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.”
This study describes the formulation and properties of active fluids that contain mixtures of microtubules, polymer solutions, microtubule motor proteins, and ATP. These fluids are isotropic materials that display spontaneous self-organized flow patterns, sometimes persisting over hours, that are a consequence of motor-driven microtubule sliding and polymer-induced microtubule bundling. The project, organized by Zvonimir Dogic’s lab, was a multi-institutional collaboration involving labs from UC Santa Barbara, Hampton Univ., Worcester Polytechnic Inst., Harvard, and Brandeis.
Chandrakar P., et al., Engineering stability, longevity, and miscibility of microtubule-based active fluids. Soft Matter 10.1039/D1SM01289D (2022)
Jeff received the 2019 Kazuhiko Kinosita Award in Single-Molecule Biophysics from the Biophysical Society. The award is named after Prof. Kazuhiko Kinosita, Jr. who was a much-admired pioneer of single-molecule biophysics, famous for his creative and intellectually rigorous approach to science. His research revealed key features of how molecular motors operate and how cells make ATP. Students will enjoy this public lecture from the January 2015 Single Molecule Biophysics conference in which Prof. Kinosita talks about his work:
Branched actin filament networks formed by the Arp2/3 complex play an essential role in force production in eukaryotic cells. Branched networks are not static components of the cytoskeleton. Instead the times and locations of network assembly and disassembly are tightly controlled by regulatory proteins. Ph.D. student Siyang Guo used single-molecule fluorescence methods to show how the Abp1 protein positively regulates branched actin networks. Remarkably, Apb1 functions by two distinct mechanisms. The protein stimulates the formation of networks by stabilizing the binding of Arp2/3 complex to the sides of actin filaments, a precursor to branch formation. However after branches form bound Abp1 works differently: it protects the network from GMF, the “pruning shears” protein that chops off branches during network disassembly. Taken as a whole, the study gives deeper insight into the multiple layers of regulation that control cytoskeleton pattern formation and dynamics. This project is part of a long-term collaboration on cytoskeletal regulation with Bruce Goode’s lab.
Abp1 promotes Arp2/3 complex-dependent actin nucleation and stabilizes branch junctions by antagonizing GMF
Siyang Guo, Olga S. Sokolova, Johnson Chung, Shae Padrick, Jeff Gelles, Bruce L. Goode Nature Communications (2018) 9:2895.