The actin cytoskeleton is an essential network that utilizes actin-binding proteins to perform different cellular functions. Maintenance of this network is crucial since dysregulation can lead to development defects and diseases. In this dissertation, we focus on unraveling the molecular mechanisms of two class-I myosin paralogs, myosin-1C (myo1C) and myosin-1D (myo1D), that can impact Drosophila Left-Right asymmetry. De novo overexpression of either paralog in symmetric tissues results in opposing tissue rotation, dependent on the paralog. De novo overexpression of recombinant chimera proteins show that the motor domain dictates the directionality. Interestingly, only myo1D glides actin filaments in a directional bias via in vitro gliding assays on a supported lipid bilayer. Despite myo1C’s lack of directionality at a protein level, there must be an intrinsic property in its motor domain to promote directionality at cellular to organismal levels. We performed steady-state and transient kinetics to identify differences in key steps of the paralogs’ ATPase cycle, and performed biophysical assays for validation. We discovered myo1C has a rate-limiting phosphate release followed by slow MgADP release, dwelling in the cycle for longer periods, suggesting its function as a molecular tether. Contrarily, myo1D has a rate-limiting MgADP release and can power small unilamellar vesicle transport, supporting a function as a transporter. Amazingly, both paralogs have nanomolar MgADP affinity and are the first class-I myosins identified as high duty ratio motors. In addition, I participated in a collaboration project unraveling biochemical differences of two smooth muscle gamma actin (ACTG2) mutations that result in human visceral myopathy: R40C and R257C. I performed in vitro gliding assays using smooth muscle myosin-II sub-fragment 1 (SMM-S1), where stabilized R40C polymerized into small filaments, and stabilized R257C filaments shred upon SMM-S1 gliding. Both defects decrease in the presence of Tropomyosin (Tpm) 1.4, suggesting it serves as a “protective shield”. All actin variants glide at the same velocity on SMM-S1, with and without Tpm 1.4. Results from both studies demonstrate how molecular characterizations of actomyosin determinants can lead to understanding the effects at cellular to organismal levels.