Quiz 3 Material Cytoskeleton MFs Ac1n Polymeriza1on8Based Mo1lity MFs Ac1n Motor Protein8Based Mo1lity MTs Ac1n8Myosin Systems MTs MT Motors Mitosis Mitosis Cytokinesis Cilia IFs Mitochondria Structure Genome Origins Mitochondria Protein Import Ac3n Polymeriza3on8Based Mo3lity MFs DATA CBIO3400 As the filaments grow they exert a pushing force on the membrane Thermal fluctuations allow for insertion of actin subunits at the plus end near the membrane resulting in pushing force Brownian Mo on Brownian mo on occurs lament end to make space for new subunit addi1on ac1n polymeriza1on can Actin polymerization can generate force comparable to what motor proteins produce 5 10 pN per subunit addition without hydrolysis of ATP generate force comparable to what motor proteins produce w o ATP hydrolysis as laments grow they exert pushing force on membrane pushing force generated by thermal uctua1ons that allow ac1n subunits to insert end near membrane ac1n lament binds force great enough to push something along Brownian motion of the end of the filament could make enough of space for addition of a new subunit 4 Drugs Toxins that Can A ect the Cytoskeleton Polymerizing 3 jasplakinolide stabilizes ac1n dimers 4 phalloidin stabilizes FIac n Depolymerizing 1 cytochalasm binds 2 end no growth on end but s1ll have depolymeriza1on of 8 end latrunculin binds GIac n monomer binding blocked no polymeriza1on RocketILike Mo on of Pathogenic Bacteria Like Listeria DATA Rocket like motion of pathogenic bacteria e g Listeria but also viruses and vesciles enter mammalian cell via CBIO3400 endocytosis escape phagosome enter host cytosol rapid prolifera1on rapid movement due to ac1n recruitment genera1on of ac1n tail laments forma1on of protrusions of host PM engulfed by neighboring cell bacteria use ac1n to invade other cells ac1n comet tail entry into a mammalian cell by phagocytosis escape from the phagosome into the host cytosol and rapid proliferation rapid movements based on generation of tails of actin filaments using actin and ABPs of the host cell formation of protrusions of the host plasma membrane that are engulfed by neighboring cells Image by Daniel Portnoy http mcb berkeley edu index php option com mcbfaculty name portnoyd Arp2 3 Enriched Leading Edge CBIO3400 nucleates ac1n forms DATA branches will see MFs when there s a lot of Arp2 3 short highly branched ac1n laments found leading edge of membrane nd Arp2 3 ALL branch points net lament disassembly behind leading edge net lament assembly leading edge disassembly done by co lin DATA CBIO3400 ActA S mulates Arp2 3 ActA stimulates Arp2 3 In vitro studies showed that ActA directly binds to the Arp2 3 complex ActA directly binds Arp2 3 1 Arp2 3 alone has rela1vely Arp2 3 alone has relatively weak ability to stimulate actin assembly weak ability to s1mulate ac1n assembly 2 ActA binds Arp2 3 enhances its ability to nucleate ac1n assembly ActA binds to Arp2 3 and enhances its ability to nucleate actin assembly 50 fold 3 Ac1n preferen1ally Thus actin will polymerize preferentially near the surface of the polymerizes near surface of pathogen pathogen b c ActA is anchored to membrane ActA also binds monomeric actin ActA also binds GIac n profilin and VASP binds pro lin which exchanges ac1n s GDP for GTP ac1vates GIac n allowing polymeriza1on In Vitro System Mimics Rocket Mo on of Pathogens DATA DATA A completely synthetic in vitro system that mimicks the rocket motion of pathogens CBIO3400 CBIO3400 ActA8expressing bacterium or ActA8coated bead necessary in vitro ingredients GIac n ATP co lin ADF depolymerizer Arp2 3 complex branching ac1vity CapZ pro lin end blocking capping protein not essen1al but increases rate of mo1lity ActA Bead ActA expressing bacterium or ActA coated bead G Actin ATP Cofilin ADF Arp2 3 complex branching activity CapZ End blocking protein Profilin not essential increases the rate of motility Mammalian Cells Have WASP Related to ActA WASP NIWASP func1onally analogous to ActA share sequence homology w domain of ActA all of these proteins s1mulate Arp2 3 ac1ve WASP binds protein unfolds allows interac1ons to occur binds Arp2 3 complex some pathogenic bacteria capable of moving via rocket mechanism lack ActA instead recruit WASP of host onto own surface WASP binds signal proteins like GTPases on one side ac1n polymeriza1on components on other side DATA WASP Regula on of Ac nIBased Mo lity Leading Edge Regulation of actin based motility at the leading edge CBIO3400 N8terminal domain The N terminal domain of WASP is regulatory binds regulators including regulatory a Rho like G protein Cdc42 binds regulators including Rho8 The C terminal domain is catalytic like G8protein Cdc42 binds Arp2 3 binds Arp2 3 Cdc42 ac1vated by signaling pathways PM Cdc42 is activated by signaling pathways at the plasma membrane C8terminal domain cataly1c Binding to Cdc42 activates WASP by unfolding and exposing its C terminal binding Cdc42 ac1vates WASP catalytic domain by unfolding exposing its C8terminal cataly1c domain Rho GTP ac1vates e ector proteins like WASP GTPase BD Rho BD DATA CBIO3400 Membrane associated small G proteins integrate extracellular signals and actin cytoskeleton Ac n Polymeriza on Steps CBIO3400 1 Hormone comes in as signal 2 GTP domain ac1vated 3 GTP domain recruits WASP 4 WASP ac1vated Arp2 3 complex 5 Ac1ve Arp2 3 complex binds side of exis1ng lament causing polymeriza1on New lament growth toward cell membrane Capping protein added ATP ac1n converts to ADP ac1n i e treadmilling of ac1n laments Co lin ADF binds ADP ac1n part of lament 7 8 6 9 10 Pro lin binds ADP ac1n monomers re8charging them so they can be recycled for ac1n polymeriza1on 8 treadmilling of actin filaments yellow actin ATP Pollard Ann Rev Biophys Mol Struct 2007 DATA 1 3 signaling pathways converge to activate WASp proteins 4 WASp activates the Arp2 3 complex 5 Active Arp2 3 complex binds to the side of an existing filament 6 new filament growth towards the cell membrane Actin binding proteins including 7 capping protein 9 ADF cofilin 10 profilin modulate these events Cancer mammalian cells broblasts that express mutant cons1tu1vely ac1ve small G8proteins Rho Rac Cdc42 oncogenes these are gain8of8func1on muta1ons that result in excessive ac1vity of mutated protein GTPase dead muta1on can s1ll bind GTP but cannot hydrolyze it always on can also mutate to always o by preven1ng exchange of Front of cell extends forward Rac Cdc42 GDP for GTP involvement of Rac Rho