SummaryThe contractile actin cortex is a thin layer of actin, myosin, and actin-binding proteins that subtends the membrane of animal cells. The cortex is the main determinant of cell shape and plays a fundamental role in cell division [1–3], migration [4], and tissue morphogenesis [5]. For example, cortex contractility plays a crucial role in amoeboid migration of metastatic cells [6] and during division, where its misregulation can lead to aneuploidy [7]. Despite its importance, our knowledge of the cortex is poor, and even the proteins nucleating it remain unknown, though a number of candidates have been proposed based on indirect evidence [8–15]. Here, we used two independent approaches to identify cortical actin nucleators: a proteomic analysis using cortex-rich isolated blebs, and a localization/small hairpin RNA (shRNA) screen searching for phenotypes with a weakened cortex or altered contractility. This unbiased study revealed that two proteins generated the majority of cortical actin: the formin mDia1 and the Arp2/3 complex. Each nucleator contributed a similar amount of F-actin to the cortex but had very different accumulation kinetics. Electron microscopy examination revealed that each nucleator affected cortical network architecture differently. mDia1 depletion led to failure in division, but Arp2/3 depletion did not. Interestingly, despite not affecting division on its own, Arp2/3 inhibition potentiated the effect of mDia1 depletion. Our findings indicate that the bulk of the actin cortex is nucleated by mDia1 and Arp2/3 and suggest a mechanism for rapid fine-tuning of cortex structure and mechanics by adjusting the relative contribution of each nucleator.

Summary Cell locomotion and endocytosis are powered by the rapid polymerization and turnover of branched actin filament networks nucleated by Arp2/3 complex [1]. While a large number of cellular factors have been identified that stimulate Arp2/3 complex-mediated actin nucleation, only a small number of studies so far have addressed which factors promote actin network debranching [2–4]. Here, we investigated the function of a conserved homologue of ADF/cofilin, glia maturation factor (GMF) [5, 6]. We found that S. cerevisiae GMF (also called Aim7) localizes in vivo to cortical actin patches, and displays synthetic genetic interactions with ADF/cofilin. However, GMF lacks detectable actin binding or severing activity, and instead binds tightly to Arp2/3 complex. Using in vitro evanescent wave microscopy, we demonstrated that GMF potently stimulates debranching of actin filaments produced by Arp2/3 complex. Further, GMF inhibits nucleation of new daughter filaments. Together, these data suggest that GMF binds Arp2/3 complex to both ‘prune’ daughter filaments at the branch points and inhibit new actin assembly. These activities and its genetic interaction with ADF/cofilin support a role for GMF in promoting the remodeling and/or disassembly of branched networks. Therefore, ADF/cofilin and GMF, members of the same superfamily, appear to have evolved to interact with actin and actin-related proteins, respectively, and to make mechanistically distinct contributions to the remodeling of cortical actin structures.

The cellular actin cortex is the cytoskeletal structure primarily responsible for the control of animal cell shape and as such plays a central role in cell division, migration, and tissue morphogenesis. Due to the lack of experimental systems where the cortex can be investigated independently from other organelles, little is known about its composition, assembly, and homeostasis. Here, we describe novel tools to resolve the composition and regulation of the cortex. We report and validate a protocol for cortex purification based on the separation of cellular blebs. Mass spectrometry analysis of purified cortices provides a first extensive list of cortical components. To assess the function of identified proteins, we design an automated imaging assay for precise quantification of cortical actomyosin assembly dynamics. We show subtle changes in cortex assembly dynamics upon depletion of the identified cortical component profilin. Our widely applicable integrated method paves the way for systems-level investigations of the actomyosin cortex and its regulation during morphogenesis.