fluorescent proteins in current biology
DESCRIPTION
AACIMP 2011 Summer School. Neuroscience Stream. Lecture by Pavel Belan.TRANSCRIPT
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Bogomoletz Institute of PhysiologyKiev
Ukraine
Dr. Pavel Belan
Fluorescent proteins in current biology
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Lecture Plan
• Development of optical microscopy
• Basics of fluorescence
• Basics of molecular biology
• Green Fluorescent Protein (GFP) in the current biology
• Gene delivery techniques
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Replica of microscope by Van Leeuwenhoek
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Microscopic section through one year old ash tree
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Compound microscope
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Current optical microscope
Olympus web site
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iMIC Digital Microscopes Intravital 2P
Copyright © 2006 - 2011 TILL Photonics GmbH
State-of-the-Art microscopes
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• The rapid advances made in developing live-cell imaging microscopes based upon low light level electron multiplying CCD cameras and different confocal systems, allows imaging fluorescent probes and optical highlighters over the entire visible and near-infrared spectral regions with near real-time precision and submicron space resolution.
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DIC image of cultured hippocampal neurons
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Fluorescent image of double labeled cultured hippocampal neurons
Biochimica et Biophysica Acta 1802(1):143-50. 2010
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Basic principles of fluorescence
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Marking proteins by fluorescent tags
Fluorescent tag
Protein of interest
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Problems with fluorescent tags
Cell
impermeability
unspecificity
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How can we specifically tag proteins by fluorescent markers?
How to do that in living cells?
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Gene expression
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Reporter gene expression
Gene under study
Reporter fluorescent protein
Protein under study
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Himeric gene expression
FP gene
Gene under study
Himeric fluorescent protein
Gene under study
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Plasmid construction
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"Bioluminescent dinoflagellates (Lingulodinium polyedrum) lighting a breaking wave at midnight. The blue light is a result of a luciferase enzyme (like firefly luciferase, but the enzyme in L. polyedrum shares no similarity with that of the firefly enzyme). Under the right conditions, the dinoflagellates become so numerous that the water takes on a muddy reddish color (hence the name "Red Tide").
Image of bioluminescent tide event at a beach in Carlsbad California
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
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Aequorea victoria
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
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Studies of protein expression using GFP как главный репортерный белок
GFP
It is possible to report gene expression in single living cells
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First study describing Green Fluorescent Protein (GFP) as a protein expression marker
Science 1994:Vol. 263. no. 5148, pp. 802 - 805DOI: 10.1126/science.8303295
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Olympus website
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Diversity of Fluorescent proteins
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
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Examples of Labeling by Different Fluorescent Proteins
http://www.conncoll.edu/ccacad/zimmer/GFP
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Pyramidal neurons of «rainbow» rats
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J Biol Chem. 2011 Mar 30. [Epub ahead of print]
Mitochondrial dynamics in axons of hippocampal pyramidal neurons
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llustrated is the photoconversion of a PS-CFP2 fluorescent protein fusion product with human beta-actin using a 405-nanometer diode laser for imaging and conversion, as well as the argon-ion 488-nanometer spectral line for imaging and tracking of the photoconverted protein.
Photoconversion of a PS-CFP2 fluorescent protein as a way to study protein polymers
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Examples of in vivo FP expression
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How can we introduce plasmids that are large circular DNA molecules into the cells?
Transfection and infection as methods to introduce gene constructs
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Direct microinjection into cultured cells or nuclei
Physical Transfection Methods
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Electroporation
Biolistic particle delivery
Physical Transfection Methods
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Single Cell Electroporation (SCE)
Physical Transfection Methods
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Various transfection technologies based on chemical reagents
Chemical Transfection Methods
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Flowchart of the main procedures of Ca2+ phosphate transfection protocol
Chemical Transfection Methods
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Formation of optimal DNA/Ca2+-phosphate precipitate and subsequent dissolution to stop transfection
Chemical Transfection Methods
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Viral infection as a way to deliver genetic materials into the cells
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Virus infection technique
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Rat neocortical neurons infected with recombinant Sindbis virus-enhanced green fluorescent protein in vivo
A) Image of the injection site (arrow) and surrounding area (postnatal day [P] 11). (B) Higher magnification images (P 14). Left, layer 2/3 pyramidal neurons. Middle, layer 5 interneuron. Right, layer 5 pyramidal neurons. (C) 2-photon laser scanning microscopy image of a layer 2 pyramidal neuron in a section from a mouse (P 36).
Lern. Mem. 7, pp. 433-441, 2000.
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(a1) Overlay of fluorescent and phase-contrast images illustrating a pair of transfected (green) and nontransfected neurons. (a2) Double whole-cell patch-clamp recording of the pair of neurons shown in (a1). Upper trace shows presynaptic current and lower trace the corresponding postsynaptic current. (b) A pair of neurons both transfected with EGFP.
Transfection of hippocampal neurons in low-density microisland cultures
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Fast protein translocation study by means of GFP
Eur. J. Neurosci. (2010) 32:347
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• Conclusions• The battery of fluorescent proteins, tools for their
genes delivery and imaging tools enables the cell biologists to readily monitor protein dynamics in living cells, continuing to provide numerous new insights into the behavior of proteins, organelles, and cells. In so doing, these remarkable probes have ushered in a new era of cell biology in which both steady-state and kinetic microscopy techniques can be used to decipher pathways and mechanisms of biological processes.
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• Basics of FRET
• Combining FRET with molecular biology techniques
• Introduction to optogenetics
Lecture Plan
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Can we study inter- and intramolecular interection in living
cells using GFPs?
Do we have enough spatial resolution with our conventional
microscope systems?
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Optical microscopy spacial resolution
Compound optical microscopes are limited in their ability to resolve fine details by the properties of light. Optical microscope resolution depends upon the microscope objective and refraction properties of medium between the object and objective lens altogether characterized by the objective numerical aperture, AN. At the certain wavelengths of light (λ), the maximal resolution (d) is given by:
In case of λ = 550 nm (green light) and air as the medium, (the highest practical AN is 0.95), the maximal resolution is about 0.3 micrometers. At the same time due to aberrations, even the best optical microscope is limited to a resolution of around 0.5 micrometers.
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http://www.olympusfluoview.com/applications/fretintro.html
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http://www.olympusfluoview.com/applications/fretintro.html
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The efficiency of FRET, EFRET, which is defined as probability of the occurrence of energy transfer per donor excitation event, is a steep function of the distance between the fluorophores, r, as given by the following equation:
EFRET
where Ro is the Forster distance
http://www.olympusfluoview.com/applications/fretintro.html
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Experimental Setup for Ratiometric FRET Imaging
DualView
CCD camera
Inverted Microscope
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The MAG Biosystems™ Dual-View utilizes a single beamsplitter to split the incident beam from the microscope into two independent beams. One beam contains all the emission reflected off of the beamsplitter; the other contains all the emission transmitted through the beamsplitter. Each of these emission channels is projected onto half of the CCD array at exactly the same point in time. Simultaneous multichannel imaging is essential to achieve quantitative emission ratiometric analysis.
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http://www.olympusfluoview.com/applications/fretintro.html
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Cameleons have been devised by Roger Tsien and others. They are based on fluorescence resonance energy transfer (FRET) between two fluorescent molecules that are linked by a short stretch of calmodulin, a protein that changes its shape in the presence of calcium. In the absence of calcium the two fluorescent proteins are
well separated. These calcium sensors are called Cameleons because they change color and have a long tongue (calmodulin) that retracts and extends in and out of its mouth when it binds and releases calcium.
Cameleon – FRET based Ca2+ probe
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Cameleon – основанный на FRET Ca2+ зонд
Tsien’s Lab
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Design and plasma membrane expression of VSFP2s
PLoS ONE, doi:10.1371/journal.pone.0000440
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Protein kinase G activity measured by FRET
Tsien’s Lab
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FRET summary movie
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• Conclusions• Fluorescent proteins together with current biophysical
and molecular biology approaches are the powerful tool allowing to study practically any type of inter- and intramolecular interection in living functional cells and thereby they can be used to decipher pathways and mechanisms of biological molecular signaling processes.
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• Can we not only study molecular signaling processes in living cells using optically active proteins but also control these processes in cell specific manner?
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Optogenetics
Nature web site
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Conclusions
• Optogenetics is a new experimental methodology based upon the combination of genetic and optical methods to control specific mainly electrical events in targeted cells of living tissue.
• Optogenetics allows to almost instantaneously switch on and off certain neuronal groups in order to study their functions and relationships with other cells of animal body.
• Optogenetics also allows to correct pathological changes of signaling processes being a prerequisite for treatment of many neurodegenerative disorders.