![]() ![]() One z-axis plane fluorescence image wasĬnm67Δ cells. NUCLEAR TIME LAPSE VIDEO MOVIEAcquisition interval, 1 min movie speed, 10 frames per Two delayed mitoses can be seen in the second half of the time-lapse Movie 1: Nuclearĭynamics as seen in Hhf2-GFP–labeled wild-type cells followed for >10 (144′) First distal budĮmergence of the daughter cell. To that of the mother indicates completion of cell separation. (106′) A sudden change of the orientation of the daughter cell relative Mother and by a constriction of the bud neck (indicated with arrows). The longitudinal axis of the daughter cell relative to that of the (92′) Onset ofĬytokinesis can be observed by a slight change of the orientation of Movements indicating that the spindle is still intact. This time point the separated chromosomal masses show slow coordinated (69′) Final separation of chromosomal masses. This can be seen by the fast change in position of Typically accompanied by bending and rapid oscillations of the nucleus (62′–67′) The labeledĬhromosomal material transforms into an hourglass shape. Orientation, elongation, and penetration. Nucleus becomes bar shaped, elongates along the mother–bud axis, and (2′–57′) During the positioning phase the nucleus (0′) At onset of bud emergence the nucleus is Hhf2-GFP–labeled wild-type cells observed by in vivo time-lapse Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs. However, successful nuclear segregation in cnm67Delta cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. ![]() We postulate that in cnm67Delta cells Spc72-gamma-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. Concomitantly, Spc72 protein, the cytoplasmic anchor for the gamma-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. We monitored dynamics of green fluorescent protein-labeled nuclei and microtubules over several cell cycles. ![]() Saccharomyces cerevisiae cnm67Delta cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. ![]()
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