Uyeda, T. Q. | Search bioRxiv

bioRxiv2025

Conformational dynamics of actin filaments crosslinked with alpha-actinin and their roles in suppressing cofilin-induced helical shortening and cluster formation

Actin is a conserved cytoskeletal protein essential for morphogenesis, motility, and division. Its versatility arises from filament assembly and regulation by actin binding proteins. Among these, alpha-actinin organizes filaments into bipolar or unipolar networks, whereas cofilin binds preferentially to ADP-actin regions and forms clusters to shorten the half helical pitch (HHP). Here, we investigated the molecular mechanism of how alpha-actinin alters filament and protomer conformations and influences cofilin binding. Using all-atom molecular dynamics simulations, principal component analysis, and high-speed atomic force microscopy, we show that alpha-actinin crosslinking stabilizes actin filaments in the canonical helical state, thereby preventing the cofilin-induced helical shortening required for cooperative filament decoration. Stabilization occurs without significant changes in protomer twist and rise and subdomain geometry and maintains a flattened protomer conformation that restricts twisting needed for cofilin cooperative binding. By contrast, the isolated alpha-actinin-1 actin binding domain mutant (ABD-E235K), comprising two calponin homology domains (CH1-CH2), transiently binds to actin filaments and induces local transitions from the canonical double-helical filament to the single- and parallel-helical protofilament states, weakly affecting cofilin binding and cluster formation through a distinct structural and binding mechanism. Together, these findings reveal a mechanistically distinct function of full-length alpha-actinin and its isolated ABD and support a stepwise mechanism in which cooperative cofilin binding to double-helical actin filaments requires initial binding to actin regions with shortened HHP, followed by protomer twisting and further helical shortening. By stabilizing the canonical filament architecture or perturbing filament organization, alpha-actinin suppresses these structural transitions and thereby perturbs cofilin cooperativity. TeaserAlpha-actinin stabilizes actin helices, suppressing cofilin cooperative binding through distinct structural mechanisms.

biophysics↗

bioRxiv2023

Ras Activation by Hydrostatic Pressure is Enhanced by GAP and GEF in vitro

Hydrostatic pressure (HP) is a necessary stimulus for cell differentiation and growth in cultured chondrocytes. Assuming that Ras activation is involved in HP-induced reactions, if cellular Ras activity is increased by HP, Ras itself, Raf, Ras regulators including GTPase activating proteins (GAP) and guanine nucleotide exchange factors (GEF), then the upstream receptor and/or interactions between them should have HP sensitivity. Based on this hypothesis, we first attempted to examine whether Ras is activated by HP, and in the affirmative, identify which factors displayed HP sensitivity using an in vitro system to measure Ras activity. This in vitro system included mRaichu, a FRET-based Ras activity probe, to which two-point mutations were introduced to reduce Ras-independent signals. This improved Raichu was used to investigate the HP sensitivity of two components, the GAP domain (GAPd) derived from p120GAP and the GEF domain (GEFd) derived from hSOS-1. It was found that HP weakly activated Ras activity in the absence of GAPd and GEFd, presumably by facilitating GDP dissociation from Ras. A low concentration of GAPd enhanced HP-induced Ras activation by 16.3% whereas high concentrations of GAPd removed HP sensitivity, suggesting that HP partially dissociates GAPd from the GAPd-Ras-GDP complex and reduces the fraction of inactive Ras. Moreover, a broad concentration range of GEFd also enhanced HP-induced Ras activation. Given that HP also increased Ras activity under a condition mimicking cellular Ras activity, we propose that Ras activation is involved in the differentiation and growth stimulation of chondrocytes by HP. Statement of SignificanceThe Ras-cycle has been implicated in the regulation of growth and differentiation of eukaryotic cells. Here, we investigated the relationship between hydrostatic pressure (HP) and components of the Ras-cycle: Ras-Raf, the GAP domain, and the GEF domain. Generally, HP tends to weaken protein-protein and protein-ligand interactions, but in this study, a seemingly positive response was observed: HP-induced Ras activation. Additionally, this response was enhanced by the GEF domain and the GAP domain. Thus, given the multiple cellular functions of Ras and the responses of the Ras-cycle to HP, this study will help clarify the molecular mechanism by which HP modulates cellular functions, particularly in chondrocytes, which are subjected to repetitive HP stimuli in vivo.

biophysics↗