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5. Cell adhesion and the actin cytoskeleton

The cell cortex The cytoskeleton networks The actin cytoskeleton Adhesion proteins The extracellular matrix The cell architecture is regulated by the adhesion zones

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The cell cortexActin microfilaments

Protein complexes membranesMedalia et al. 2002 Science 298: 1209-1213100 nm cryoelectron tomography

5 nm0.2 µm

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Cells possess two or three cytoskeleton networks made of polymerized proteins : an actin cytoskeleton (microfilaments), a tubuline one (microtubules) and intermediate filaments

The cycle of microfilament polymerisation-depolymerisation is coupled to ATP hydrolysis by actinThe cycle of microtubule polymerisation-depolymerisation is coupled to GTP hydrolysis by tubulinIntermediate filaments are controlled by protein phosphorylation and dephosphorylation

About hundred proteins control the growth and dissociation rates or are associated to microfilaments and microtubules : actin binding proteins, microtubule associated proteins, bundling proteins, molecular motors ...

microfilaments microtubules

Intermediate filaments

The cell cytoskeleton networks

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The actin cytoskeleton

Microfilaments are actin polymers, an ATP binding and ATP hydrolysing protein. The cycle of microfilament polymerisation-depolymerisation is coupled to ATP hydrolysis by actin

Microfilaments constitute the membrane cortex which allows the deformation of the plasma membrane. Stress fibers are actin microfilaments that link adhesion focal points. Actin microfilaments are reversibly linked to the plasma membrane by specific proteins (talin, catenin, ezrin ...)

Mechanical forces are exerted by actin polymerization at the plasma membrane and between actin microfilaments by molecular motors

reflection interference contrast microscopy actin immunofluorescence

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Structure of actin microfilaments

4 nm

barbed end

pointed end

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Actin polymerization kinetics : 1) no hydrolysis

At steady state, V = (1/a)dL/dt = konC – koff

Critical concentration :Cc = koff/kon = Keq

Cc+end = Cc

-end

+ end

- end

Cc

V(polymerizationrate)

C (monomer concentration)

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Actin polymerization kinetics : 2) ATP hydrolysis

ATP hydrolysis

AATPAADP

Pi

+ end

- end

CcATP

V(polymerizationrate)

C (monomer

concentration)

+ end

- end

CcADP

At steady state, during treadmillingV = VT(+) + VD(-) = kon

T.CT – koffT + kon

D.CD – koffD

CD ~ 0.1 CT , koffT < koff

D and konD < kon

T

In steady state CTc koff

D/konT

2 sec

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Experimental polymerization dynamics

CTc koff

D/konT 0.25/11.6 = 0.02 µM

Physiological actin concentration : about 100 µMMost actin is complexed to thymosin [ActinT] 10 µM

The theoretical actin microfilament elongation rate is :dL/dt = a.VT = a.kon

T.[ActinT] < (4 nm).(12 µM-1.s-1).(10 µM) = 0.48 µm.s-1

At the pointed end :-dL/dt = a.VD = a.koff

D = (4 nm).(0.25 s-1) = 1 nm.s-1

To be compared to the actual velocity of cell edge movements : up to 10 µm.s-1

Fujiwara et al. PNAS 2007 104 : 8827–8832

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In vivo evidence of microfilament treadmilling by FRAP experiments

Injection of rhodamine-actin

Localized photobleaching

Transport (treadmilling )

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How cytoskeleton molecules exert forces ?

1. Actin polymerization : the elastic brownian ratchet model(Mogilner and Oster 1996 Biophys. J. 71 : 3030-45)

Stall force

Mechanical work = ATP hydrolysisF. = G/N = G° + RT ln{[actinATP]}

= kBT ln{kon[actinATP]/koff}

actinATP + filamentN filamentN+1 + Pi

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How cytoskeleton molecules exert forces ?

2. Actin microfilament pressure at nucleation zones(C Sykes & J Prost (2005)P.N.A.S., 102, 7847-52)

Actin nucleation at the bead surface Concentric growth of actin microfilaments Shear stress accumulation Breaking the actin gel, resulting in asymmetric growth

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Visualization of F-actin network movement in motile keratocytes with FSMYam et al. JCB 178:1207-1221, 2007

Actin filament dynamics

30x real time

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How cytoskeleton molecules exert forces ?

3. Molecular motors sliding on microfilaments

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Regulation of cytoskeleton dynamics

↑ Stress fibers

↑ Filopodia↑ Lamellipodia

Ridley & Hall 1992Allen et al. 1997

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Plasma membrane Actin cytoskeleton

Example : microvilli at the plasma membrane Brush border cells of the intestinal epithelium

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Cell adhesion and the actin cytoskeleton

The cell cortex The cytoskeleton networks The actin cytoskeleton Adhesion proteins The extracellular matrix The cell architecture is regulated by the adhesion zones

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The extracellular matrix and cell adhesion molecules

The extracellular matrix is made of macromolecules (proteins and polysaccharides) synthesized and secreted by cells. It provides a specific mechanical and chemical environment for the cells.

Cell adhesion molecules are proteins expressed at the surface of cells that mediate binding to other cells or to the extracellular matrix.

Extracellular matrix proteins have a complex structure

Proteins of the ECM have a very large multi-domain structure They often contain growth factor-like domains or bind growth factors

A) Fibronectin. Encoded by a single gene but alternatively spliced at three regions [blue circles and box and V (variable) segment] to generate 12 proteins in rodents and 20 in humans. FN3 domains are widespread in ECM proteins. Binding sites for other matrix proteins are marked. The heparan sulfate–binding site can interact with proteoglycans or with syndecan, an integral-membrane proteoglycan. Integrin-binding sites; RGD (indicated by an asterisk) and LDV (Leu-Asp-Val, indicated by a pound sign). FN is a proangiogenic molecule, whose function depends on both the RGD site and the two alternatively spliced FN3 domains. FN also binds the proangiogenic growth factors VEGF and HGF. B) Fibrillin-1. Fibrillins include EGF-like domains, found in many ECM proteins, as well as TB (TGFb-binding, denoted by T) and hybrid (H) domains, specific to fibrillins and LTBPs. Binding sites for other matrix proteins and growth factors are marked. C) LTBP-1. Four-gene family with structures related to fibrillins. Known binding sites for TGF-b/LAP latent complex (SLC, blue), fibrillin, and FN are marked. RGD (asterisk) sequences in fibrillins and LTBPs may bind integrins. D) Thrombospondin-1 (TSP-1). TSPs contain TSP1 repeats (also found in other ECM proteins), EGF-like repeats, and a VWC domain, known in other proteins to bind BMPs. TSP3 repeats (purple) and C-terminal domains are unique to TSPs and bind multiple Ca2+ ions. The RGD (asterisk) sequence is known to bind to integrins. TSPs 1 and 2 have the structure shown, and both have antiangiogenic activity located in the TSP1 repeats, which bind to the CD36 receptor (39)

RO Hynnes (2009) the extracellular matrix : not just pretty fibrils Science 326 : 1216-19

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Fibrillin (2 µm)

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Cell adhesion molecules and cell adhesion structures

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Some family members

Ca2+ or Mg2+ dependence

Type Cytoskeleton association Cell structures

Cell-Cell Adhesion

Classical cadherins E, N, P, VE yes homophilic actin filaments (via catenins) adherens junctions

Desmosomal cadherins desmoglein yes homophilic Intermediate filaments (via desmoplakin, plakoglobin, and other proteins)

desomosomes

Ig family members N-CAM no both unknown no

Selectins (blood cells and endothelial cells only)

L-, E-, and P-selectins

yes heterophilic actin filaments no

Integrins on blood cells αLβ2 (LFA-1) yes heterophilic actin filaments no

Cell-Matrix Adhesion

Integrins many types yes heterophilic actin filaments (via talin, filamin, α-actinin, and vinculin)

focal adhesions

α6β4 yes heterophilic intermediate filaments (via plectin) hemidesmosomes

Transmembrane proteoglycans

syndecans no heterophilic actin filaments no

Classes of cell adhesion molecules

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Madin-Darby Canine Kidney cells, an epithelium model

Polarized cell : two compartments around the cell

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Tight junctions separates apical from basolateral compartments

Electron micrographs of cells in an epithelium in which a small, extracellular, electron-dense tracer molecule has been added to either the apical side (on the left) or the basolateral side (on the right). In both cases, the tracer is stopped by the tight junction. (photo Daniel Friend)The sealing strands hold adjacent membranes

together. They are composed of transmembrane proteins (claudins, occludins) that make contact across the intercellular space and create a seal.

24http://www.cytoo.com/

The cell architecture is regulated by the adhesion zones

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Golgi apparatus (in red) in RPE1 cells in standard culture conditions

Golgi apparatus (in red) in RPE1  cells onfibronectin micropatterns (in green)

Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity.Thery M, Racine V, Piel M, Pepin A, Dimitrov A, Chen Y, Sibarita JB, Bornens M. Proc Natl Acad Sci U S A 103(52):19771-6. 2006.