AbstractMitotic spindle formation in the pathogenic budding yeast, Cryptococcus neoformans, depends on multitudes of inter-dependent interactions involving kinetochores (KTs), microtubules (MTs), spindle pole bodies (SPBs), and molecular motors. Before the formation of the mitotic spindle, multiple visible microtubule organizing centers (MTOCs), coalesce into a single focus to serve as an SPB. We propose a ‘grow-and-catch’ model, in which cytoplasmic MTs (cMTs) nucleated by MTOCs grow and catch each other to promote MTOC clustering. Our quantitative modeling identifies multiple redundant mechanisms mediated by a combination of cMT-cell cortex interactions and inter-cMT coupling to facilitate MTOC clustering within the physiological time limit as determined by time-lapse live-cell microscopy. Besides, we screened various possible mechanisms by computational modeling and propose optimal conditions that favor proper spindle positioning - a critical determinant for timely chromosome segregation. These analyses also reveal that a combined effect of MT buckling, dynein pull, and cortical push maintain spatiotemporal spindle localization.Author summaryCells actively self-assemble a bipolar spindle to facilitate chromosomal segregation. Multiple MTOCs, on the outer nuclear envelope, cluster into a single SPB before spindle formation during semi-open mitosis of the budding yeast Cryptococcus neoformans. Eventually, the SPB duplicates and organizes the spindle to position it within the daughter bud near the septin ring during anaphase. In this work, we tested various computational models to match physiological phenomena in an attempt to find plausible mechanisms of MTOC clustering and spindle positioning in C. neoformans. Notably, we propose an MT ‘grow-and-catch’ model that relies on possible redundant mechanisms for timely MTOC clustering mediated by (a) minus end-directed motors that crosslink and slide anti-parallel cMTs from different MTOCs on the nuclear envelope and (b) a Bim1 mediated biased sliding of cMTs along the cell cortex toward the septin ring that pulls MTOCs in the presence of suppressed dynein activity. By combining an analytical model and stochastic MT dynamics simulations, we screened various MT-based forces to detect steady spindle positioning. By screening the outputs of various models, it is revealed that proper spindle positioning near the septin ring requires MT buckling from the cell cortex.
Subgroup II PAK-mediated phosphorylation regulates Ran activity during mitosis
