lateral localization precision (~10 nm) [3]. Axial position can be accessed up to the fi rst 600 nm within the sample, but with lower localization precision. This unique property of absolute axial localization permits a direct combination of axial protein localization which improve 3D co-localization experiments at the nanoscale. In particular the 3D complex architecture of biological structures such as focal adhesion and podosomes can be revealed. Podosomes are adhesion structures involved in the degradation of the extracellular matrix and formed by macrophages and monocyte/macrophage-derived cells. We are investigating how podosomes are organised at the nanoscale, and how this organisation regulates protrusion force generation. We will present the 3D organization of proteins surrounding the F-actin core of podosomes in human macrophages revealed by our DONALD nanoscope

[1] T. Ruckstuhl et al.,Forbidden light detection from single molecules, Analytical chemistry, 2000[2] T. Barroca et al., Full-fi eld Near-Field Optical Microscope for Cell Imaging, PRL, 2012[3] N. Bourg et al. Direct Optical nanoscopy with axially localized detection, Nat. Phot., 2015

3D molecular architecture of podosomes revealed by Supercritical Angle Fluorescence

77.

P30Armando Maestro1

1 Cavendish Laboratory, University of Cambridge, JJ Thompson Avenue, CB3 0EH Cambridge, UK

Clathrin-mediated endocytosis is the main mechanism by which proteins are controllably transported across the plasma membrane, and is thus vital to cellular life. It is a beautiful example of controlled multi-scale choreography, involving scales of components from the molecular (10’s Å) to the vesicle (100’s nm), and long-range processes of membrane-mediated interaction possibly also coming into play. This complexity has up to now prevented a full understanding of this key cellular process, despite the fact that very precise knowledge exists on specifi c aspects of molecular detail. In-vitro experiments have been very insightful so far, but remain lacking a physical description of this collective mechanism that relies on protein aggregation and self-assembly coupled to membrane processes that involve non-equilibrium thermodynamic conditions. Our fi nal goal is to disentangle the molecular mechanism by which the recruitment of clathrin by the membrane is triggered by the binding of endocytic adaptor proteins to particular lipid domains: the ones constituted by phosphatidyl inositol PtdIns4,5P2 (PiP2). By fluorescence microscopy and surface experiments monitoring the lateral pressure of the lipid monolayer, we addressed in a controlled in-vitro experiment how the uptake and change of conformation of adaptor proteins, concretely the heterotetrameric AP2 complex, that triggers the recruitment of clathrin is regulated by the asymmetry in the distribution of PiP2 in the plane of the monolayer. Besides, by exploring the relaxation response after applying an external deformation (for instance, shear) the dynamical behaviour related to the creation of a clathrin coat has been identifi ed.

Unravelling the complex choreography of endocytosis through in-vitro experiments

78.

P31Laura Maddalena1, Maria F Garcia-Parajo1,2 and Carlo Manzo1

1 ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, 08860 Castelldefels (Barcelona), Spain2 ICREA-Institucio Catalana de Recerca i Estudis Avancats, 08010 Barcelona, Spain

The kinetics of biochemical interactions occurring among specifi c molecules on the cell membrane regulates a variety of processes underlying biological function, such as adhesion, migration, pathogen recognition, and signaling. These interactions alter the diffusion of membrane molecules, producing deviation from pure Brownian behavior consisting in trapping, confi nement, anomalous diffusion and ergodicity breaking [1].

Advanced biophysical techniques - including single particle tracking (SPT) - have revealed the link between the lateral diffusion of molecular components and the occurrence of such interactions [2]. However, resolving the kinetics of multi-particle processes such as molecular clustering and oligomerization is still challenged by the high local density, the spatial proximity of molecules and the fast time scale of interactions.

In order to overcome these limitations and directly visualize multi-particle interactions, we have realized a multicolor SPT setup capable of simultaneously resolving and tracking four spectrally distinct q-dots with a single excitation laser. Filter-based separation of the emission of the four fluorescent probes with minimum spectral overlap and cross talk allows us to measure molecular diffusion at densities as high as ~10 μm−2 while preserving localization precision of ~20 nm.

We are currently performing system calibrations and implementing a software platform for the channels registration and automatic particle tracking [3, 4]. The multicolor SPT setup will be used to investigate the clustering kinetics of the pathogen receptor DC-SIGN and its relation with anomalous diffusion and weak ergodicity breaking [5, 6]. Future developments of the setup will involve the extension of the number of spectral components (up to eight different probes) and their detection by means of a spectral decomposition scheme and machine-learning-based color assignment.

References[1] C. Manzo, M. F. Garcia-Parajo. A review of progress in single particle tracking: from methods to biophysical insights, Rep. Prog. Phys. 78 (2015) 124601.[2] J. A. Torreno-Pina, C. Manzo, M. F. Garcia-Parajo. Uncovering homo-and hetero-interactions on the cell membrane using single particle tracking approaches, J. Phys. D: Appl. Phys. 49 (2016) 104002.[3] W.S. Rasband, Image J, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016.[4] I.F. Sbalzarini , P. Koumoutsakos. Feature point tracking and trajectory analysis for video imaging in cell biology, Journal of Structural Biology 151 (2005) 182–195.[5] C. Manzo, J. A .Torreno-Pina, B. Joosten, I. Reinieren-Beeren, E. J Gualda, P. Loza-Alvarez, C. G Figdor, M. F Garcia-Parajo, A. Cambi. The Neck Region of the C-type Lectin DC-SIGN Regulates Its Surface Spatiotemporal Organization and Virus-binding Capacity on Antigen-presenting Cells, Journal of biological chemistry 287 (46), 38946-38955.[6] C. Manzo, J. A. Torreno-Pina, P. Massignan, G. J. Lapeyre, M. Lewenstein, and M. F. Garcia Parajo. Weak Ergodicity Breaking of Receptor Motion in Living Cells Stemming from Random Diffusivity, Phys. Rev. X 5, 011021 (2015).

A multicolor single particle tracking approach to reveal molecular interactions in a dense milieu.