Actin Motility 10/6/2010

 
Actin

• 42,000 mw monomer

– Most abundant protein in most eukaryotic cells

– Binds ATP, forms a polarized, helical filament

• In vertebrates, 6 genes

– Over 96% identical

• Actin-related proteins 1,2 and 3

– Overall structural similarities, low homology at a.a. level

– Arp1: involved in dynein/dynactin

– Arps 2 and 3: Arp2/3 complex

Actin drugs

• Filament disrupting drugs

– Cytochalasin binds to barbed (+) end of filaments to prevent monomer

assembly

– Latrunculin B

• Filament stabilizing drugs

– Phalloidin: peptide binds to groove of preformed filaments; has

negligable off-rate

Actin filaments have intrinsic polarity

• actin filaments decorated with the motor domains of myosin II form a
characteristic arrowhead pattern

•The barbed end (+) end is the fast growing end, the pointed end (-) is the slow
growing end

Preferred addition of actin monomers from the barbed or + end

Actin filament assembly

• Occurs in three steps

– Monomer activation
– Nucleation
– elongation
Actin dynamics in vitro do not reflect what happens in cells

• Actin filaments turn over much more rapidly in cells (in vivo) than in the test
tube (in vitro)

• Actin filaments in crawling cells are always arranged with their barbed ends
facing the membrane

• Actin filaments appear to be nucleated near the leading edge, and then move
in a retrograde fashion toward the cell center

Actin filament nucleation and organization is mediated by actin-binding proteins

• Actin nucleating factors: reduce the rate-limiting step of filament

polymerization

– Arp2/3 complex

• Actin capping proteins: bind barbed or pointed end and control monomer
 addition

– Capping protein, gelsolin

• Monomer-binding proteins: bind actin monomers and prevent spontaneous

polymerization of actin filaments

– Profilin, thymosin

Actin filament nucleation and organization is mediated by actin-binding proteins

Together, actin-binding proteins direct the polymerization of actin, which drives

the cell forward

• In response to an external signal, Arp2/3 is activated, and binds the sides of

existing actin filaments

– Arp2/3 activated by WASP

– Wasp activated by G proteins Rac and Cdc42

• Arp2/3 brings together actin monomers, which create a free barbed end

• Actin monomers readily add to the free barbed end, which is quickly capped
to limit filament length

Learning about actin polymerization from nasty bugs

• Lysteria and Shigella: genes on the cell surface (ActA and IcsA) nucleate
actin polymerization

– ActA/IcsA bound to beads nucleate actin tails in Xenopus extracts

• Requires Arp2/3 complex

• Mimic WASP activation of Arp2/3

• Bead motility shown to be reconstituted in vitro using Arp2/3, actin, capping
protein, VASP, cofilin

Intracellular pathogens: a model for lamellapodial extension?

A brief recap:

• Actin polymerizes into polarized filaments

– “+” end fast growing, slow depolymerizing

– “-” end slow growing, fast depolymerizing

– ATP > ADP-actin

• Monomer sequestering Proteins

– Actually favor polymerization in presence of uncapped filaments

• Capping proteins: prevent monomer addition

• Nucleating factors: negate rate limiting step