L3 Actin Microfilaments PDF

BS31004 Cell Shape and Movement L3: Actin Microfilaments Alan Prescott [email protected]  Actin Cytoskeleton Important roles in – cell motility contractility shape changes cytokinesis cell polarity phagocytosis ma...

BS31004 Cell Shape and Movement L3: Actin Microfilaments Alan Prescott [email protected]  Actin Cytoskeleton Important roles in – cell motility contractility shape changes cytokinesis cell polarity phagocytosis macropinocytosis Actin very abundant - 20% of mass in skeletal muscle 5% of total proteins in non-muscle Actin in cell shape changes and cell movement in non-muscle cells Actin assembly accompanies membrane ruffles, lamellipodia formation and also cell movement – How is actin assembly initiated ? – What regulates actin assembly ? – How does actin drive cell movement?  Actin proteins Highly conserved protein 375 amino acids 43 kDa globular monomer, two domains, G-actin At least 6 isoforms in mammals, encoded by separate genes  Actin proteins Three classes of actin defined Class 1 - Non-muscle b, g and smooth muscle g-actin. Class 2 - Skeletal, cardiac and vascular a-actin Class 3 - Actin-RPV, centractin, lower eukaryote actins.  Actin Isoform Localisation in non-muscle Cells g-actin - at cell periphery (sub-plasmalemmal array) a-actin - in stress fibres. b-actin - in membrane ruffles; involved in cell movement which requires distortion of the plasma membrane. Upregulation of b-actin accompanies increased cell movement  Immunolocalisation of actin isoforms b-actin Phalloidin  ACTIN STRUCTURES IN CELLS Transient/labile surface structures on cells that are due to assembly of actin: - Lamellipodia – Filopodia – Ruffles Stable actin structures: - Microvilli Actin associated junctions: - focal complexes - focal adhesions Examples of microfilament-based structures. Stable Actin Filament Structures Microvilli contain bundles of actin filaments stabilised by cross-linking proteins Microvilli are finger-like projections of the cell surface. Barbed-end (+ end) Important in epithelia such as the intestine where they increase cell surface area for absorption. Cross-links villin, fimbrin espin Bundles of 20-30 actin filaments. All oriented with barbed-ends at tip Cross-linked by the bundling proteins villin, fimbrin and espin  ACTIN FILAMENTS - CONTROL AND REGULATION Polarized filaments: “+” and “-” ends, kinetics differ Actin assembly requires energy – from nucleotide (ATP-ADP) hydrolysis in cells Actin binding proteins regulate monomer to filament ratio – by exploiting the difference in the assembly characteristics at either end Cell signalling alters actin assembly – small GTPases, rac/rho/cdc42 Actin assembly into filaments  pure actin nucleates as trimers and elongates by addition of monomers forms actin filaments, F-actin usually in bundles or networks 6-8 nm diameter polar - subunits oriented same way right-handed double-helical polymers Actin Filament Polarity “Arrowhead” decoration Actin filament polar- ends show + + Barbed-end different kinetics slow growing-end = minus-end fast growing-end = plus-end Polarity demonstrated by decoration with fragments of myosin (S1) Produces a characteristic arrowhead pattern: barbed-end = plus-end, critical conc. x10 lower pointed-end = minus-end - - Pointed-end  Actin assembly Subunits add and come off both ends, but different critical concentrations operating at either end: + - Plus end: Low critical Minus end: higher critical concentration, only need ~ concentration, need over 0.1µM (about 8µg/ml) 0.8µM to start adding monomeric actin to start adding subunits subunits So between 0.1-0.8 µM, subunits will add at plus end and drop off at the minus end, giving treadmilling The two ends of a myosin-decorated actin filament grow unequally.  Formation of New Actin Filaments Three sources: 1. De novo nucleation 2. Uncapping 3. Severing existing filaments Actin binds ATP tightly binds to ATP and Mg++ (ATP binding site in cleft between the two domains) ATP is then hydrolysed to ADP after polymerisation assembly competent Structures of monomeric G-actin and F-actin filaments.  Formation of New Actin Filaments Pure actin assembles spontaneously into filaments: Assembles from ATP bound G-actin ATP hydrolysed to ADP after polymerisation, lags behind = ATP cap ATP hydrolysis = ADP-actin = depolymerisation state Actin polymerisation The three phases of in vitro G-actin polymerization.  Filament Elongation Elongation favoured at barbed-end (+ end) Critical concentration for the two ends varies for ATP-actin Lower at barbed-end (+ end) so growth mainly occurs here net addition of subunits to barbed-end and net loss at pointed-end flux of subunits through the filament Treadmilling Actin treadmilling. Actin assembly in 3 stages ACTIN-BINDING PROTEINS: Sequestering proteins  Actin Assembly in vivo Actin polymerisation favourable However in cells like fibroblasts, 50% of actin is G- actin, and G-actin is above the critical concentration - How does a cell maintain a pool of G-actin? Regulatory proteins control assembly and keep G-actin concentration high: Profilin Capping protein Thymosin-b4  Actin Assembly in vivo Profilin - binds G-actin/ATP state - thus lowering critical conc. (0.1 to 0.007µM) - thus limits elongation to the barbed-end by suppressing spontaneous nucleation  ACTIN BINDING PROTEINS - Profilin Ubiquitous protein Major but not exclusive function is the binding of actin 1. Binds ATP-actin and the complex associates with the barbed end (+) of actin filaments. This effectively reduces the critical concentration. 2. UNIQUE PROPERTY - a nucleotide exchange factor: ADP-actin >>> ATP-actin (ADP-Actin = 3.7µM; ATP-Actin = 0.3-0.6µM) Dual effects upon actin assembly. Too much and too little profilin is detrimental to filament formation. Knockouts in mice and yeast show it to be an essential protein.  Actin Assembly in vivo Capping protein - caps the barbed-end, stops elongation Thymosin-b4 sequesters G-actin making it polymerisation- incapable (another multigene family, b4 is commonest mammalian isoform) Pointed-end < critical concentration, thus driving disassembly  ACTIN BINDING PROTEINS : ß-thymosin Enriched at the cell periphery Exclusive actin binding protein 100 x greater affinity for ATP-actin than ADP-actin ATP-actin affinity is 0.6µM This means that when the barbed end (+) becomes uncapped, revealing the high affinity actin binding site, there is a pool of actin ready to assemble immediately at these sites. Regulation of filament formation by actin-binding proteins.  ACTIN ENDS - SUMMARY Growth is predominantly at the plus end, less growth at the minus end Critical concentration differs FOR ATP ACTIN at the two ends - 0.1 µM versus 0.6µM - gives treadmilling (Tubulin assembly similar but nucleotide involved is GTP) This is the key to dynamic regulation of actin and tubulin networks in cells  Free Ends and Free Subunits Free G-actin mostly in the ATP form, and therefore actin assembly-competent Free G-actin forms complexes with actin binding proteins as a mechanism to regulate actin assembly F-actin ends bind CAPPING proteins, providing one mechanism to regulate G-actin addition and therefore F-actin growth Capping proteins. ACTIN-BINDING PROTEINS: polymer modifying proteins ACTIN BINDING PROTEINS over 50 known SEVERING-CUTTING CAPPING + AND - ENDS CROSSLINKING, BUNDLING G- AND F-ACTIN PREFERENCE ATP/ADP-ACTIN PREFERENCE Actin binding proteins-regulators of assembly & branching Actin binding proteins-regulators of assembly & cross-linkers Figure 16-42 Molecular Biology of the Cell (© Garland Science 2008)  Actin filament assembly is facilitated by formins Figure 16-38 Molecular Biology of the Cell (© Garland Science 2008)  Filamin forms a cross-linked actin network at the cell cortex Figure 16-51 Molecular Biology of the Cell (© Garland Science 2008) ERM proteins link the actin filaments to the plasma membrane Ezrin Radixin Moesin Figure 16-53 Molecular Biology of the Cell (© Garland Science 2008) Actin cross-linking proteins.  Actin Structures in Cells - filopodia, lamellipodia, ruffles and stress fibres Actin organisation in a migrating cell Rho-GTPases - Rac lamellipodium Cdc42 Rho Regulation of the state of actin in cells Actin is in a constant state of flux Changes in actin configuration mediated locally by signalling Signalling mediated by small GTPases Rac, Rho and cdc42  How the role of Rac/Rho/cdc42 in actin regulation was discovered Serum-starved Swiss 3T3 cells : serum addition (to push cells into cycle) gave dramatic changes in the actin cytoskeleton : membrane ruffling and stress fibre formation C3 transferase (Clostridium botulinum exoenzyme) prevents these actin changes. ADP ribosylates Rho, inactivating this small GTPase protein. Rho, Rac, cdc42 are members of the Ras superfamily of proteins – N17forms are dominant negative – V12 forms are constitutively active Regulating actin structures hormone  Actin assembly at the leading edge of the cell ARP2/3 complex induces filament branching Fish “keratocytes” - model systems for studying movement Combination of video microscopy followed by immunoelectron microscopy The Arp 2/3 Complex : – Actin related proteins in combination with 5 other regulatory proteins such as p21 – Nucleates actin filaments – Binds to the sides of actin filaments – Activated by WASP (Wiskott-Aldrich syndrome protein) and others Actin depolymerising factor (ADF)/cofilin : – Bind F- and G-actin, preferring ADP forms – Enhance depolymerisation by increasing rate constant for dissociation from pointed end – Sever actin filaments Fish keratocytes The Arp2/3 Complex: Nucleates actin filaments de novo consists of 7 polypeptides - Arp2 and Arp3 - actin related p40, p35, p21, p18 and p14(ARPC1-5) binds to pointed-end (- end), caps it and stabilises it enabling assembly at barbed-end binds to side of pre-existing filaments to produce branching high concentration at leading edge activated by signalling and regulatory proteins (NPFs-nucleation and promoting factors, eg WASP,WAVE)  Arp2 and Arp 3 share structural homology to actin Figure 16-34a Molecular Biology of the Cell (© Garland Science 2008) Nucleotide-binding pocket Actin binding site Barbed end activated by WASP ARP2/3 complex provides nucleation site for actin polymerisation  Hijacked by parasites: Listeria, Shigella, Helicobacter and several other pathogens hi-jack the actin machinery to promote infectivity  Listeria actin tails Freeze-etch S1 myosin decorated  Nucleation-promoting factors: Rapid filament assembly at the plasma membrane  Spatial and temporal regulation in response to external stimuli Signalling pathways for Arp2/3 activation - cell surface receptors Rho-family GTPases (cdc42, Rac, Rho) WASP/Scar(WAVE) Cell surface receptors - Receptor tyrosine kinases such as PDGF (platelet - derived growth factor) and EGF (epidermal growth factor) PIP2 (phosphatidylinositol (4,5) biphosphate) Serpentine receptors-G-protein coupled Figure 17.44 Summary of signal-induced changes in the actin cytoskeleton. Figure 16-98a Molecular Biology of the Cell (© Garland Science 2008) Figure 16-98b Molecular Biology of the Cell (© Garland Science 2008) Figure 16-97 Molecular Biology of the Cell (© Garland Science 2008) Steps in cell movement. Contribution of Cdc42, Rac, and Rho to cell movement.  Rac - Lamellipodium formation Rac Scar/WAVE Arp2/3 complex Capping protein Cofilin Loss of Arp2/3 Actin disassembly Modified from Svitkina and Borisy. 1999. Trends Biochem. Sci. 24:432  Dendritic Nucleation Model ARP2/3 nucleates new filaments, caps pointed-ends and anchors them to pre-existing filaments Arp2/3 complex at Y-junctions Modified from Svitkina and Borisy. 1999. Trends Biochem. Sci. 24:432  Dendritic Actin Network Arp2/3 complex Y-junction From Svitkina and Borisy. 1999. JCB 145:1009 Actin associations at a cell edge Actin dynamics and cell protrusions are controlled by: cell surface receptors Rho-family GTPases, Cdc42, Rac WASP and Scar related proteins Arp2/3 complex capping protein profilin cofilin (ADF)