Transcript Actin
Lecture 3 Actin and myosin in non-muscle cells; Cell motility Outline: Actin polymerization in vitro Regulation of actin dynamics in cells Actin organization Cell motility Paper: Self-polarization and directional motility of the cytoplasm Actin highly conserved 375 aa, 43 kD protein the most abundant protein in non-muscle cells 1-5% roles: cell shape, polarization, locomotion, division; vesicle traffic monomer = G-actin polymer = F-actin, microfilaments inhibitors: latrunculin, cytochalasin; phalloidin platelet dynamics resting activated retraction Dramatic morphological changes result from reorganization of actin cross-linked to plasma membrane Actin Structure pointed two-stranded helix barbed Actin highly conserved, binding proteins are not Actin polymerization dynamics in vitro Assays to measure: 1) viscometry 2) sedimentation 3) fluorescence spectroscopypyrene actin assembly assay Pyrene actin assembly assay couple to C-374 of actin pyrene *steady state fluorescence polymer elongation nucleation time Mg++, KCl *[free actin] =Cc= 0.1 mM steady state - treadmilling of subunits + D D D D D T DPi DPi T T D T D Cc(- end) = 0.8 mM > Cc(+ end) = 0.1 mM filament turnover rate: t1/2 = 30 min rate limiting step = dissociation of ADP actin from minus end Actin dynamics in vivo Parameters: 1. Spatial and temporal control of polymerization/depolymerization 2. Turnover 3. Movement of actin filaments - myosins Model systems: 1. Fibroblast 2. Keratocyte - epithelial cell 3. Listeria monocytogenes - intracellular bacterial pathogen Listeria monocytogenes Spatial Control microinject fluorescently-labeled actin t= 0 t= 1 min t= 5 min polymerization occurs at leading edge of keratocyte, rear surface of Listeria Filament Turnover microinject “caged” fluorescently-labeled actin illuminate in specific location with UV light to release caging group caging group resorufin resorufin t=0 t = 30 sec actin stays in same place as cell moves forward t = 1 min Measure rate of fluorescence decay = actin turnover rate t1/2 = 30 sec in vitro t1/2 = 30 min Actin Cc = 0.1 mM in vivo t1/2 = 0.5 min [Actin]= 500 mM Actin dynamics in vivo are controlled by actin binding proteins + - 4 D D D D D D DPi D DPi T T T 1 1. monomer pool 2. nucleation 3. elongation 4. depolymerization 3 2 Regulation of the monomer pool Thymosin b4 M.W. 5000 binds 1:1 - enough to buffer all the actin sequesters actin from polymerizing localization - diffuse Profilin M.W. 14,000 binds 1:1 - can buffer 20% of actin promotes nucleototide exchange and polymerization binds PIP2 and proline-rich sequences localization - diffuse and leading edge, Listeria surface Nucleation Arp2/3 complex 7 subunits, include actin-related proteins 2 and 3 promotes actin polymerization at listeria surface accelerates actin polymerization in pyrene actin assembly assay (with activator, eliminates lag phase) binds (-) ends and filament sides - branching function localization - lamellipodia Activators: Listeria: Act A cells: WASP family proteins immunoEM of Arp2/3 at actin branch points in leading edge Elongation Profilin promotes (+) end growth Capping factors: CapZ (Capping protein) - (+) end tropomodulin - (-) end gelsolin - (+) end • can stabilize or destabilize filaments, • prevent elongation Depolymerization gelsolin M.W. 87,000 Ca++-dependent severing ADF/cofilin M.W. 19,000 binds G- and F-actin accelerates (-) end depolymerization 25-fold + ADF Question: what controls depolymerization in Listeria tails? Listeria + cytoplasmic egg extract motility in vitro immunodeplete gelsolin or ADF/cofilin and observe effects control gelsolin Rosenblatt et al., 1997 ADF/Cofilin Important Breakthrough: • Reconstitution of Listeria motility from purified components required: • Actin and ATP • Arp2/3 complex • ADF/cofilin • Capping protein stimulators: • VASP -binds ActA, actin, profilin • Profilin • -actinin depolymerization: ADF/cofilin + - D D D D D D D DPi DPi end availability: CapZ, gelsolin T T T monomer regulation: thymosin b4, profilin nucleation: Arp2/3 complex Organization of actin filaments Myosins Cross-linking proteins Membrane attachments Assemblies: cell cortex, stress fibers, contractile ring, cell protrusions, microvilli stationary cell - stress fibers Dividing cell Dictyostelium amoeba locomoting cell - filopodia and lamellipodia Cell Motility Swimming Microtubule-based – cilia, flagella Crawling Actin-based purposes: 1) 2) 3) 4) wound healing - epidermal cells immune response - leukocytes –migrate to sites of infection development – neural crest cells; neuronal process extension cancer cell metastasis –malignancy determinant 4 processes coordinated: protrusion anchorage forward movement tail retraction protrusion Actin polymerization at leading edge - local force Proposed mechanisms: 1) “thermal rachet” - actin polymerization pushes 2) myosin I - movement of actin filaments 1) thermal rachet - membrane fluctuations 2) myosin I - dependent myosin I could also transport assembly factors to membrane anchorage Adhesion plaques: connect cell to substratum prevent leading lamella from retracting forward movement Observations: Actin networks stationary with respect to substratum Cell body and nucleus rotates myosin II required Proposed mechanisms: 1) sarcomere-like contractions in rear 2) transport along actin arrays crawling Dictyostelium amoeba tail retraction passive - cell snaps loose from adhesion plaques How is cell polarity established? cell loaded with Ca++ -sensitive dye Fura-2