Mechanics of MTOC clustering and spindle positioning in budding yeast Cryptococcus neoformans

Abstract

The mitotic spindle formation in pathogenic budding yeast, Cryptococcus neoformans, depends on multitudes of intertwined interactions primarily between kinetochores, microtubules (MT), spindle pole bodies (SPBs) and various molecular motors. Prior to spindle formation microtubule organizing centers (MTOCs), embedded on the outer nuclear envelope (NE), coalesce into a single SPB.We propose a 'grow-and-catch' model, in which cytoplasmic MTs (cMTs) nucleated by MTOCs grow and catch each other, to facilitate MTOC clustering. Our quantitative modeling analysis supported by experiments, for the first time, identifies multiple redundant mechanisms mediated by cMT-cell cortex interactions via dynein and Bim1, act in synchrony for timely clustering of MTOCs. Implementing a similar stochastic model of 'grow-and-catch' kinetochores, here we demonstrate that pre-clustered kinetochores and highly biased MTs are a pre-requisite for timely capture of duplicated kinetochores. Further, in the absence of an error correction mechanism, the mitotic division culminates in a biased and asymmetric distribution of the nuclear mass. The model predicts that either a marginal delay in MT nucleation from the daughter SPB or an enhanced MT nucleation from the mother SPB can independently account for the asymmetry as observed in the experiment.

Cryptococcus neoformansSlu7 ensures nuclear positioning during mitotic progression through RNA splicing

“…Recent studies in C. neoformans have shown that the spatial distribution of dynein and Bim1 plays an essential role in nuclear migration (Varshney et al, 2019). As the cell cycle progresses, a localized dynein patch is formed in the daughter cortex along the axis of symmetry, which pulls the nucleus inside the daughter bud (Chatterjee et al, 2021). In our transcriptomic data, we observe that BIM1 transcript was at higher levels in slu7kd when compared wildtype while DYN1 transcript levels were unaffected.…”

“…Recent biochemical and cryo–EM studies have reported the role of LIS1/Pac1 in dynein regulation in S. cerevisiae (Markus et al, 2011; Huang et al, 2012; DeSantis et al, 2017). Studies in Cryptococcus neoformans have demonstrated the mechanics of nuclear migration and spindle positioning by time–lapse live cell imaging and quantitative modelling (Kozubowski et al, 2013; Varshney et al, 2019; Chatterjee et al, 2021). Transcriptional profiling of C. neoformans cell cycle revealed periodic changes in the expression of 1134 out of 6182 transcripts tested (Kelliher et al, 2016).…”