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How Arp2/3 Complex Nucleates Actin Filaments

Background: Arp2/3 and actin-based motility

The Arp2/3 com­plex builds crosslinked actin net­works that drive cell move­ment, mak­ing it pos­si­ble for immune cells to catch bac­te­ria, or neu­rons migrate and wire up to the right place in the brain.  Arp2/3 builds these net­works by bind­ing to the sides of exist­ing actin fil­a­ments and nucle­ates growth of new fil­a­ments from their sides, result­ing in a den­dritic fil­a­ment array.

Arp2/3 complex nucleates a network of actin filaments that produce force at the membrane 

To under­stand how Arp2/3 nucle­ates fil­a­ments, we focused in on the Arp2 and Arp3 sub­units of the 7-subunit pro­tein com­plex.  These Arps (Actin Related Pro­teins) have a strik­ingly sim­i­lar struc­ture to actin, includ­ing the ATP bind­ing pocket, so we looked to see if they bound and hydrol­ysed ATP.

ATP binding by Arp2/3

We mea­sured the bind­ing kinet­ics of Arp2/3 to ATP, and found that both Arp2 and Arp3 bind ATP, but which much weaker affin­ity than actin binds ATP.  This allowed us to use a kinetic trick—loading actin with ATP, but keep­ing the ATP con­cen­tra­tion very low then mix­ing with a large amount of ADP, (or AMP-PNP, a non-hydrolyzable ana­logue of ATP) just before start­ing a nucle­ation exper­i­ment.  In this way, we could see if chang­ing the nucleotide on Arp2/3 changed its abil­ity to nucle­ate, while keep­ing ATP on actin.  We found that Arp2/3 needs ATP (and not ADP or AMP-PNP) to nucle­ate new fil­a­ments, sug­gest­ing that it might be hydrolyzed dur­ing nucle­ation.

Look­ing for ATP hydrol­y­sis on Arp2/3 is dif­fi­cult, because when acti­vated Arp2/3 nucle­ates actin poly­mer­iza­tion, and actin itself hydrol­y­ses ATP when it poly­mer­izes.  The chal­lenge is to mea­sure ATP hydrol­y­sis on minute (nM) con­cen­tra­tions of Arp2/3 com­plex in a solu­tion con­tain­ing large (uM) con­cen­tra­tions of ATP-hydrolyzing actin.

ATP hydrolysis by Arp2/3

The trick we used to mea­sure ATP hydrol­y­sis on Arp2/3 was to replace nor­mal ATP with a radio-labeled ver­sion (32P on the Ɣ-phosphate), and crosslink this cova­lently to the Arp2 and Arp3 sub­units of the com­plex.  We washed it so that the only 32P in the reac­tion was on the Arp2/3, so we could then run the sam­ples out on a gel to sep­a­rate the pro­teins, and look for loss of the 32P label­ing over time (which indi­cates cleav­age of the Ɣ-phosphate and ATP hydrol­y­sis).  Because the only 32P-ATP in the reac­tion is phys­i­cally attached to the Arp2/3 com­plex, we don’t have to worry about ATP hydrol­y­sis by actin con­found­ing the exper­i­ment.  When we per­formed a nucle­ation reac­tion with this labeled Arp2/3,  we found that Arp2 rapidly hydrol­y­ses ATP when Arp2/3 nucle­ates new fil­a­ments, and we didn’t see any hydrol­y­sis of ATP on Arp3.

Investigating the Mechanism of Filament Nucleation

The fact that ATP hydrol­y­sis occurs rapidly on Arp2 upon nucle­ation meant we could use ATP hydrol­y­sis as a probe for acti­va­tion of the Arp2/3 com­plex. These are the com­po­nents of the nucle­ation reac­tion:

Nucleation Components

Com­po­nents of the Arp2/3 fil­a­ment nucle­ation reac­tion

We put all of the fac­tors required to nucle­ate actin fil­a­ments (monomeric actin, actin fil­a­ments, and a nucle­at­ing factor(VCA)) in var­i­ous com­bi­na­tions to see if they acti­vated ATP hydrol­y­sis on Arp2 (using Latrun­culin A and Phal­loidin to keep the monomers monomeric and the poly­mers from depoly­mer­iz­ing.)  Here’s what we found:

ATP hydrolysis requirements 

  • The first line is the nor­mal poly­mer­iza­tion reaction—nothing sur­pris­ing there.
  • The sec­ond line shows that with­out actin monomers, there’s no ATP hydrol­y­sis on Arp2.
  • The third line shows that adding actin monomers does trig­ger ATP hydrol­y­sis on Arp2 even when those monomers are pre­vented from poly­mer­iz­ing by Latrun­culin A—i.e. one monomer is all that’s required to trig­ger Arp2 to hydrolyze ATP (in the pres­ence of the other com­po­nents), you don’t need poly­mer­iza­tion.
  • The fourth line is par­tic­u­larly inter­est­ing.  Here we took Arp2/3, mixed it with fil­a­ments then sheared the fil­a­ments to break them.  Arp2/3 then attaches to the ends of the fil­a­ments (cap­ping), much like it is attached to the end of the new fil­a­ment it nucle­ates when it forms branches.  This cap­ping also trig­gers Arp2 to hydrol­yse ATP, but with­out the other com­po­nents (the mother fil­a­ment and VCA)!


This is explained in more detail in the paper, but the bot­tom line is that it explains how Arp2/3 is work­ing, imply­ing that Arp2 is behav­ing like an actin monomer does in a fil­a­ment.

How we think Arp2/3 nucleates new actin filaments

When actin monomers poly­mer­ize to make a fil­a­ment, the pro­tein con­for­ma­tion of the monomer changes and causes it to hydrolyze ATP.  Here’s a model show­ing this, with the con­for­ma­tional change shown by the lit­tle red levers that extend out to con­tact the core of the fil­a­ment (actin shown in white):

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Model for how a con­for­ma­tional change in actin within a fil­a­ment trig­gers ATP hydrol­y­sis

(down­load ani­ma­tion)

The idea is that Arp2 is doing the same thing, but that the Arp2 is put in a filament-like con­for­ma­tion by the assem­bly of a nucleus (Arp2-Arp3-monomer).  The clear­est evi­dence for this is the trig­ger of ATP hydrol­y­sis by cap­ping, which directly cre­ates the Arp2-Arp3-monomer arrange­ment (actin shown in white, Arp2 in blue, Arp3 in pink):

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Model for how Arp2/3 docks onto the pointed end of an actin fil­a­ment, putting Arp2 and Arp3 into a filament-like con­for­ma­tion and trig­ger­ing ATP hydrol­y­sis on Arp2

(down­load ani­ma­tion)

And the Latrun­culin result (that when fil­a­ments and VCA are present, you just need one monomer) implies that when  nucle­ation is trig­gered by the for­ma­tion of this Arp2-Arp3-monomer nucleus, with VCA bring­ing in the monomer.

Putting this together gives us the model for fil­a­ment nucle­ation (actin shown in white, Arp2 in blue, Arp3 in pink):

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Model for how Arp2/3 nucle­ates actin fil­a­ments: VCA brings an actin monomer to the Arp2/3 com­plex, cre­at­ing an Arp2-Arp3-actin nucleus and trig­ger­ing ATP hydrol­y­sis on Arp2. Actin poly­mer­izes from this nucleus to form the daugh­ter fil­a­ment.

(down­load ani­ma­tion)

Function of ATP hydrolysis

We used ATP hydrol­y­sis to probe the nucle­ation mech­a­nism, and didn’t directly address the func­tion of ATP hydrol­y­sis itself. It may be, like actin, involved with later dis­as­sem­bly events (for actin this is really deter­mined by phos­phate (Pi) release, which is down­stream of, and occurs much later thanm ATP hydrol­y­sis).


[1] Acti­va­tion of Arp2/3 com­plex: addi­tion of the first sub­unit of the new fil­a­ment by a WASP pro­tein trig­gers rapid ATP hydrol­y­sis on Arp2.
Dayel MJ, Mullins RD.
PLoS Biol. 2004 Apr;2(4):E91. Epub 2004 Apr 13.
PMID: 15094799
Free Open-access arti­cle

[2] Arp2/3 com­plex requires hydrolyz­able ATP for nucle­ation of new actin fil­a­ments.
Dayel MJ, Holleran EA, Mullins RD.
Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):14871–6.
PMID: 11752435
Free Open-access arti­cle

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3D Movies of the model

I made some 3D ani­ma­tions to show how I think things might fit together using the actual struc­tures of Arp2/3 and actin.  Click on the thumb­nails below to take a look (right-click the thumb­nails and select save-as to down­load the movies). Stills from the movies are avail­able here.


Arp2/3 Flexing motion showing how Arp2 and Arp3 might come together to mimic the arrangement of two actin monomers in a filament

The Arp2/3 com­plex, show­ing a flex­ing motion that enables Arp2 and Arp3 to come together to mimic the arrange­ment an actin dimer.


Filament Capping by Arp2/3 showing how Arp2/3 caps the ‘pointed’ end of actin filaments

Fil­a­ment Cap­ping by Arp2/3 com­plex: Arp2/3 caps the ‘pointed’ end of actin fil­a­ments.

Filament Nucleation by Arp2/3 showing a summary of all the results described above: how VCA brings an actin monmer to a filament-bound Arp2/3, forming an Arp2-Arp3-actin nucleus, from which the daughter filament polymerizes. The drop in affinity with Arp2/3 when Arp2 hydrolyzes ATP, and the loss of monomer binding energy helping VCA to release.

Fil­a­ment Nucle­ation by Arp2/3 com­plex show­ing a sum­mary of all the results described above: VCA brings an actin monomer to a filament-bound Arp2/3 form­ing an Arp2-Arp3-actin nucleus from which the daugh­ter fil­a­ment poly­mer­izes. The drop in affin­ity between VCA and Arp2/3 when Arp2 hydrolyzes ATP, and the loss of monomer bind­ing energy, help VCA to release.

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