Cryo-EM of human Arp2/3 complexes provides structural insights into actin nucleation modulation by ARPC5 isoforms

Abstract

The Arp2/3 complex regulates many cellular processes by stimulating formation of branched actin filament networks. Because three of its seven subunits exist as two different isoforms, mammals produce a family of Arp2/3 complexes with different properties that may be suited to different physiological contexts. To shed light on how isoform diversification affects Arp2/3 function, we determined a 4.2 Å resolution cryo-EM structure of the most active human Arp2/3 complex containing ARPC1B and ARPC5L, and compared it with the structure of the least active ARPC1A-ARPC5-containing complex. The architecture of each isoform-specific Arp2/3 complex is the same. Strikingly, however, the N-terminal half of ARPC5L is partially disordered compared to ARPC5, suggesting that this region of ARPC5/ARPC5L is an important determinant of complex activity. Confirming this idea, the nucleation activity of Arp2/3 complexes containing hybrid ARPC5/ARPC5L subunits is higher when the ARPC5L N-terminus is present, thereby providing insight into activity differences between the different Arp2/3 complexes.

Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction

“…This central role of ArpC3 in the formation and maintenance of the branch is in accordance with previous studies showing that ArpC3 KO mice and S. pombe are not viable 42,43 . ArpC5 does not form any contact with either the mother or the daughter filament, in line with previous reports suggesting its role to be mainly regulatory 30 .…”

“…3). This flexibility could be due to alternate conformations of this region of the subunit, caused by the existence of the two isoforms ArpC5 and ArpC5L, in line with a recent study 30 . Hence, we restrained the movements of this subunit in the MD modeling.…”

Novel cryo-electron tomography structure of Arp2/3 complex in cells reveals mechanisms of branch formation

“…S5a). ArpC5 does not form any contact with either the mother or the daughter filament, in line with previous reports suggesting its role to be mainly regulatory [25]. Correspondingly, in total 5 monomers (M2, M4-M7) in the mother actin filament are positioned to contact the Arp2/3 complex.…”

“…In the inactive complex and the recent in vitro structure of the Dip1-activated S. pombe Arp2/3 complex, the ArpC5 N-terminus has been shown to bind the side of subdomain 3 of Arp2 [7,25,29]. In our structure, no density for this interaction can be observed (Figure 4b).…”

“…S3). This flexibility could be due to alternate conformations of this region of the subunit, caused by the existence of the two isoforms ArpC5 and ArpC5L, in line with a recent study [25]. Hence, we restrained the movements of this subunit in the MD modelling.…”