Decoding the functions of myosin II isoforms with super-resolution microscopy 1) Background and key gaps in our understanding. Cells modify their shape and surroundings to drive processes vital for eukaryotic life, including cell division, cell migration, and muscle contraction. Forces generated by the molecular motor, myosin II, drive these processes. Thus, how myosin II assembles filaments capable of generating force inside of cells is central to our understanding of both developmental processes and the force-dependent progression of diseases such as cancer. Previous studies have provided elegant details of how a single filament of myosin II assembles. Steric hindrance limits the number of myosin II molecules that can be added to a single filament. In order to increase the scale of force generation inside of cells, myosin II filaments are organized into arrays referred to as ?stacks?. While it is well documented that both muscle and non-muscle isoforms of myosin II are found within stacks, we do not know how stacks assemble. 2) Description of recent progress by the PI. At the end of his post-doctoral work, the PI showed that super-resolution microscopy could be used to resolve the structure of a non-muscle myosin IIA (NMIIA) filament and that a stack of filaments somehow grows from a single filament at the edge of a migrating cancer cell (Burnette et al, JCB 2014). The first independent paper from the Burnette lab subsequently defined the steps through which a NMIIA filament physically grows into a stack in a mechanisms we call ?expansion?, and that this is regulated by the motor activity of NMIIA, the density of surrounding actin filaments, and Rho GTPase signaling (Fenix et al, MBoC 2016). Expansion occurs at the edge of migrating cells during interphase and in the contractile ring during cell division. The second paper from the Burnette lab showed a force balance between myosin II-based contractility and adhesion was controlling the shape of the cleavage furrow (Taneja et al, Scientific Reports 2016), similar to how the leading edge of a crawling cell obtains its shape (Burnette et al. JCB 2014). We now have data suggesting NMIIA and NMIIB play distinct roles during cytokinesis. NMIIA is required for the proper formation of the contractile ring and initial ingression of the cleavage furrow, and NMIIB is required for the completion of cytokinesis, as well as maintaining the integrity of the cell cortex throughout mitosis. 3) Overview of future research program. We propose to continue our research on how myosin II filaments create larger contractile arrays by addressing three main themes. 1) We will continue to use migrating cells as a model system to investigate the molecular mechanisms controlling the assembly/disassembly of NMII filament-stacks. 2) We will also explore the different roles of NMIIA and NMIIB during cytokinesis with a particular focus on their cooperation in creating the contractile arrays in the cleavage furrow and cell cortex. 3) Finally, we will further test the universality of our expansion model by investigating how muscle myosin II isoforms create filament- stacks within cardiac muscle cells. Our ultimate goal is to develop a universal model of myosin II filament-stack formation that can be applied to the study of diverse contractile systems.
Studies investigating the mechanisms by which myosin II-based contractile systems form have significant promise to gain a mechanistic understanding of pathologic processes and subsequently identify targets for intervention, providing a health benefit to the public at large. Specific examples of aberrant processes include dissemination of cells from a primary tumor leading to cancer metastasis and thickening of the blood vessel wall from the aberrant migration of smooth muscle cells leading to vascular disease. The line of research proposed here is designed to develop a molecularly detailed and predictive model of how contractile systems generate more force to drive cell division and movement, with a goal of distinguishing mechanisms used by normal vs. aberrant cells.
| Manalo, Annabelle; Schroer, Alison K; Fenix, Aidan M et al. (2018) Loss of CENP-F Results in Dilated Cardiomyopathy with Severe Disruption of Cardiac Myocyte Architecture. Sci Rep 8:7546 |
| Fenix, Aidan M; Neininger, Abigail C; Taneja, Nilay et al. (2018) Muscle-specific stress fibers give rise to sarcomeres in cardiomyocytes. Elife 7: |
| Taneja, Nilay; Rathbun, Lindsay; Hehnly, Heidi et al. (2018) The balance between adhesion and contraction during cell division. Curr Opin Cell Biol 56:45-52 |
| Lehtimäki, Jaakko I; Fenix, Aidan M; Kotila, Tommi M et al. (2017) UNC-45a promotes myosin folding and stress fiber assembly. J Cell Biol 216:4053-4072 |
