GMF Severs Actin-Arp2/3 Complex Branch Junctions by a Cofilin-like Mechanism

Abstract

Summary Background Branched actin filament networks driving cell motility, endocytosis, and intracellular transport are assembled in seconds by the Arp2/3 complex and must be equally rapidly debranched and turned over. One of the only factors known to promote debranching of actin networks is the yeast homologue of GMF, which is structurally related to the actin filament-severing protein cofilin. However, the molecular mechanism underlying debranching, and whether this activity extends to mammalian GMF, have remained open questions. Results Using scanning mutagenesis and TIRF microscopy, we show that GMF depends on two separate surfaces for debranching. One is analogous to the G-actin and F-actin binding site on cofilin, but we show using fluorescence anisotropy and chemical crosslinking that it instead interacts with actin-related proteins in Arp2/3 complex. The other is analogous to a second F-actin binding site on cofilin, which in GMF appears to contact the first actin subunit in the daughter filament. We further show that GMF binds to Arp2/3 complex with low nanomolar affinity and promotes the open conformation. Finally, we show that this debranching activity and mechanism are conserved for mammalian GMF. Conclusions GMF debranches filaments by a mechanism related to cofilin-mediated severing, but in which GMF has evolved to target molecular junctions between actin-related proteins in Arp2/3 complex and actin subunits in the daughter filament of the branch. This activity and mechanism are conserved in GMF homologues from evolutionarily distant species.

The other side of the coin: Functional and structural versatility of ADF/cofilins

“…Fitting the GMF-Arp2/3 complex structure into the model of the branch junction constructed from electron tomography (Rouiller et al, 2008) shows that besides interaction with the Arp2 subunit GMF using an interface similar to the F-site of ADF/cofilins can also interact with the SD2 and SD1 region of the adjacent actin subunit in the Arp2/3 complex generated daughter filament (Luan and Nolen, 2013). The structural data is consistent with recent findings that the activity of GMF requires both the G-and the F-sites and that GMF promotes the dissociation of Arp2/3 complex generated branches using a cofilin-like severing mechanism (Ydenberg et al, 2013). Moreover, the model revealed that the movement of GMF at the branch junction towards the first actin subunit of the daughter filament results in a steric clash, which could destabilize the Arp2-actin contacts or could interfere with the association of G-actin to the branch junction (Luan and Nolen, 2013).…”

“…In contrast to Abp1, GMF interferes with the activity of the Arp2/3 complex (Boczkowska et al, 2013;Luan and Nolen, 2013;Ydenberg et al, 2013). This property of GMF is conserved between yeast and mouse (Chaudhry et al, 2007;Gandhi et al, 2010;Nakano et al, 2010).…”

Actin network disassembly powers Listeria monocytogenes dissemination

“…Mammalian TWF1, and potentially TWF2, displays capping and severing activity in vitro (Poukkula et al, 2011). Yeast (Saccharomyces cerevisiae) Gmf1, a homolog of GMFB, displays ARP2/3-specific de-branching activity (Gandhi et al, 2010;Nakano et al, 2010), and its in vitro debranching activity was recently shown to be conserved in mammalian GMFc (Ydenberg et al, 2013). CAP1 is thought to participate in the dissociation of the cofilin-ADP-actin complex (Mattila et al, 2004) and was recently shown to directly enhance cofilin-mediated filament severing (Chaudhry et al, 2013;Normoyle and Brieher, 2012).…”

Cellular functions of the ADF/cofilin family at a glance

“…However, although cofilin debranching activity has been studied and does occur, another cofilin-like molecule termed glia maturation factor (GMF, of which there are different isoforms) appears to be more specific for the Arp2/3 complex branches and is more efficient than cofilin in mediating debranching (Haynes et al, 2015;Poukkula et al, 2014;Ydenberg et al, 2013).…”