APBs involved in regulating actin dynamics (Cont.) 2. How high rates of actin polymerization are maintained at the protruding edge? Thymosin  -4 Profilin Arp2/3 Capping Severing Lodish 5th Ed. Chapter 19, p

How is Arp2/3 activated Wiskott-Aldrich Syndrome protein – implicated in immune dysfunction –Scar/WAVE = WASp-related proteins Purified ARP2/3 complex has low nucleating acitivity –Enhanced when WASp added –Enhanced further when pre-incubated w. F-actin –VCA (verprolin cofilin acidic) Is proline rich, VCA domain essential for nucleation –Verprolin binds an actin monomer, A binds ARP2/3 –F-actin enhances its binding to ARP2/3 N-terminus C-terminus

Activation of WASp Is auto inhibited until activation WASp is activated when the proline rich and GBD domain binds PIP2 and active Cdc42 (small GTPase) Further activation = ATP hydrolysis within ARP1 or ARP2 New filaments grow from the side of pre-existent filaments Growth stops when + end is capped

Treadmilling in vitro is much slower than in vivo Treadmilling rate in vitro is limited by the depolym. rate (-end) = 0.2 monomers/sec Resulting in a max growth rate at +end of ~ 0.05 um/min. BUT some cells can move = >10 um/min i.e. x200 greater To achieve rates of protrusion observed in vivo - need to supply + end with subunits/second ! Capping proteins 1. Generate more + ends 2. Increase size of G-actin pool 3. Restrict polymerization to where it’s needed Severing proteins Increase numbers of + and – ends Increase G-actin pool

Actin binding proteins that cap and sever F-actin Capping proteins inhibit addition or loss of G-actin from the end (+ or -) of the actin filament Actin filaments in muscle are very stable - capped by capZ at + end and tropomodulin at - end Table 19-2

The dynamics of actin filaments is greatly affected by ABPs that bind to filament ends Filaments that are capped at the + end are less dynamic and more stable than uncapped filaments Low concentrations of these ABPs (relative to actin monomers) are sufficient In non muscle cells capping is regulated by PIP2 –Decrease in PIP2 (when cleaved) increases capping –An increase in PIP2 (shortly after initial signal) leads to uncapping

Increase actin filament dynamics by generating a large number of shorter filaments Gelsolin can: –inc. number of growing +ends –Inc. depolymerization from the -end Low stoichiometry –e.g. 1 gelsolin molecule for every 400 actin monomers (in a filament) is sufficient! Severing proteins regulate the length and dynamics of actin filaments

Mechanism of severing Severing proteins, e.g. gelsolin are activated in by an increase in intracellular calcium following a signaling event. After Ca2+ inc. and severing F-actin, gelsolin caps the + end Shortly after this, PIP2 increases and gelsolin dissociates from the + end allowing polymerization

Gelsolin regulates the biomechanical properties of actin gels Gelsolin solates actin gels allowing cytoplasmic streaming Cytoplasm “gelates” or solidifies at the tip Filamin and  -actinin are important for gelation –Abundant =1% tot. protein in cell Very sensitive to increases in [Ca2+]i (10 -7 M) during signaling Cytoplasmic extracts in amoeba, leukocytes, fibroblasts can still stream Cytoplasmic streaming Amoeba proteus

The stability of actin filaments is regulated by ABPs that bind to their sides Need to be present in high concentractions e.g. –Tropomyosin binds to the sides of 7 adjacent actin monomers Tropomyosin stabilizes the actin filaments and excludes other ABP’s from binding, therefore its activity must be regulated In muscle, the position of tropomyosin is altered to expose myosin II binding sites, only when contraction is triggered

It causes the actin filaments to twist more tightly, and so weakens interactions between neighboring actin subunits Cofilin binds preferentially to ADP actin, so its activity is restricted to “older” regions of F- actin (closer to -end). Cofilin increases the rate of depolymerization from the - end of F-actin by 25 times Cofilin de-stabilizes actin filaments

Mechanism of ADF/Cofilin

CofilinActinMerge Cofilin is located ~1-2um behind the leading edge Distribution of cofilin in a fish keratocyte Svitkina and Borisy, 1999

ADF/cofilins increase disassembly (x25) from - ends Nucleotide exchange Fast + end assembly of a few uncapped filaments Capping The rate of polymerization is increased ADF/cofilins increase the ATP G-actin pool Capping and severing work synergistically to increase the rate of treadmilling The funneled treadmilling process (Carlier and Pantaloni, 1997) sustains an efficient, rapid, rate of actin polymerization Regulated capping of +ends increases the pool of ATP-G actin Un-capping

The combined effect of capping, severing and depolymerizing F- actin is important for sustaining rapid protrusion From: “Control of actin dynamics” Carlier 1998

The combined effect of capping, severing and depolymerizing F- actin is important for sustaining rapid protrusion From: “Control of actin dynamics” Carlier 1998 (For notes )

Mullins 1998

FACT: In a steadily migrating cell lamellipodium width is ~ constant, - due to balance between assembly at the front and disassembly further back Treadmilling occurs within the whole population of F-actin

Arp2/3ABP-120  -actinin At front edge ~4um behind front edge Location of actin-binding proteins reflects their function