Molecular Mechanisms of Non-muscle Myosin II Contractility

/ABSTRACT Non-muscle myosin II (NMII) contractility is critical to number cellular processes from development to disease. NMII is an ATP-dependent molecular motor that functions as a dimer composed of two heavy chains, which are made of an ATPase/motor domain that binds actin and a coiled-coil tail domain. It is also bound to two accessory proteins, the essential light chain which plays structural roles, and the regulatory light chain which is the target of phosphorylation, integrating the molecule into a myriad of signaling pathways. Phosphorylation of the regulatory light chain leads to a relief of an autoinhibition, opening up the molecule and making it competent to bind actin, however, by itself it is a poor motor protein. The second required step of activation is oligomerization into higher ordered, bi-polar filaments. This oligomerization is thought to be regulated by the tail domain where again, phosphorylation is hypothesized to be the main driver of this transition. While decades of research have revealed much about these biochemical and biophysical properties, we questioned whether there were other, yet-to-be revealed, mechanisms that may contribute to NMII’s regulation. The overarching goal of this proposal is to understand the mechanisms that regulate NMII contractility. In Project 1, we explore a potential novel NMII binding protein, Split Discs (Spdi). Spdi’s human homolog, SPECC11L has been implicated in a spectrum of cranial-facial pathologies, highly suggestive of abearent cranial neural crest cell migration. SPECC1L was initially characterized as actin-microtubule crosslinking proteins, however data from my lab suggests that its target is NMII and actin. It is our hypothesis that Spdi binds NMII to regulate its contractility. Through a series of biochemical characterization and cell biology experiments where we elucidate the mechanism by which Spdi associates with NMII, and employ an ex-vivo developmental model to understand how its regulation of contractility affects collective cell migration. In Project 2 we focus on the role of acetylation on the regulation of NMII filament assembly. While phosphorylation has long been thought of as the major driver of NMII dynamics and behavior, recent data from my lab suggests that acetylation may play an equally important role in this process. We will identify the enzymes involved in this acetylation-deacetylation cycle, and elucidate the role this post-translational modification has on force generation. The proposed research spans the molecular and biochemical, to cell biology and development, integrating high-resolution microscopy and capitalizing on Drosophila and the broad genetic tools they provide. Given the high degree of conservation and its universality to number of critical cellular processes the results we obtain here will have broad implications for how NMII is regulated across species and will bring new insights to how NMII is integrated in cellular signaling paradigms. Project Number: 1R35GM164067-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Derek Applewhite | Institution: REED COLLEGE, PORTLAND, OR | Award Amount: $430,383 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award - D Study Section[MRAD] View on NIH RePORTER: https://reporter.nih.gov/project-details/11331697