| Summary: | How cells temporally and spatially form complex cellular structures within a crowded cytoplasm is a fundamental question in biology. The actin cytoskeleton is a dynamic cellular network that self-organizes into a range of networks to regulate diverse cellular processes, such as cytokinesis, polarization, endocytosis, and cell motility. Understanding how these diverse F-actin networks can be assembled and maintained from their shared pool of actin monomers at the right place and time is a long-standing question in our lab. The actin cytoskeleton assembles dynamic networks of filamentous actin (F-actin) from shared G-actin monomers through the action of actin nucleators, such as formin and Arp2/3 complex. Additionally, the individual and combined presence and functions of a wide variety of actin binding proteins (ABPs) work to define an actin network's identity-regulating assembly and disassembly, network density, and architecture-all of which lead to specialized, unique network functions. Therefore, in order to fully understand each network, it is crucial to understand the mechanistic principles that drive ABP functions, both alone and in concert. Previous studies in our lab have demonstrated that steady-state actin networks' size and density are maintained through actin nucleator's competition for actin monomers, and that this competition is uniquely tuned by the monomer binding protein, profilin. Our lab showed that profilin favors formin-mediated actin assembly, while directly inhibiting Arp2/3 complex branching; yet, the exact mechanism underlying this inhibition is unclear. Here, we built an optimal fluorescently-tagged fission yeast Arp2/3 complex and demonstrated it rapidly nucleates branching in vitro, with no significant defects when incorporated into fission yeast cells. This construct will now be used to directly examine the mechanism by which profilin inhibition of branching occurs, along with many other mechanistic investigations. As previous work to directly visualize Arp2/3 complex has been limited-and with no direct visualization of the fission yeast protein-this new construct will prove invaluable to the field. Additionally, in looking to understand the mechanisms of actin nucleators, we found that the fission yeast contractile ring formin, Cdc12, is specifically tailored for its function during cytokinesis. By building chimera proteins where the FH1 and FH2 domains of Cdc12 were replaced with formins with different or similar nucleation or elongation properties, we discovered that nucleation rate is the driving factor behind efficient contractile ring assembly. Work in our lab has shown the importance of competitive and cooperative interactions of actin binding proteins (ABPs) [28, 133]. Here, we have expanded this work and found a competitive interaction between the crosslinking proteins fimbrin and α-actinin, in which they compete for binding both in vitro and in actin patches and the contractile ring [29]. Further, we showed that the side-binding protein tropomyosin can synergize with α-actinin to make it a better competitor against fimbrin [29]. Much work has been done by our lab and others to understand how fission yeast actin net- work assembly is regulated, yet little has been done to understand the mechanistic properties of fission yeast actin disassembly. Here, we engineered and purified fission yeast disassembly proteins. We found that fission yeast twinfilin and CAP1 synergize for rapid barbed and pointed end depolymerization of F-actin in vitro, similar to homologs in other systems. Further, while our lab's previous work looked at what drives actin cytoskeleton self- organization during steady state, how network identity and self-organization is initially established in fission yeast is unclear. Here, we developed and optimized a microfluidic system for fission yeast, allowing us temporal control for actin network perturbations previously not available in this organism. Through temporally controlled treatment and washout with Latrunculin A, an actin sequestering protein, we were able to mimic an initial 'disassembled' rate that was rapidly reversible to monitor initial network assembly and self-organization. We observed that Arp2/3-mediated F-actin networks were always the first to re-assemble. Interestingly, formin-mediated F-actin networks did not re-assemble in the absence of Arp2/3- mediated networks, suggesting that endocytic actin patches or their associated proteins are required for the initial establishment of formin-mediated networks.
|
|---|
