Actin Lecture Notes PDF

Cytoskeleton overview. Actin Outline: The cytoskeleton Basics of actin assembly Actin Nucleation Factors Actin Binding Proteins Vladimir Sirotkin, PhD Dept. Cell and Dev. Biology 3majorcl...

Cytoskeleton overview. Actin Outline: The cytoskeleton Basics of actin assembly Actin Nucleation Factors Actin Binding Proteins Vladimir Sirotkin, PhD Dept. Cell and Dev. Biology 3majorclasses The Cytoskeleton MreB Actin filaments response Cell Division magggg state Cell Motility Cell Contractility Cell Shape Endocytosis Microtubules mitosis Mitosis Cell Polarity Organelle Transport Cell Motility Intermediate Filaments exskin dynamic less strutffes Mechanical Integrity Structural Scaffolds Tissue Organization Functions of the actin cytoskeleton Cell migration resoptick Cell division – cytokinesis changes Cell contractility (example: muscle) Cell shape Endocytosis and phagocytosis Cell adhesion Intracellular motility (driven by myosins) Bacterial invasion: Listeria nyjacked gt an tomove used around bacteria Microvilli Stress fibers Lamellipodia Contractile ring & filopodia  Actin cytoskeleton is highly dynamic Amoeba vs. fission yeast (S. pombe) Cell migration Cell division – cytokinesis Cell contractility Cell shape Endocytosis and phagocytosis Cell adhesion Intracellular motility Bacterial invasion: Listeria itfkment uses Microvilli Stress fibers Lamellipodia Contractile ring & filopodia  Actin molecule G-actin (monomeric) FEE MW: 42 kDa Binds 1 molecule of ATP or ADP (hydrolysis occurs after addition to polymer) Binds Mg2+ or Ca2+ The subunit is oriented with plus 4 2 end at the bottom. 4parts ATPase 1 Actinis a protein 3 ATPbinding Yetiimitates Pollard and Earnshaw 36-5  Actin origin From Holmes et al. (1995) FASEB J. Actin in eukaryotes and prokaryotes molecule Actite From van den Ent et al. (2002) EMBO J.  Tesses.is Goodson, H. V. et al. J Cell Sci 2002;115:2619-2622 Actin-related proteins that have cytoskeletal functions Actin common family Arp1 – part of dynactin complex Yellthhavein MT-based transport Arp2/Arp3 – part of Arp2/3 complex Actin assembly Note similar fold but different surfaces: Yellow – identical to actin Green – conservative substitutions Blue – nonconservative substitutions Red - inserts invagination of assemble Makeoverall 0 they form Pollard and Earnshaw 36-7 Actin Filament structure hexices two Pollard and Earnshaw 4-5 Pyrene-actin assay for monitoring actin assembly important very I pyggypization Ifffferized 5% pyrene labeled actin monomers you Tation adoprase MgCl2 KCl Natged measuring senses theforce  Actin polymerization: nucleation and elongation aeiitiio.fr L i afftngargYY.IE eye ther wIow Profilin: Capping Protein: blocks nucleation stops & pointed end barbed end elongation elongation Pollard and Earnshaw 4-6 Actin filament nucleation, elongation, and nucleotide hydrolysis ATP hydrolysis is followed by phosphate release, so that actin subunits remain ADP-bound Pollard and Earnshaw 36-10  Actin filament polarity Youthis works K=2.0 Decoration by The plus/barbed S1 (myosin-II end grows and motor domain) shrinks faster reveals actin than the filament polarity minus/pointed end. end they ing K=2.0 Thryffeo Pollard and Earnshaw 36-9 Critical Concentration: Subunit concentration above which polymerization will occur. The concentration of actin monomer that is in equilibrium with polymer once polymerization has reached steady state equilibrium. [polymer] Critical Concentration, Co the in affzero 0 [Actin monomer]  Extrapolation of critical concentration, kon and koff rate constants from elongation rate measurements. Elongation rate = kon*[monomer] – koff Hence, C0= koff / kon addition that Elongation rate plus end megtergation Co minus end 0 koff why [ATP-Monomer]  Actin filament polarity K=2.0 S1 decoration In ATP critical reveals actin concentration is filament polarity lower for barbed end than for pointed end. In ADP critical concentration is the same for both ends differeas K=2.0 The Easy Pollard and Earnshaw 36-9  Actin filament treadmilling in the presence of ATP and monomer concentration intermediate between critical concentrations for barbed and pointed ends. + End Rate of Filament Assembly - End 0 critical concentration overenthrough them dissoation Actin Monomer Concentration Inefferof assoiaction one  Actin-Binding Proteins regulate actin assembly Bind monomers Nucleate assembly Cap ends (block assembly) Sever filaments Crosslink bundles Crosslink in mesh Stabilize filaments Motor proteins Fig 17-32 Monomer binding proteins regulate nucleotide exchange Thymosin prefers ATP-actin. Cofilin prefers ADP-actin. Profilin promotes nucleotide exchange. APPOSES olizerzation PY1 p Pollard and Earnshaw 36-6 Monomer binding proteins thymosin and profilin Thymosin Profilin blocks blocks nucleation nucleation & elongation & elongation at the minus but not the plus end Profilin also binds PIP2 & proline-rich sequences (on formins and VASP) Alberts 16-30  Actin nucleation factors metistering weO Arp2/3 complex makes branched filaments Formins make unbranched filaments WH2 repeat proteins Spire and Cordon-bleu (NEW!) make unbranched filaments Alternatives to nucleation: uncapping or severing of preexisting filaments  Actin nucleation factors: Arp2/3 complex Nucleation Promoting Factor: site WASp Arp2/3 complex makes branched filaments Arp verifies 1 µm Svitkina and Borisy, 1999  Actin Nucleation Factors: Arp2/3 complex assembles branched actin networks whletoioeo opfx.it aponits https://youtu.be/1tUBRCkCnm0 movie Goode Lab  Regulation of activity of Nucleation Promoting Factors WASp and Scar/WAVE WASp Scar/WAVE  Dendritic nucleation model for actin assembly at the leading edge incinersitis graphics by J. Kuhn; modified from Pollard et al. (2000) Annu Rev Biophys Biomol Struct. 29: 545-76. Watch the narrated animation: https://youtu.be/okPsrHzrC5g  Actin Nucleation Factors: Formin Family anathes (mDia1) From Higgs (2005) TiBS  Actin nucleation factors Formins make Profilin unbranched filaments enhances elongation by formin one Example: video Actin Contractile Ring Kamasaki et al., 2007  Actin Nucleation Factors: Formins assemble linear actin networks Goode Lab  Formins and myosin-II assemble actin contractile ring for cytokinesis noisinin Formin and myosin-II localize to medial cortical nodes Formin generates linear filaments Filaments are captured by myosin-II in adjacent nodes Myosin-II moves along filaments, pulling nodes together into a ring Myosin-II motor activity powers ring constriction  Actin-Binding Proteins regulate actin assembly Bind monomers Nucleate assembly Cap ends (block assembly) Sever filaments Crosslink bundles Crosslink in mesh Stabilize filaments Motor proteins Fig 17-32  Actin Capping Proteins Pollard and Earnshaw 36-13 Barbed End capping proteins compete with formins for barbed-end binding.  Actin Severing Proteins Cofilin Gelsolin Gelsolin is activated by Ca2+ and inactivated by PIP2. Cofilin prefers ADP-actin From Pollard and Earnshaw 36-14 Actin filament cross-linking and bundling proteins ABD = Calponin Homology domain Pollard and Earnshaw 36-15 Actin filament cross-linking and bundling proteins Alberts 16-41 Alberts 16-40 iodaotAlberts 16-42  Actin Binding proteins:viii filament side binding and membrane attachments Side binding proteins: Tropomyosin: ▪ Together with troponin, is important for regulation of muscle contraction. ▪ Exists in non-muscle cells. ▪ Acts as gate keeper: permits or blocks binding of other ABPs. IMPORTANT CONCEPT: some ABPs compete for binding F-actin Coronin. Arp2/3 complex inhibitor. Caldesmon, calponin, nebulin and several others. From Clayton et al. Curr. Biol. (2010)  Actin Binding proteins: filament side binding and membrane attachments Actin membrane attachments: Spectrin. Classic example - cell cortex in red blood cells. Dystrophin/utrophin. Dystrophin defects result in muscular dystrophy. ERM proteins (ezrin/radixin/moesin). Talin. Present in focal adhesions. Cell cortex in red blood cells:  Extracelullar and intracellular signals trigger actin assembly Slime mold chemotaxis Rho-family GTPases: Cdc42 Rac Rho Pollard and Earnshaw 36-20 Filopodia Ruffles Stress Fibers Mechanisms include activation of formins and NPFs Phosphoinositides regulate: ▪ NPFs ▪ Some formins ▪ ERM ptoteins ▪ Capping Protein Pollard and Earnshaw 36-19 ▪ Profilin and cofilin  Anti-actin Drugs Phalloidin (from the mushroom Amanita phalloides) stabilizes actin filaments by binding along the sides of the filament. Used to visualize F-actin. Jasplakinolide (from marine sponge Jaspis johnstoni) stabilizes filaments. Membrane permeable. Cytochalasins B and D (made by molds) block assembly at the plus end. Latrunculin A (from Red Sea sponge Negombata or Latrunculia) binds G-actin, blocks assembly. Negombata Amanita magnifica phalloides CK-666 inhibits Arp2/3 complex SMIFH2 inhibits formins mangegose From www.ars.usda.gov Actin filament assembly propels the formation of lamellipodia, dorsal ruffles, and filopodia at the leading edge of crawling cell. Any questions?