Fig. 3

Cut7-GFP is found at the initial contact points of MTs. a Reassembly time as a function of the distance between the SPBs for cut7.24^ts cells (strain CF.391) at non-permissive temperature (37 °C), n = 34 cells. Pink data points denote cells in which the spindle was not reassembled within 10 min. b Time-lapse images of a cut7.24^ts cell expressing mCherry-tubulin (strain CF.391) at non-permissive temperature (37 °C), in which the spindle did not reassemble. c Spindle reassembly in a cell expressing Cut7-3GFP (green), mCherry-tubulin (magenta), and Sid4-mCherry (magenta; strain LW042). Merged time-lapse images (left column) and separate channels (central and right column, both in gray scale) are shown. Note the accumulation of Cut7 at the site of MT contact (arrowhead). d Signal intensity profiles of mCherry-tubulin (left) and Cut7-3GFP (right) measured along the MT contour extended into the cytoplasm (see example in the inset on the left), at times in min:s noted in the legend on the right (strain LW042). The arrowhead marks the peak of Cut7-3GFP signal in the MT contact region. The intensity profiles were measured on the images shown in panel c. e Spindle reassembly in a cell expressing GFP-tubulin (green) and Ndc80-tdTomato (a kinetochore marker, magenta; strain AH01). Note that spindle reassembly including MT alignment occurs without kinetochores being present close to the MT contact point. In these experiments, cold treatment was performed as described in [29], and images were acquired by using a DeltaVision RT system. In b, c, and e, images are maximum-intensity projections, time is given in min:s; scale bars, 1 μm