cytoskeleton and locomotion lecture eph 2014 láng, orsolya md, phd dept. genetics, cell &...

Cytoskeleton and locomotion

www.dgci.sote.huLecture EPh 2014

Láng, Orsolya MD, PhDDept. Genetics, Cell & Immunobiology, Semmelweis University

Main components of the eukaryotic cytoskeleton

Microfilaments:

actin

7nm

Microtubules:

tubulins (

25 nm

Intermediate filaments:

lamin

cell specific prot. (e.g. vimentin)

8-12 nm

+ Associated proteins (e.g. motor proteins)

Filamentous structures in the cytoplasm of walled bacteria

Microfilament like:

MreB

Microtubule like:

FtsZ, TubZ

Intermediate filament like:

Crescentin, Parm

weak sequence homology butcrystallography appreciate their striking structural properties and similarity to eukaryotic cytoskeletal elements

Nature Cell Biology12, 731–733 (2010)Nature Cell Biology12, 731–733 (2010)

Tissue level:

muscle movement

Cellular level:

determines shape of the cell

motility of the cells

cell adhesion

mitosis, meiosis

Subcellular level:

anchors organelles

organization of organelles

provides tensile strength

movement of chromosomes

organizing cell polarity

Intracellular movement of vesicles

- Endocytosis – clathrin-mediated endocytosis and phagocytosis

Function of the cytoskeleton

Dynamic

Adaptable

Stable

Strong

Cytoskeletal filaments are dynamic and adaptable

Stability of cytoskeletal filaments

Strong cytoskeletal filaments

Intermediate filaments- resistant to stretching forces

Microfilaments

( Garland Science Molecular Biology of the Cell 2008)

Molecular structure of actin

F-actinG-actin

ATPATPADPADP

ADPADP ATPATP

PiPolymerization - slow

Depolymerization

+

-

Drugs: cytochalasin – inhibition (cap at + end) phalloidin - stabilizer

Polymerazition of actin

Treadmilling of actin filament

Video: http://csls-text.c.u-tokyo.ac.jp/flash/0611_1.html

Molecular Biology of the Cell (© Garland Science 2008)

Actin and accessory proteins

3 groups: 3 groups:

banding and cross linking proteins banding and cross linking proteins

regulatory proteins: regulatory proteins: polymerization/depolymerization, polymerization/depolymerization, severing proteins,capping proteins severing proteins,capping proteins

Motor proteinsMotor proteins -- sliding on MF (myosin)sliding on MF (myosin)

Organisation of Organisation of actin filamentsactin filaments

SlidingSliding

Actin binding proteins (ABPs)

Molecular Biology of the Cell (© Garland Science 2008)

Organisation of actin filaments

Molecular Biology of the Cell (© Garland Science 2008)

Cross-linking proteins I.

Contractile bundle Parallel bundle

Molecular Biology of the Cell (© Garland Science 2008)

Cross-linking proteins II.

Gel-like network

plasma membrane

Regulatory proteins

-- ++

CofilinCofilinSeverinSeverinGelsolinGelsolin

CAP39CAP39

SeverinSeverin

GelsolinGelsolin

VillinVillin

CapZCapZ

TropomodulinTropomodulin

Profilin: G actin pool

Thymosin: actin sequestring

Formin: actin polymerizing protein

Actin polymerisation – moition I.Actin polymerisation – moition I.

(Lodish, H. et al. Mol. Cell Biol. 2000, 767)(Lodish, H. et al. Mol. Cell Biol. 2000, 767)

Acrosomal reaction

Listreia Monocytogenes infection

Actin polymerisation – motion II.Actin polymerisation – motion II.

Actin – membrane links

F-ActinF-Actin

IntegrinIntegrinFilaminFilamin

ProfilinProfilin- G-aktin- G-aktin

Myozin I.Myozin I.Arp2/3Arp2/3

membranemembrane

Model of actin nucleation

WASP = Wiscott-Aldrich syndr. prot.WASP = Wiscott-Aldrich syndr. prot.

Proline-richprotein

Profilin-mechanism

(Lodish, H. et al. Mol. Cell Biol. 2000, 767)(Lodish, H. et al. Mol. Cell Biol. 2000, 767)

TT44 = = thymozin thymozin 44

Filamin – Membrane link

filaminfilamin

actinactin

actin filamentactin filament actininactinin

vinculinvinculin

paxillinpaxillin

talintalin

integrinintegrinfibronectinfibronectin

Structure of focal contact

++

Thrombocyte: Thrombocyte: filaminfilamin

Muscle: Muscle: dystrophindystrophin

Epithel: Epithel: ezrinezrin

A plasma membrane – cortex links

SpectrinSpectrintetramertetramer

GlycophorinGlycophorin

AnkyrinAnkyrin

(Lux SE, 1979 Nature 281:426)

Microvilli

At bottom: (spectrin, myosin, intermediate filaments)It is called: terminal web

Motor proteins: myosins

General structure:

Globular head and fibrillar tail

Heavy chains and light chains

Head: motor domain with ATP-ase activity

ADP- straight

Direction: + end motors

Molecular Biology of the Cell (© Garland Science 2008)

Myosin II molecule

Distribution of myosines in the migrating Distribution of myosines in the migrating DyctiosteliumDyctiostelium and in dividing celland in dividing cell

myozin Imyozin I(green)(green)

myozin IImyozin II

(red)(red)

(Fukui, Y. Mol. Cell Biol 2000, 785))

Microtubules

Molecular Biology of the Cell (© Garland Science 2008)

Molecular structure of tubulin dimers and microtubules

GTP

Molecular Biology of the Cell (© Garland Science 2008)

Polymerisation of microtubules

Drugs: Colchicine and colcemid– inhibition (binds subunit) Taxol - stabilizer

Molecular Biology of the Cell (© Garland Science 2008)

Polymerisation of microtubules

Dynamic instability

and treadmilling Video: http://csls-text.c.u-tokyo.ac.jp/flash/0612_1.html

Molecular Biology of the Cell (© Garland Science 2008)

Role of tubulin in nucleation of microtubules at - end

Structural MAP-sStructural MAP-s-- MT-assemblyMT-assembly-links to MF and to IF links to MF and to IF

(eg. tau, MAPs1 and MAPs2)(eg. tau, MAPs1 and MAPs2)

Motor proteinsMotor proteins -- sliding on MTsliding on MT(e.g. kinesin and dynein family)(e.g. kinesin and dynein family)

Enzymes, signal moleculesEnzymes, signal molecules- glycolytic enzymes- glycolytic enzymes

- kinases- kinases

Shape and polarityShape and polarityof the cellof the cell

Vesicular transportsVesicular transports

Assembly of moleculesAssembly of molecules

Microtubule associated proteins (MAPs)

Structure ofStructure of motor-proteinsmotor-proteins

motor domain

motor domain

assoc.polypeptides

assoc.

polypeptides

assoc.

polypeptides

„stalk”

„stalk”

Kinesin Myozin Dynein

Motor proteins

++

--

dyneindynein

++

kinesinkinesin

light chainlight chain

heavyheavychainchain

?

++--

kinesinkinesin

dyneindyneincAMPcAMP cAMPcAMP

pigment cellspigment cells

ATPATPADPADP

ATPATP

ADPADPADPADP

ATPATP

ADPADP

ADP-PiADP-Pi

KinesinKinesin

Interphase cellInterphase cell

Dividing cellDividing cell

NeuronNeuron

centrosomecentrosome

Basal bodyBasal body

CillaCilla

spindlespindle

centrosomecentrosome

axonaxon

Microtubular systems in the cells

-Centrosome

-Cilia / flagellum

-Mitotic system

- Vesicular transport

In cells: MTOC = Microtubule organizing center

(Brinkley, B.R. Encyclop. Neurosci. 1987, 665)

-tubulin-tubulin

9x3 microtubules A,B,C

2 centrioles at a right angle

Organisator:

Gamma-tubulin in pericentriolar matrix

- end of forming microtubule

Molecular Biology of the Cell (© Garland Science 2008)

Centrosome

Cilia

ciliacilia

flagellumflagellum

Paramecium

AABB dynein-armsdynein-arms

nexinnexin

tubulin tubulin (13 ill. 11 protofilaments)(13 ill. 11 protofilaments)

Structure of cilia

Molecules composing the cilia

70% α and β tubulin dynein arms - outer - 9 polypeptides - ATP-ase

- inner – composition varies radial spokes - 17 polypeptides

more than more than 250250 types of molecules types of molecules

Role of the dynein arms in beating cilia

Telescopic effect Beating

Microtubules of mitotic spindle and kinetochore

How motor proteins can organise the position of cell organelles (ER, Golgi) ?

(Hirokawa, N. Science 1998, 279:519

Dynein – membrane relationsDynein – membrane relations

(Hirokawa, N. Science 1998, 279:519)

Intermediate filaments

Molecular Biology of the Cell (© Garland Science 2008)

Structure of an intermediate filaments

Monomer

Parallel dimer

Antiparallel tetramer

Protofilaments

Intermedaite

filaments

Mechanical characterization of cytoskeleton components

defo

rmat

ion

force

actin filament

intermedier filamenti.e. vimentin

microtubule

= rupture

Role of intermedier filaments

Buffer against external mechanical stress

Tissue specificity !!!

Epithel – keratin

Connective tissueMusclesNeuroglia

Neurones(axon) - neurofilaments

} vimentin-like

Exception:Nucleus – lamines (A,B,C) →(lamina fibrosa)

Vimentin

Desmin

Glial protein

HH22N-N- --COOHCOOH

keratinskeratins

vimentinvimentin

neurofilam. prot.neurofilam. prot.

nuclear protnuclear prot..

helical domainhelical domain

Domain structures of intermedier filamentumsDomain structures of intermedier filamentums

HeadHead TailTail

Molecular Biology of the Cell (© Garland Science 2008)

Cross bridges are formed by non-

helical C terminus

few cross bridges

Axon of a neuron

Neurofilament (NF-H)

Glial cell

Glial filaments

Axon cross-section

Cell locomotion/ movement

Cellular level:

Ciliar movement

Amoeboid mocevent

Mesenchymal migration

Collective migration

Tissue level:

muscle movement – skeletal muscle

Cilia - flagellum

Amoeboid movement

chemoattractant

Composition of thick filament in a sarcomer

Composition of sarcomer

Working unit bordered by two Z-lines/disc Z line – -actinin + desmin Thin filament – -helical actin

molecules + tropomyosins + troponin Thick filament – myosin II molecules

+ MBP (myosin binding proteins)Other proteins: titin, distrophin

Troponin:Troponin: Tn-T binds tropomyosin Tn-C binds Ca2+(4 Ca2+/mol = calmodulin) Tn-I inhibitor

Skeletal muscle - Sarcomere

Skeletal muscle - Sarcoplasmic reticulum

X-linked recessive inheritance

Duschenne muscular distrophy

Useful videos and linkes:

http://csls-text.c.u-tokyo.ac.jp/active/06_01.htmlhttp://www.microscopyu.com/moviegallery/livecellimaging/index.htmlhttp://www.cellmigration.org/science/