Lecture 3

1. 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

2. monomer = G-actin polymer = F-actin, microfilaments 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 inhibitors: latrunculin, cytochalasin; phalloidin

3. platelet dynamics resting activated retraction Dramatic morphological changes result from reorganization of actin cross-linked to plasma membrane

5. Actin Structure pointed two-stranded helix barbed

6. Actin highly conserved, binding proteins are not

7. Actin polymerization dynamics in vitro Assays to measure: 1) viscometry 2) sedimentation 3) fluorescence spectroscopy- pyrene actin assembly assay

8. couple to C-374 of actin pyrene *steady state fluorescence  polymer elongation nucleation Mg++, KCl time Pyrene actin assembly assay *[free actin] =Cc= 0.1 mM

9. steady state - treadmilling of subunits + - D D D D-Pi T D D D-Pi 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

10. 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

11. Listeria monocytogenes

12. t= 0 t= 1 min t= 5 min Spatial Control microinject fluorescently-labeled actin polymerization occurs at leading edge of keratocyte, rear surface of Listeria

13. resorufin caging group resorufin Filament Turnover microinject “caged” fluorescently-labeled actin illuminate in specific location with UV light to release caging group

14. t = 0 t = 30 sec t = 1 min actin stays in same place as cell moves forward Measure rate of fluorescence decay = actin turnover rate t1/2 = 30 sec

15. in vitro in vivo t1/2 = 30 min t1/2 = 0.5 min Actin Cc = 0.1 mM [Actin]= 500 mM Actin dynamics in vivo are controlled by actin binding proteins

16. 3 4 2 1 1. monomer pool 2. nucleation 3. elongation 4. depolymerization + - D D D D-Pi T D D D D-Pi T D T

17. 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 Regulation of the monomer pool

20. 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

22. immuno- EM of Arp2/3 at actin branch points in leading edge

23. Elongation Profilin promotes (+) end growth • Capping factors: • CapZ (Capping protein) - (+) end • tropomodulin - (-) end • gelsolin - (+) end • can stabilize or destabilize filaments, • prevent elongation

24. ADF/cofilin M.W. 19,000 binds G- and F-actin accelerates (-) end depolymerization 25-fold + ADF Depolymerization gelsolin M.W. 87,000 Ca++-dependent severing

25. control gelsolin ADF/Cofilin Rosenblatt et al., 1997 Question: what controls depolymerization in Listeria tails? Listeria + cytoplasmic egg extract  motilityin vitro immunodeplete gelsolin or ADF/cofilin and observe effects

26. 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 • a-actinin

27. + - D D D D-Pi T D D D D-Pi T D T end availability: CapZ, gelsolin depolymerization: ADF/cofilin nucleation: Arp2/3 complex monomer regulation: thymosin b4, profilin

28. Organization of actin filaments Myosins Cross-linking proteins Membrane attachments Assemblies: cell cortex, stress fibers, contractile ring, cell protrusions, microvilli

29. stationary cell - stress fibers

31. Dividing cell

32. Dictyostelium amoeba

33. locomoting cell - filopodia and lamellipodia

35. Cell Motility Swimming Microtubule-based – cilia, flagella Crawling Actin-based purposes: 1) wound healing - epidermal cells 2) immune response - leukocytes –migrate to sites of infection 3) development – neural crest cells; neuronal process extension 4) cancer cell metastasis –malignancy determinant

36. 4 processes coordinated: protrusion anchorage forward movement tail retraction

37. protrusion Actin polymerization at leading edge - local force Proposed mechanisms: 1) “thermal rachet” - actin polymerization pushes 2) myosin I - movement of actin filaments

38. 1) thermal rachet - membrane fluctuations 2) myosin I - dependent myosin I could also transport assembly factors to membrane

40. anchorage Adhesion plaques: connect cell to substratum prevent leading lamella from retracting

41. 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

42. crawling Dictyostelium amoeba

43. tail retraction passive - cell snaps loose from adhesion plaques

44. How is cell polarity established? cell loaded with Ca++ -sensitive dye Fura-2