1) Background and key gaps in our understanding. Cellular force generation drives processes vital for eukaryotic life, including cell division, cell migration, and muscle contraction. Thus, the basic principles underlying cellular force generation are central to both developmental biology and the progression of force-dependent diseases like cancer. Cells generate force by assembling cellular contractile systems. Contractile systems emerge from the collective action of individual components within a larger molecular assembly. Our first investigation into cellular contractile systems focused on the class of molecular motors responsible for generating contractile forces, myosin II. The force-generating form of myosin II is a filament. A given myosin II filament can contain multiple, distinct myosin II paralogs (i.e., hetero-filaments), wherein each individual myosin II paralog has unique biophysical properties. Previously, the specific function(s) of myosin II-containing hetero-filaments within contractile systems was unknown. Our work during the ESI MIRA funding period has revealed multiple roles for hetero-filaments in the context of cell division and muscle contraction. Future work should address how hetero- filaments are regulated, influence mechanical output, and cooperate with other contractile system components. 2) Description of recent progress by the PI. In non-muscle cells, we reported that hetero-filaments serve multiple functions. Myosin IIA filaments seed the formation of myosin IIB filaments in both interphase and mitosis/cytokinesis (Fenix et al. 2016, Taneja et al., 2020). The presence of myosin IIA in hetero-filaments drives cortex tension (Taneja et al. 2020) and is regulated by both myosin IIA turnover and by phosphorylation of the myosin IIA regulatory light chain (Taneja and Burnette 2019, Taneja et al. 2021). Meanwhile, the presence of myosin IIB in hetero-filaments stabilizes the cell cortex during mitosis/cytokinesis and regulates cytokinetic fidelity through multiple mechanisms (Taneja et al. 2020). Within cardiac muscle cells, we reported that NMIIA/NMIIB hetero-filaments seed filaments of the muscle-specific myosin paralog, β myosin II, and are found exclusively in the sarcomere precursors known as muscle stress fibers. We also experimentally demonstrated that muscle stress fibers directly give rise to sarcomeres (Fenix et al. 2018, Taneja, Neininger et al. 2020). 3) Overview of future research program. Here, we propose to build upon our findings from the ESI MIRA by taking a multi-faceted approach aimed at three areas: A) We will continue elucidating how specific molecular components of myosin II filaments, as well as those of other contractile system proteins like α-actinin and formins, regulate cellular force production. B) We will determine the roles, and specific differences in function, of the non- muscle and muscle paralogs of myosin II, α-actinin, and potentially other sarcomeric proteins during both sarcomere form...