Trajectory Classification Method for Anchored Molecular Motor-Biopolymer Interactions in the C. elegans first Mitosis

During zygotic mitosis, forces generated at the cell cortex are required for the separation and migration of paternally provided centrosomes, pronuclear migration, proper segregation of genetic material, and successful cell division. Identification of individual cortical force generating units in vivo is necessary to study the regulation of microtubule dependent force generation throughout the cell cycle, to further understanding of asymmetric cell division, and to identify the molecular mechanism of force generation. Here we present a method to determine both the location and relative number of microtubule dependent cortical force generating units using single molecule imaging of fluorescently labelled dynein. Dynein behavior is modeled to differentiate and categorize trajectories that correspond to that which is cortically bound and interacting with a microtubule, and is cortically bound and not interacting with a microtubule. The categorization strategy recapitulates well known force asymmetries in the first mitosis of the C. elegans embryo. To evaluate the robustness of categorization, we RNAi depleted the microtubule subunit TBA-2 resulting in reduction of the number of trajectories categorized as engaged with a microtubule. This technique will be a valuable tool to provide new insight to the molecular mechanisms of dynein cortical force generation and its regulation as well as other instances wherein anchored motors interact with biopolymers (eg. Actin, tubulin, DNA).