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. 2015 Nov 5;26(22):3954–3965. doi: 10.1091/mbc.E15-04-0236

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© 2015 Sutradhar, Yadav, Sridhar, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — Dependence of nuclear migration on the number of cMTs and dynein activity in ascomycetes and basidiomycetes. (A) In silico measurements of the neck to spindle distance upon altering cMT numbers per cell during mitosis. We observed that, for a fixed density of cortical dynein, a higher number of cMTs leads to a deeper penetration of the spindle into the daughter cell (bud). With four cMTs, the observed spindle distance from the neck is close to −1.0 μm, which is similar to that observed in ascomycetes. As we increase the number of cMTs, the spindle moves closer to the neck, and when there are six cMTs, the spindle just crosses over into the daughter cell. The spindle is strongly pulled and moved deep into the daughter cell when the number of cMTs is eight or more, resembling what is observed in experiments. (B) Mean distance of the spindle from neck is plotted as observed in simulation (n = 100) and experiments (n = 30) for both C. albicans and C. neoformans. Experimental measurements were carried out in a strain that had MTOCs tagged with GFP. Differential interference contrast was used as a reference point for calculating spindle mid to neck distance. The mean distance of the spindle from the neck in C. albicans is estimated as −1.0 ± 0.22 μm in experiments, while our in silico model prediction with four cMTs turns out to be −1.0 ± 0.02 μm. On the other hand, the spindle to neck distance in C. neoformans with eight or more cMTs is found to be +0.84 ± 0.23 μm and +1.0 ± 0.05 μm from experiments and in silico measurements, respectively. (C) The spindle migration can also be affected by an alternative pathway involving cortical dyneins. An increase in the cortical dynein density for a fixed number of cMTs results in similar nuclear dynamics obtained previously by altering the cMT number. SEM is shown in red bars. (D and E) C. albicans (YJB12856) and C. neoformans (CNVY109) strains expressing Tub1-GFP were used to monitor and estimate cMTs. To rule out false positives in counting, we used high-resolution three-dimensionally rendered images to trace cMTs before estimation of their numbers. The cMTs in all stacks were taken into consideration. Two different views over the y-axis (0°, top panels; 160°, bottom panels) of the three-dimensionally rendered images are shown to improve the visibility of cMTs that may be masked by others in a given orientation. Scale bar: 2 μm. (F) The cMTs were counted in a large number of cells of C. albicans and C. neoformans. These values were plotted, and the calculated mean of cMTs per cell in each case is represented by a gray line. C. neoformans was found to contain six to 15 cMTs per cell, with an average of 8.95, while C. albicans was found to contain three to five cMTs per cell, with an average of 3.9.