The Arp2/3 complex is a multi-component molecular machine that nucleates branched actin filament networks at the leading edge of cells to promote protrusion and at sites of endocytosis to drive membrane invagination. While the process of branched actin nucleation is now well understood (including at mechanistic and structural levels), what is less well understood is how the actin networks are subsequently debranched, or ‘pruned’. Debranching is an absolutely essential step in network remodeling and turnover, which is required for cell motility and endocytosis. The branched actin structures produced by Arp2/3 complex are kinetically stable, with spontaneous dissociation occurring only after tens of minutes to hours, whereas in vivo the branches dissociate in seconds. How is this achieved?
Two separate members of the larger ADF-homology family of proteins, glia maturation factor (GMF) and cofilin, have been implicated in promoting debranching. In this paper, Gelles lab member Johnson Chung, in collaboration with Jeff and with Bruce Goode from the Brandeis Biology Dept., used multi-wavelength single molecule florescence microscopy and quantitative kinetic analysis to define the mechanisms by which these proteins promote debranching. Dr. Chung shows that “coﬁlin, like GMF, is an authentic debrancher independent of its ﬁlament-severing activity and that the debranching activities of the two proteins are additive. While GMF binds directly to the Arp2/3 complex, coﬁlin selectively accumulates on branch–junction daughter ﬁlaments in tropomyosin-decorated networks just prior to debranching events. Quantitative comparison of debranching rates with the known kinetics of coﬁlin–actin binding suggests that coﬁlin occupancy of a particular single actin site at the branch junction is sufficient to trigger debranching. In rare cases in which the order of departure could be resolved during GMF- or coﬁlin-induced debranching, the Arp2/3 complex left the branch junction bound to the pointed end of the daughter ﬁlament, suggesting that both GMF and coﬁlin can work by destabilizing the mother ﬁlament–Arp2/3 complex interface. Taken together, these observations suggest that GMF and coﬁlin promote debranching by distinct yet complementary mechanisms.”
Chung J, et al. Single-molecule analysis of actin filament debranching by cofilin and GMF.
PNAS,119, e2115129119 (2022)
From the article: “The endoplasmic reticulum (ER) is the site at which secreted proteins (such as the hormone insulin) and membrane-bound proteins are folded. ATP-dependent chaperones within the ER help proteins fold. This study describes how two key ER chaperones, BiP and Grp94, work together at a molecular level. BiP binds to Grp94, which enables Grp94 to change conformation and hydrolyze ATP. In short, BiP provides a signal to switch on Grp94 conformational changes that are required to help other proteins fold. This finding helps explain how two chaperones can work together collaboratively in protein folding. Because BiP and Grp94 are members of highly conserved chaperone families, these findings may provide insight into chaperone-assisted protein folding beyond the ER.” This project was a collaboration with members of Timothy Street‘s lab in the Brandeis Biochemistry Department.
Huang B., et al., The endoplasmic reticulum chaperone BiP is a closure-accelerating cochaperone of Grp94.
PNAS 119, e2118793119 (2022)
The dynamic assembly, remodeling, and turnover of actin networks drives cellular processes
ranging from cell motility, endocytosis, and phagocytosis to cell division, cell and tissue
morphogenesis, and neuronal pathfinding. Here, we describe a new actin regulatory activity that changes understanding of how actin networks can be turned over. In a collaborative project with Bruce Goode’s lab, postdocs Shashank Shekhar and Greg Hoeprich used microfluidics-assisted total internal reflection fluorescence (TIRF) microscopy to show that mammalian twinfilin, an evolutionary conserved ADF/cofilin-homology protein, accelerates depolymerization at newly-assembled (ADP-Pi) but not older (ADP) actin filaments, even under assembly-promoting conditions (i.e., at G-actin concentrations above the critical concentration). Our data suggest that twinfilin molecules interact processively with the barbed end of the filament as it shrinks, blocking ATP-actin subunit addition while allowing ADP-Pi subunit dissociation. These novel activities of twinfilin reveal that cells have machinery that can bypass the normal filament aging process and induce the depolymerization of barbed ends as needed. These results may explain known genetic interactions between twinfilin and cofilin, and localization of twinfilin to the tips of filopodia and stereocilia, where actin filament barbed ends are clustered.
Shekhar S, et al., Twinfilin bypasses assembly conditions and actin filament aging to drive barbed end depolymerization.
Journal of Cell Biology 220, e202006022 (2021)