i. non-muscle actin: bundles and networksburr/bio5440/lecnotespdfs/...the cdc42 g-protein (wasp...

BIO 5440 Burr, 2012 I. Non-muscle Actin: Bundles and Networks 1. Actin bundles: (“Plus” ends are attached to membranes)

a) “Tight bundles”: bundled by fascin [fibroblasts], or villin, fimbrin [microvilli of intestinal epithelial cells: fig 17-4, p716]. (Protein structures listed in figure 17-18, p 729)

Examples: fibroblasts: lamellipodia, microspikes (10 µm), filopodia (= long microspikes: 50µm) epithelial cells: Microvilli [ fig 17-4, p716] b) “Loose bundles” (contractile): bundled by α-actinin [leaves room in between parallel actin filaments for the insertion of myosin I or II] Examples: (i) fibroblast “stress fibers” (fig 19-32, p832) ( ⇒ focal adhesions/“3D” adhesions) (fig 19-33, p834; fibronectin (Fn) Fn receptor (an Integrin) Focal adhesion kinase (fak) pp. 10-12 of handout Src kinase vinculin, talin, (ii) contractile ring in mitotic cells (fig 17-34, p742) (iii) adherens belt in epithelial cells (fig 17-4, p716) 2. Actin networks: filamin [crosslinks f-actin, leading to gel formation] (fig 17-18, p 729), versus

gelsolin [Ca++-activated severing of f-actin, leading to sol formation] Examples: cell cortex; gel-sol conversions in amoeba pseudopod (Tom Stossel,

American Scientist, 78, 408-423 [1990]: pp 17a,17b of handout) 3. Actin filaments are linked to membrane proteins Examples: dystrophin; the actin-spectrin network underlying the red blood cell membrane II. Polymerization Kinetics of Actin, in vitro. (p24 of handout) 1. Critical concentration for polymerization (Cc) 2. Plus and minus ends 3. Significance of ATP hydrolysis: Cc for plus end can be different from Cc for minus end 4. Treadmilling 5. Drugs affecting polymerization/depolymerization of actin: cytochalasin (destabilizes) & phalloidin (stabilizes)

BurrTypewritten TextLecture 2(a)

BIO 5440 J.G. Burr III. Regulation of actin polymerization/depolymerization, in vivo Based on the intracellular concentration of actin (0.5mM) and ionic conditions in vivo, one would predict that nearly all cellular actin should exist as filaments. Actual measurements, however, show that as much as 40% of actin is unpolymerized. What’s going on? 1. Inhibition of f-actin assembly by thymosin β4 [forms 1:1 complex with g-actin (ATP form of g- actin); covers up ATP binding site]; profilin also forms 1:1 complex with g-actin, competes with thymosin β4 for binding to g-actin. Profilin binds on + side of g-actin, leaving the ATP-binding site

on the other side (-) open and accessible (fig 17-11, p.722). 2. Promotion of f-actin assembly by profilin [lower affinity for g-actin than thymosin β4, and permits ADP/ATP exchange; binds membranes via PIP2] (fig 17-11, p.722). 3. After actin polymerizes, it quickly (few seconds) hydrolyzes ATP, but ADP + Pi remains in binding

site. Cofilin promotes release of Pi, and severing of f-actin, leading to depolymerization. (fig 17-11, p.722) (Handout, pp 35, 36)

4. Two classes of cytosolic proteins nucleate actin filament polymerization in vivo: formins and

Arp2/3 complexes

(a) Formins: formins nucleate the polymerization of linear actin filaments that form the contractile actomyosin stress fibers which connect focal adhesion structures; many formins are regulated by Rho family G-proteins. (Figs 17-13a, 17-14) (Notes, pp 32, 33)

(b) Arp2/3 complexes associate WASp family proteins (eg, N-WASp, WAVE) to nucleate actin filament polymerization. (Fig 17-16) • When partnering with a WASp family member such as N-WASp, the Arp 2/3 complex

initiates the polymerization of linear actin filaments, which then bundle to form filopodia at the leading edge of moving cells. (Notes p. 34a)

• When acting with a WAVE partner (eg, WAVE2), which is activated via the small G-protein Rac, the Arp 2/3 complex initiates the formation of branched actin filaments, which can form lamellipodia at the leading edge of a moving cell. (Notes p. 34b)

5. The Cdc42 G-protein (WASp activation) and Rac G-protein (WAVE activation) are found at the

leading edge of a moving fibroblast where they generate lamellipodia and filopodia; Rho is confined to the rear of the cell, where it initiates formation of actin filaments that interact with myosin contractile bundles in both stress fibers and the cell cortex. (Notes pp 39, 40)






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NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 6 | JANUARY 2005 | 57

R E V I E W S

RHO-FAMILY GTPases

A subfamily of small (~21 kDa)GTP-binding proteins that arerelated to Ras and that regulatethe cytoskeleton. Thenucleotide-bound state isregulated by GTPase-activatingproteins, which catalysehydrolysis of the bound GTP,and guanine nucleotide-exchange factors, which catalyseGDP–GTP exchange.

many studies have shown that FAK inhibition blocks theresponse to cell motility cues18, recent and provocativestudies have shown that inhibition of FAK expression oractivity resulted in increased carcinoma cell migrationthrough the dissolution of N-cadherin-mediated cell–cellcontacts in HeLa CELLS19.

It is possible that this latter observation might be acell-type-specific signalling event. However, we specu-late that the ability of FAK to promote both the matura-tion and turnover of focal contacts is related to its role asboth a signalling kinase and as an adaptor/scaffold pro-tein, which places FAK in a position to modulate variousintracellular signalling pathways (FIG. 1). Namely, it is theassociation of FAK with both activators and/orinhibitors of various small GTPase proteins (Rho, Rac,Cdc42 and Ras) that enables changes in FAK activity tobe connected to alterations in the polymerization or sta-bilization of actin and microtubule filaments.Additionally, because migrating cells experience changesin forces through integrin contacts that link the ECMwith the cytoskeleton, FAK is important in the ‘sensing’of mechanical forces that are either generated internallyor exerted on cells20. FAK activation is therefore involvedin modulating ‘corrective’ cell responses to environmen-tal stimuli. FAK does this through signal-mediatedeffects on actin polymerization, the assembly or disas-sembly of focal contacts, and the regulation of proteaseactivation or secretion16,21,22.

The FERM and FAT domains of FAKFAK is a ubiquitously expressed 125-kDa protein tyro-sine kinase that is composed of an N-terminal FERM(protein 4.1, ezrin, radixin and moesin homology)domain, a central kinase domain, proline-rich regionsand a C-terminal focal-adhesion targeting (FAT)domain (FIG. 2). The FERM domain of FAK facilitates asignalling linkage from receptor tyrosine kinases such asthe epidermal growth factor receptor (EGFR) and theplatelet-derived growth factor receptor (PDGFR)23. Inanalysing cell-motility-promoting signals that are initi-ated by G-PROTEIN-COUPLED RECEPTORS (GPCRs), overexpres-sion of the FAK FERM domain blocked FAK activationand resulted in the inhibition of G-protein-stimulatedcell migration24. It is the FAK FERM domain that canbind to and promote the integrin- and FAK-mediatedactivation of other non-receptor tyrosine kinases such asETK25. Additionally, actin- and membrane-associatedadaptor proteins such as ezrin can bind to the FAKFERM domain and facilitate increased FAK activation inan integrin-independent manner26.

How the FAK FERM domain associates with varioustargets is an active area of research. FAK can becomepost-translationally modified by the covalent additionof a small ubiquitin-related modifier (SUMO) at theε-amino position of Lys152 (REF. 27). In most instances,sumoylation is associated with the nuclear import ofproteins and, correspondingly, sumoylated FAK wasenriched in the nuclear fraction of cells27. Althoughblocking nuclear export using leptomycin B promotesthe nuclear accumulation of FAK, and exogenousexpression of the FAK FERM domain exhibits strong

(PTK2) and it is located at human chromosome8q24term, a locus that is subject to amplification inhuman cancer cells9. Furthermore, elevated levels ofPTK2 mRNA have been found in studies of human car-cinoma tumours and in acute lymphoblastic leukaemias,as detected by large-scale gene expression profiling10,11.FAK protein expression is elevated in many highlymalignant human cancers12, and studies have shownthat FAK signalling can promote changes in cell shape13,14

and the formation of podosomes or invadopodia15,which leads to an invasive cell phenotype16,17. Whereas

Box 1 | Molecular architecture of focal contacts

The extracellular matrix, integrins (α- and β-transmembrane heterodimeric proteins)and the cell cytoskeleton interact at sites called focal contacts. Focal contacts are dynamicgroups of structural and regulatory proteins that transduce external signals to the cellinterior and can also relay intracellular signals to generate an activated integrin state atthe cell surface113. The integrin-binding proteins paxillin and talin recruit focal adhesionkinase (FAK) and vinculin to focal contacts (see figure). α-Actinin is a cytoskeletalprotein that is phosphorylated by FAK, binds to vinculin and crosslinks actomyosinstress fibres and tethers them to focal contacts. Zyxin is an α-actinin- and stress-fibre-binding protein that is present in mature contacts. Although the aforementioned proteinsare found in most focal contacts, the membrane-associated protein tyrosine kinase Srcand the ADAPTOR PROTEIN p130Cas associate with focal contacts following integrinclustering. Integrin-mediated FAK activation is mediated in part by matrix binding or byforce-dependent changes in cytoskeletal linkages. Several other proteins such asextracellular signal-regulated kinase 2 (ERK2) and calpain are known to be transientlypresent at focal contacts (not shown). The composition of a focal contact is thereforeconstantly varying depending on external cues and cellular responses.

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BurrTypewritten TextNature Reviews: Molecular Cell Bio0logy 6, 56-68 (Jan 2005)

BurrTypewritten TextMitra, S. et al. "Focal Adhesion Kinase"

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Molecular Detail of Adhesion Plaque Structure

Molecular architecture of focal contacts

The extracellular matrix, integrins ( - and -transmembrane heterodimeric proteins) and the cell cytoskeleton interact at sites called focal contacts. Focal contacts are dynamic groups of structural and regulatory proteins that transduce external signals to the cell interior and can also relay intracellular signals to generate an activated integrin state at the cell surface113. The integrin-binding proteins paxillin and talin recruit focal adhesion kinase (FAK) and vinculin to focal contacts (see figure). -Actinin is a cytoskeletal protein that is phosphorylated by FAK, binds to vinculin and crosslinks actomyosin stress fibres and tethers them to focal contacts. Zyxin is an -actinin- and stress-fibre-binding protein that is present in mature contacts. Although the aforementioned proteins are found in most focal contacts, the membrane-associated protein tyrosine kinase Src and the adaptor protein p130cas associate with focal contacts following integrin clustering. Integrin-mediated FAK activation is mediated in part by matrix binding or by force-dependent changes in cytoskeletal linkages. Several other proteins such as extracellular signal-regulated kinase 2 (ERK2) and calpain are known to be transiently present at focal contacts (not shown). The composition of a focal contact is therefore constantly varying depending on external cues and cellular responses.

10 Nature Reviews Molecular Cell Biology 6 (No 1), 56-68; January 2005

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ffiffiilffiffi3,ffiBlogenesls of focal sdhesions. r I Many integrins that are not bound to the exfracellular matrix @CM) are presenton the oell surfaoe in an inactive oonformation, which is oharacterized by bent' extracellular domains that mask theECM-binding pocket. This oonformation is stabilized by interactions between integrin tansmemb'rane domains,membrane-proximal extacellular domains and a salt bridge between the cytoplasmio domains. b I When talin isrecruited to the plasma membrane and aotivated in association with phosphatidylinositol phosphate kinase tJ?e-I'f(PIPKIV), it binds to the cytoplasmic tail of Fintegrins. This interaction s€parates the cytoplasmic domains andinduces the integrins to adopt the lrrimed' conformation. c I The int€grin exfacellulm domains extend and unmaskthe ligand-binding site, allowing the integrin to bind specifrc ECM molecules. The separated integrin cytoplasmiodomains and talin form a platform for the recruitrnent of other focal-adhesion proteins. Integrin-linked kinase (IIJ(),and isofomrs of particularly interesting Cys-His-rich protein @INCII) and parvin form the IPP complex @g. 2) inthe cloplasrq and this complex is recruited to focal adhesions through intcraotions with otlrer factors, suoh aspaxillin. Other proteins suoh as vinculin and focal adhesion kinase (FAK) are recruited to the nasoent focal conplexin a sequential manner. The rnaturation of focal adhosions involves clustering ofactive, ligand-bound integrins altdtho ura$ly of a mult[[,o8h ooEgl€x tlnt is crpqble of linting intsgrins to lhe .etin cytoslaldon lndcommunicating with signelling pathways.

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Example of the roles of inside-out and outside-in signaling in cell behavior: Entry of neutrophils into infected tissue Neutrophils (a type of white blood cell) circulating in the blood encounter chemokines such as CXCL8 bound to the luminal extracellular matrix of capillary endothelial cells in infected tissue. CXCL8 binds to its receptor on the surface of the neutrophill; the receptor then initiates signaling events that lead to activation of talin and consequent binding of talin to the cytosolic tails of inactive integrins, thereby converting the integrins (LFA-1 in this case) from the inactive (bent) form to the primed, high affinity state (extended form) (see the figure on preceding page in your notes) (this is inside-out signaling). When the high affinity LFA-1 integrin binds its ligand (ICAM-1) on the surface of the capillary endothelial cells, the activated integrins initiate outside-in signaling that results in the white blood cell behavior known as ‘diapedesis’—the invasive migration of the neutrophil out of the capillary lumen, through the capillary endothelium, and migration into the infected tissue.

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1 Inside-out signaling initiated by chemokine receptor leads to activation of the integrin LFA-1 (conversion to high affinity)

2 Outside-in signaling initiated by the LFA-1 integrin (after binding to ICAM-1) leads to diapedesis by the cell.

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i. non-muscle actin: bundles and networksburr/bio5440/lecnotespdfs/...the cdc42 g-protein (wasp...

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