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Supplemental videos for:
Finley, K. and Berman, J. 2005. Microtubules in Candida albicans hyphae drive nuclear dynamics and connect cell cycle progression to morphogenesis. Eukaryotic Cell, 4(10):1697-1711
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Dynamics of Nop1 nuclear migration in C. albicans hyphae. A nucleus migrates to and divides across the presumptive septum (presumptum). Both nuclei move rapidly away from each other until the mother nucleus pauses near the hyphal neck, at which time the movement of both nuclei is negligible. The mother nucleus then migrates slowly into the basal cell. Also shown here, but not addressed in the paper, is the post-mitotic behavior of the daughter nucleus. It moves forward in the germ tube prior to mitosis. After separation, dNop1 migrates back toward the primary septum, while the granddaughter nucleus migrates forward out of the field of view. Strain, YJB6449 (Nop1-YFP); objective, heated (37°C) 60X; illumination, single DIC and 4-image YFP Z-stack (1µm steps) acquired at 2.5-minute intervals; playback, 10 frames per second (fps, 1500x real time). Bar, 10µm. Time, hh:mm.
Short spindles migrate to and elongate across presumpta. As the hypha elongates, the SPB duplicates and then migrates into the germ tube. The duplicated SPBs adopt a short spindle conformation prior to reaching the presumptum. Spindle elongation then occurs across the presumptum. mSPB is positioned at the hyphal neck when the spindle disassembles. Strain, YJB8952 (Tub2-GFP); objective, heated (37°C) 60X; illumination, single DIC and 4-image GFP Z-stack (1µm steps) acquired at 2.5-minute intervals; playback, 10 fps (1500x real time). Bar, 10µm. Time, hh:mm.
Nuclear dynamics with respect to the true septin ring. Nop1 enters the germ tube only after Cdc3 localizes to the presumptum. Cdc3 persists at the presumptum until well after mitosis. Strain, YJB8860 (Nop1-YFP, Cdc3-GFP); objective, heated (37°C) 60X; illumination, single DIC and 4-image GFP Z-stack (1µm steps) acquired at 5-minute intervals; playback, 5 fps (1500x real time). Bar, 10µm. Time, hh:mm.
Spindle dynamics with respect to the true septin ring. Cdc3 localizes to the presumptum just after the SPB duplicates. Strain, YJB8961 (Tub2-GFP, Cdc3-YFP); objective, heated (37°C) 60X; illumination, single DIC and 4-image GFP Z-stack (1µm steps) acquired at 5-minute intervals; playback, 5 fps (1500x real time). Bar, 10µm. Time, hh:mm.
Short spindles migrate to the presumptum by repetitive MT sliding events. Spindle displacements coincide with lateral MT interactions with the cortex. Displacements also occur at the same time as and in the same direction as MT growth. Strain, YJB8895 (Tub2-GFP); Objective, heated (37°C) 60X; illumination, single GFP image acquired at 3-second intervals; DIC image acquired at the beginning of observation; playback, 10 fps (30x real time). Bar, 10µm. Time, mm:ss.
A second example of repetitive MT sliding events displacing a short spindle.
Anaphase B contributes more to nuclear movement than anaphase A. Nop1 position is clearly a function of SPB position and divides while the spindle is relatively short with respect to overall spindle length. Both mNop1 and dNop1 remain closely associated with the spindle poles throughout mitosis, suggesting that sister chromatids lose cohesion early in anaphase. Strain, YJB8548 (Nop1-CFP, Tub2-GFP); objective, unheated 60X; illumination, single DIC, CFP and YFP images acquired at 30-second intervals; playback, 10 fps (300x real time). Bar, 10µm. Time, mm:ss.
SPB-bound MTs grow and shrink at their distal ends. Fluorescent speckles are evident along the length of the MT. This movie includes the distal end of the hypha and the MT while it is within that region. The positions of the speckles are fixed with respect to the SPB and the distal ends. Strain, YJB8952 (Tub2-GFP); Objective, unheated 100X; illumination, single GFP image acquired at 3-second intervals; DIC image acquired at the beginning of observation; playback, 10 fps (30x real time). Bar, 10µm. Time, mm:ss.
Free MTs are regulated during the cell cycle. Free MTs are prevalent in young hyphae with non-migratory nuclei (Fig 9A.mov and Fig 9B.mov), but are less prevalent after forward migration begins (Fig 9C.mov) and throughout mitosis (Fig 9D.mov). As spindles disassemble, the number of free MTs increases dramatically (Fig 9E.mov). Strain, YJB8952 (Tub2-GFP); Objective, unheated 100X; illumination, single GFP image acquired at 1 (Fig 8A, B) or 3-second (Fig 8C-E) intervals; DIC image acquired at the beginning of observation; playback, 10 fps (10x or 30x real time, respectively). Bars, 10µm. Time, mm:ss.
Free MTs are also cell cycle regulated in yeast cells. From left to right: a single unbudded cell, cells with short spindles, a spindle disassembles in a mother-daughter pair. Strain, YJB8952 (Tub2-GFP); Objective, heated (30°C) 60X; illumination, single GFP image acquired at 3-second intervals; playback, 10 fps (30x real time). Bar, 10µm. Time, mm:ss.