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PDF Printing 600 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) 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) 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) EJ.) ( r.* .'h4il Acl bfttrr a.l:*, (a) 'a: Adheionol plaque Ttt 11'?l (.) f. lol3 Ca\\r ',ttJ t*o r tX-t"\; $u gftutJ: G) t rw^ {ea* ft t\ arut t'' ( " tP)' n 1 G {.* {n'r\ "JLl'* '.-5 I g\t*'\ * 6P nt4wr It . \o*\ oJlrrdts''r e" (t A" chcrr* ( C.**d." I ( L'r{ lr Eenser .*ll t) q (b) t . ltfb) REVIEWS many studies have shown that FAK inhibition blocks the Box 1 | Molecular architecture of focal contacts response to cell motility cues18,recent and provocative studies have shown that inhibition of FAK expression or activity resulted in increased carcinoma cell migration through the dissolution of N-cadherin-mediated cell–cell Actin stress fibres contacts in HeLa CELLS19. It is possible that this latter observation might be a cell-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 as Myosin both a signalling kinase and as an adaptor/scaffold pro- Myosin Myosin tein, which places FAK in a position to modulate various intracellular signalling pathways (FIG. 1).Namely,it is the association of FAK with both activators and/or Zyxin α inhibitors of various small GTPase proteins (Rho, Rac, -A V c α in tin -A Cdc42 and Ras) that enables changes in FAK activity to c in V ctin u in lin c in α u be connected to alterations in the polymerization or sta- -A α lin ctin -A bilization of actin and microtubule filaments. in ctin in Focal contact V Additionally, because migrating cells experience changes in c proteins FAK FAK u in forces through integrin contacts that link the ECM lin Pa axillin p130 p130 xillin with the cytoskeleton, FAK is important in the ‘sensing’ P T Plasma Talin Src Cas Cas Src alin of mechanical forces that are either generated internally membrane or exerted on cells20.FAK activation is therefore involved in modulating ‘corrective’ cell responses to environmen- tal stimuli. FAK does this through signal-mediated effects on actin polymerization, the assembly or disas- Integrins sembly of focal contacts, and the regulation of protease activation or secretion16,21,22. αββ α The FERM and FAT domains of FAK FAK is a ubiquitously expressed 125-kDa protein tyro- Extracellular matrix sine kinase that is composed of an N-terminal FERM (protein 4.1, ezrin, radixin and moesin homology) The extracellular matrix, integrins (α- and β-transmembrane heterodimeric proteins) domain, a central kinase domain, proline-rich regions 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 and a C-terminal focal-adhesion targeting (FAT) interior and can also relay intracellular signals to generate an activated integrin state at domain (FIG. 2).The FERM domain of FAK facilitates a the cell surface113.The integrin-binding proteins paxillin and talin recruit focal adhesion signalling linkage from receptor tyrosine kinases such as kinase (FAK) and vinculin to focal contacts (see figure). α-Actinin is a cytoskeletal the epidermal growth factor receptor (EGFR) and the 23 protein that is phosphorylated by FAK, binds to vinculin and crosslinks actomyosin platelet-derived growth factor receptor (PDGFR) .In stress fibres and tethers them to focal contacts. Zyxin is an α-actinin- and stress-fibre- analysing cell-motility-promoting signals that are initi- binding protein that is present in mature contacts. Although the aforementioned proteins ated by G-PROTEIN-COUPLED RECEPTORS (GPCRs), overexpres- are found in most focal contacts, the membrane-associated protein tyrosine kinase Src sion of the FAK FERM domain blocked FAK activation and the ADAPTOR PROTEIN p130Cas associate with focal contacts following integrin and resulted in the inhibition of G-protein-stimulated clustering. Integrin-mediated FAK activation is mediated in part by matrix binding or by cell migration24.It is the FAK FERM domain that can force-dependent changes in cytoskeletal linkages.
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