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, p786-791

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 20-200 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 (10-7M) 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

Cofilin Actin Merge

Cofilin is located ~1-2um behind the leading edge

Distribution of cofilin in a fish keratocyte

Svitkina and Borisy, 1999

ADF/cofilins increasedisassembly (x25) from - ends

Nucleotide exchange

Fast + end assemblyof a few uncapped filaments

Capping

The rate of polymerizationis 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

Arp2/3 ABP-120 -actinin

At front edge

~4um behind front

edge

Location of actin-binding proteins reflects their function