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)

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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. Several other proteins such as bind to and promote the integrin- and FAK-mediated extracellular signal-regulated kinase 2 (ERK2) and calpain are known to be transiently activation of other non-receptor tyrosine kinases such as present at focal contacts (not shown). The composition of a focal contact is therefore ETK25.Additionally, actin- and membrane-associated constantly varying depending on external cues and cellular responses. adaptor proteins such as ezrin can bind to the FAK FERM domain and facilitate increased FAK activation in an integrin-independent manner26. (PTK2) and it is located at human chromosome How the FAK FERM domain associates with various RHO-FAMILY GTPases 8q24term, a locus that is subject to amplification in targets is an active area of research. FAK can become A subfamily of small (~21 kDa) human cancer cells9.Furthermore, elevated levels of post-translationally modified by the covalent addition GTP-binding proteins that are PTK2 mRNA have been found in studies of human car- of a small ubiquitin-related modifier (SUMO) at the related to Ras and that regulate ε the cytoskeleton. The cinoma tumours and in acute lymphoblastic leukaemias, -amino position of Lys152 (REF.27).In most instances, 10,11 nucleotide-bound state is as detected by large-scale gene expression profiling . sumoylation is associated with the nuclear import of regulated by GTPase-activating FAK protein expression is elevated in many highly proteins and, correspondingly, sumoylated FAK was proteins, which catalyse malignant human cancers12, and studies have shown enriched in the nuclear fraction of cells27.Although hydrolysis of the bound GTP, that FAK signalling can promote changes in cell shape13,14 blocking nuclear export using leptomycin B promotes and guanine nucleotide- 15 exchange factors, which catalyse and the formation of podosomes or invadopodia , the nuclear accumulation of FAK, and exogenous GDP–GTP exchange. which leads to an invasive cell phenotype16,17.Whereas expression of the FAK FERM domain exhibits strong

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

© 2005 Nature Publishing Group 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.

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

More Detail on 65inside-outttand oooutside-inttsignalling - -

I *nrstivr nt$to b Fr$,rnodrtstc c Antittc rtsts tbrtn fwtnl Irxtrndfd fwwrl {llgnnd4ound forml t*n f $tttor"A Lrrtroue{l*l*rr qr'rrrrl I r^ irlf;g1r*r;r !i t...*t" {-[u*rerirrg {.,F n{F

t f,ytoptarm iij$tfriiltbhsf th* uet{n ffi tl{ rlErullirry ffiffiilffiffi3,ffi

Blogeneslsof focal sdhesions. r I Many integrinsthat arenot boundto the exfracellularmatrix @CM) arepresent on the oell surfaoein an inactive oonformation,which is oharacterizedby bent' extracellulardomains that maskthe ECM-bindingpocket. This oonformationis stabilizedby interactionsbetween integrin tansmemb'ranedomains, membrane-proximalextacellular domainsand a salt bridgebetween the cytoplasmiodomains. b I When talin is recruitedto the plasmamembrane and aotivatedin associationwith phosphatidylinositolphosphate kinase tJ?e-I'f (PIPKIV), it binds to the cytoplasmictail of Fintegrins.This interactions€parates the cytoplasmicdomains and inducesthe integrinsto adoptthe lrrimed' conformation.c I The int€grin exfacellulm domainsextend and unmask the ligand-bindingsite, allowing the integrin to bind specifrcECM molecules.The separatedintegrin cytoplasmio domainsand talin form a platform for the recruitrnentof otherfocal-adhesion proteins. Integrin-linked kinase (IIJ(), andisofomrs of particularly interestingCys-His-rich protein @INCII) and parvin form the IPP complex@g. 2) in the cloplasrq and this complexis recruitedto focal adhesionsthrough intcraotions with otlrer factors,suoh as paxillin. Otherproteins suoh as vinculin and focal adhesionkinase (FAK) arerecruited to the nasoentfocal conplex in a sequentialmanner. The rnaturationof focal adhosionsinvolves clustering ofactive, ligand-boundintegrins altd tho ura$ly of a mult[[,o8h ooEgl€x tlnt is crpqbleof linting intsgrinsto lhe .etin cytoslaldon lnd communicatingwith signellingpathways.

NatureReviews Molecular Cell Biologt 7 (No 1),20-31; January 2006

(b) ftEfs) t\ Ir (b) 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.

1 Inside-out signaling initiated by chemokine receptor leads to activation of the 2 Outside-in signaling initiated by integrin LFA-1 (conversion to high affinity) the LFA-1 integrin (after binding to ICAM-1) leads to diapedesis by the cell.

Low-affinity LFA-1 Capillary 2 Lumen High-affinity LFA-1 1

Capillary Endothelial Cells

Infected tissue Neutrophil engaging in diapedesis.

118 (c)

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 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)