how does actin polymerization drive protrusion?
DESCRIPTION
Polymerization at tip?. Expansion of actin meshwork?. Increase in hydrostatic pressure?. How does actin polymerization drive protrusion?. Hypothesis #1. Hypothesis #2. Hypothesis #3. Evidence for #1: The Acrosome reaction. Stages during fertilization of a sea-urchin egg. - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/1.jpg)
How does actin polymerization drive protrusion?
Polymerization at tip?
Expansion of actin meshwork?
Increase in hydrostatic pressure?
Hypothesis #1
Hypothesis #2
Hypothesis #3
![Page 2: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/2.jpg)
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
![Page 3: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/3.jpg)
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.
![Page 4: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/4.jpg)
• 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 protrusionMyosin I
at leading edge
![Page 5: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/5.jpg)
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
![Page 6: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/6.jpg)
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
![Page 7: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/7.jpg)
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 sequesterers Inactive 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.
![Page 8: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/8.jpg)
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
![Page 9: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/9.jpg)
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
![Page 10: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/10.jpg)
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
![Page 11: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/11.jpg)
Role of profilin during the Acrosome reaction
![Page 12: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/12.jpg)
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.
![Page 13: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/13.jpg)
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.
![Page 14: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/14.jpg)
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)
![Page 15: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/15.jpg)
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
![Page 16: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/16.jpg)
• 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
![Page 17: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/17.jpg)
Svitkina and Borisy 1999
Distribution of Arp2/3 in a moving cell
Arp2/3
Actin
Overlay
Keratocyte Immunogold labeling of Arp2/3
![Page 18: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/18.jpg)
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
![Page 19: How does actin polymerization drive protrusion?](https://reader035.vdocuments.us/reader035/viewer/2022081420/5681334b550346895d9a50ea/html5/thumbnails/19.jpg)
• 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