1. How does actin polymerization drive protrusion? Polymerization at tip? Expansion of actin meshwork? Increase in hydrostatic pressure? Hypothesis #1 Hypothesis #2 Hypothesis #3

2. Evidence for #1: The Acrosome reaction Elongation of the acrosomal process results from a burst of actin polymerization at the tip. This allows the sperm to penetrate the jelly coat surounding the egg Stages during fertilization of a sea-urchin egg

3. Evidence for #2: Gel Swelling mechanism of protrusion 1. Protrusion in Dictyostelium starts as a bleb and actin fills in behind. 2. During the acrosome reaction: –Increased osmolarity, decreases rate of acrosomal actin filament elongation. –Decreases in osmolarity, increase rate of acrosomal actin polymerization.

4. Myosin I “walks” toward + end while associated with the plasma membrane Actin filaments slide rearwards, relative to membrane This may provide space for actin monomers to add to + ends Evidence for #3:Myosin I driven protrusion Actin filament sliding mechanism of protrusion Myosin I at leading edge

5. To understand how actin polymerization drives protrusion we need to know: 1. Where the nucleation of actin filaments occurs 2. How high rates of actin polymerization are maintained at the protruding edge 3. How polymerization generates a protrusive force –To be covered later in this course

6. APBs involved in regulating actin dynamics 1. Dynamics Thymosin  -4 (G-actin sequesterer) Profilin (Increases rate of polymerization) Gelsolin (Increases rate of actin filament turnover) Capping proteins(Increases rate of polymerization) Arp2/3 (Nucleation ) Lodish 5th Ed. Chapter 19, p786-791

7. Thymosin  -4 and Profilin are monomer sequestering proteins Fact: The Cc for actin filament polymerization is 0.1uM, the total actin concentration in a cell is 200uM. 40% of actin in cells is unpolymerized. Why ? Active sequesterersInactive sequesterers Proteins in the cytoplasm sequester or bind to actin monomers - preventing them from polymerizing. Factors which influence the binding of these proteins to actin monomers will affect the rate of actin polymerization.

8. Microinjection of excess TB4 into cells causes loss of stress fibers Although actin stress fibers are relatively stable turnover of actin monomers is occurring. Monomers leaving a stress fiber will be rapidly sequestered by TB4. Gradually the stress fiber will disappear. The equilibrium is shifted toward increasing monomer concentration at the expense of f-actin. BeforeAfter

9. Profilin increases the rate of actin polymerization Profilin binds to actin opposite the ATP binding cleft* –* allows exchange of ADP for ATP, contrasts with T  -4 Profilin-actin complex to binds readily to the + end of the actin filament (affinity of complex > than single actin monomer A conformational change in the complex occurs after binding to +end actin filament, causing profilin to fall off

10. Profilin competes with T  -4, for actin monomers When a small amount of profilin is activated it completes with thymosin for G-actin and rapidly adds it to the +end of F-actin The activity of profilin is regulated by: –phosphorylation, binding to inositol phospho-lipids The activity of profilin is increased close to the plasma membrane by binding to: – acidic membrane phospholipids, certain proline rich proteins that localize at the plasma membrane

11. Role of profilin during the Acrosome reaction

12. Functions of the actin cytoskeleton dependent on polymerization The acrosome reaction. The rapid formation of an acrosomal process penetrates the thick jelly coat of the sea urchin egg allowing nuclear fusion between sperm and egg. Before fertilization short actin filaments lie in a pocket at the head of the sperm together with many profilin-actin complexes Upon contact with the egg, the acrosomal vesicle is exocytosed, uncovering + ends of actin filaments. At the same time, profilin (of the profilin-actin complex) is activated resulting in the rapid addition of G-actin to the exposed +ends of the pre-existing actin filaments This results in an explosive elongation of the acrosomal process The acrosomal process contacts the egg plasma membrane and fuses with it. The sperm and egg nuclei fuse.

13. Experiment to demonstrate the location of newly polymerized actin New actin polymerization occurs within the actin cortex that lies just beneath the plasma membrane Actin polymerization in this location can form a variety of surface structures –Microvilli, filopodia, lamellipodia Nucleation of actin filament growth is regulated by external signals Nucleation is initiated by a comlex of 7 proteins called the ARP2/3 complex All actin is labeled in a lamellipodium 1. A fibroblast was microinjected with rhodamine (red) labeled actin monomers 2. Cell was fixed shortly after microinjection. 3. The cytoskeleton was stained with fluorescein phalloidin (green). Only newly polymerized actin is labeled Conclusion: Newly polymerized actin is found at the leading edge.

14. The role of Arp2/3 in protrusion Arp2/3 is a highly conserved complex of 7 proteins, including 2 actin related proteins (Arp2 and Arp3) Identified first in the cortical (submembranous) actin of amoebae Found in highly dynamic actin structures in many cell types –e.g. Listeria (actin tails), edge of lamellipodia, cortical actin patches (yeast)

15. Arp2/3 nucleates actin filament assembly Arp2/3 is present at high (~ 10uM) concentrations in motile cells e.g. leukocytes Arp2 and Arp3 are 45% similar to actin monomers

16. Arp2/3 nucleates actin filament by binding to the - end of the actin filament Arp2/3 can bind to the sides of pre-existing actin filaments, resulting in the development of a branching mesh of actin filaments Nucleation is more efficient when ARP2/3 is bound to the side of an actin filament Arp2/3 provides a “template” for actin filament growth

17. Svitkina and Borisy 1999 Distribution of Arp2/3 in a moving cell Arp2/3 Actin Overlay Keratocyte Immunogold labeling of Arp2/3

18. How is Arp2/3 activated WASp, Wiskott-Aldrich Syndrome protein, mutated protein leads to bleeding, immunodeficiency - is rich in proline WASp is activated when it binds PIP2 and active Cdc42 (small GTPase) VCA domain of WASp is necessary for Arp2/3 activation - binds actin and ARP2/3, -- increases affinity of ARP2/3 to side of filament Other (proline rich) activators of ARP2/3 include VASP (Vasodilator-stimulated phosphoprotein), Scar/WAVE family proteins

19. VCA domain of WASp becomes more compact when bound to G-actin A conformational change occurs so that ARP2 and ARP3 move closer together, to form a template for actin filament growth In budding yeast and Dictyostelium Myosin I may bind (via SH3 domains) ARP2/3 – possibly transporting it to the protruding edge. A conformational change occurs when Arp2/3 activated