Lecture 4Structure and Function of Actin & MyosinOutline:Actin Structure and RegulationMyosin Structure and RegulationFunctions of Actin and Myosin in CellsPaper: Control of microtubule dynamics by the antagonistic activities of XMAP215 and XKCM1 in Xenopus egg extractsStationary cell - stress fibersPlatelet Dynamicsresting activated retractionDramatic morphological changes result from reorganization of actin cross-linked to plasma membraneActinroles: cell shape, polarization, locomotion, division; vesicle traffichighly conserved 375 aa, 43 kD proteinthe most abundant protein in non-muscle cells : 1-5%monomer = G-actinpolymer = F-actin, microfilamentsinhibitors: latrunculin, cytochalasin; phalloidinActin Structurebarbedpointedtwo-stranded helix+--Cc(- end) = 0.8 µM > Cc(+ end) = 0.1 µM D D D D D T ATP+Steady state actin polymer- Treadmilling of subunits T DDTfilament turnover rate: t1/2 = 30 minrate limiting step = dissociation of ADP actin from minus end ADP DActin dynamics in vivo2. Keratocyte - epithelial cell3. Listeria monocytogenes - intracellular bacterial pathogen1. Spatial and temporal control of polymerization/depolymerization2. Turnover3. Movement of actin filaments - myosinsParameters:Model systems:1. Fibroblast- D D D D D T T+ T D D 11. monomer pool22. nucleation33. elongation44. depolymerization D DRegulation of the monomer poolThymosin β4M.W. 5000binds 1:1 - enough to buffer all the actinsequesters actin from polymerizinglocalization - diffuseProfilinM.W. 14,000binds 1:1 - can buffer 20% of actinpromotes nucleototide exchange and polymerizationbinds PIP2 and proline-rich sequences localization - diffuse and leading edge, Listeria surfaceNucleationArp2/3 complex (Actin Related Protein)7 subunits, include actin-related proteins 2 and 3accelerates actin polymerization (with activator, eliminates lag phase)binds (-) ends and filament sides - branching functionpromotes actin polymerization at listeria surfaceActivators: Listeria: Act A cells: WASP family proteinsimmuno-EM ofArp2/3at actinbranchpointsElongation Capping factors:CapZ (Capping protein) - (+) endtropomodulin - (-) endgelsolin - (+) endProfilinpromotes (+) end growth• can stabilize or destabilize filaments, prevent elongationDepolymerizationgelsolinM.W. 87,000Ca++-dependent severingADF/cofilinM.W. 19,000binds G- and F-actinaccelerates (-) end depolymerization 25-fold+ ADF- D D DD-Pi D DD-Pi T T+ T D D thymosin β4, profilin1. monomer poolArp2/3complex2. nucleationCapZ, gelsolin3. elongationADF/cofilin4. depolymerizationMyosin - the most studied of all proteins (!?)large family of myosin-related proteins ~14 in human heavy chain:1) large globular head: contains actin-binding and ATPase domains2) α-helical neck region - binds light chains common features: one or two heavy chains and several light chains3) tail domain - for oligomerization or cargo bindinglight chains:1) calcium-binding proteins, sometimes calmodulin2) regulate myosin activityMyosin IImuscle,stressfibersvesicles,organellesvesicles,organellesMyosin thick filaments: bipolar21Myosin motility assay1) Adsorb myosin molecules on glass coverslip in chamber2) Perfuse in labeled actin filaments and plus ends (and ATP)3) Observe by fluorescence video microscopymuscle myosin plus end motor~4.5 µm/sec++--other myosins can move toward the minus endMyosin II mechanismATPase activity stimulated by actin: from 4/hour to 20/secondATP binding, hydrolysis and dissociation of ADP-Pi produce a series of allosteric changes in myosin conformation Energy release is coupled to movementMyosin II crystal structure (S1 fragment)catalyticheadneck domain = lever armsuperimposestructures intwo differentnucleotide statescross bridgecyclecross bridgecycletroponintropomyosinMyosin mediated movement: in reality more complexly regulatedCalcium binds TroponinMagnesium ion approaches Myosin head.Troponin movement exposes binding site for Myosin head.Myosin head binds Actin filament. Magnesium activates Myosin head, releases Phosphorus from ATP, leaves ADPcauses Myosin head to contract.Magnesium and ADP released from Myosin headends contraction.Myosin head releases from Actin filament. Calcium ion released from Troponin, covers binding siteNew calcium ion approaches next Troponin moleculeNew ATP molecule approaches Myosin headbeginning the process over again35Functions of Actin and Myosin in Cells36Functions of Actin and Myosin in CellsCell MotilityCell DivisionMuscle ContractionPathogen Motility and InfectionCell Protrusions/microvilliCell CortexStress FibersrelaxedcontractedMuscle ContractionCell Division / CytokinesisDictyostelium amoebaCell MotilitySwimmingMicrotubule-based – cilia, flagellaCrawling Actin-based purposes:wound healing - epidermal cellsimmune response - leukocytes –migrate to sites of infectiondevelopment – neural crest cells; neuronal process extensioncancer cell metastasis –malignancy determinantlocomoting cell - filopodia and lamellipodiaFish Keratocytes vs. FibroblastsActin Dynamics in Moving Keratocytesactin polymerization at leading edge, treadmill to the rearActin polymerization required for movementlatrunculin-inhibits actin polymerizationprotrusionanchorageforward movementtail retractionCrawling: Coordination of 4 processesprotrusionActin polymerization at leading edge - local forcemechanisms:1) “thermal rachet” - actin polymerization pushes2) myosin I - movement of actin filaments1) thermal rachet - actin polmerization at leading membrane,depolymerization at the rear2) myosin I - dependentmyosin I could also transport assembly factors to membraneanchorageAdhesion plaques:connect cell to substratumprevent leading lamella from retractingforward movementObservations:actin networks stationary with respect to substratumcell body and nucleus rotatesmyosin II requiredProposed mechanisms:1) sarcomere-like contractions in rear2) transport along actin arrayscrawlingDictyosteliumamoebaMyosinII (Rhodamine) in Moving Keratocytestail retractionpassive - cell snaps loose from adhesion plaquesListeriamonocytogenesListeria movementListeria invasions‘comet tails’ formed by actin polymerizationCo-opts Actin for devious purposes.....what controls depolymerization in Listeria tails?Listeria + cytoplasmic egg extract leads to motility in vitroimmunodeplete gelsolin or ADF/cofilin and observe effects control -gelsolin ADF/CofilinNO actin severing NO -end depolymerizationImportant Breakthrough:• reconstitution of Listeria motility