Chromosomal attachments set length and microtubule number in the Saccharomyces cerevisiae mitotic spindle.
Bottom Line: The length of the mitotic spindle varies among different cell types.A simple model for spindle length regulation requires balancing two forces: pulling, due to micro-tubules that attach to the chromosomes at their kinetochores, and pushing, due to interactions between microtubules that emanate from opposite spindle poles.In the budding yeast Saccharomyces cerevisiae, we show that spindle length scales with kinetochore number, increasing when kinetochores are inactivated and shortening on addition of synthetic or natural kinetochores, showing that kinetochore-microtubule interactions generate an inward force to balance forces that elongate the spindle.
Affiliation: Molecular and Cellular Biology Department, Harvard University, Cambridge, MA 02138 FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138.Show MeSH
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Mentions: Wild-type, plasmid-free cells contained an average of 51 ± 1 microtubules, within the SD of microtubules previously observed (Winey et al., 1995). Cells containing an average of 23 plasmids as determined by qPCR contained significantly more microtubules, with an average of 74 ± 7 microtubules (p = 0.006, Student's t test; Figure 6, A and B). Similarly, cells containing an average of 11 plasmids contained an average of 67 ± 5 microtubules, a statistically greater number than that of wild type (p = 0.004, Student's t test) and intermediate between that of wild type and the cells with 23 plasmids (Figure 6, A and B; see Supplemental Fig6video1–Fig6video3 for representative 3D movies of spindles). Because budding yeast chromosomes do not condense to structures that are visible in EM, we used programs described in Winey et al. (1995) to identify the central, “core bundle” (interpolar microtubules) and presumptive kinetochore microtubules. The interpolar microtubules of the central spindle are defined as microtubules that are separated by up to 45 nm for lengths of ≥300 nm; they are shown in yellow in Figure 6A and form an obvious central spindle. Microtubules that do not meet this criterion are displayed in magenta and green and represent the putative kinetochore microtubules from each pole; the difference in microtubule number in both 11- and 23-plasmid-containing cells comes from additional kinetochore microtubules (p = 0.001 and 0.005 respectively, Student's t test). Figure 6A, bottom, shows only the putative kinetochore microtubules, to highlight the difference in number and spindle organization. Spindles with 11 plasmids have 58 ± 5 kinetochore microtubules, and those with 23 plasmids have 62 ± 8, compared with 38 ± 3 for wild type. The number of interpolar microtubules (yellow microtubules) is not significantly different between wild-type and plasmid-containing spindles (Figure 6B). Adding kinetochores increases the size of the spindle pole body's central plaque. Wild-type spindle pole bodies had a mean diameter of 107 ± 10 nm (n = 7). Spindle pole bodies from cells containing 11 plasmids (average diameter of 144 ± 27 nm, n = 7, p = 0.009, Student's t test) and 23 plasmids (average diameter of 163.4 ± 32.8, n = 8, p = 0.001, Student's t test) were both significantly larger than those from wild-type cells (Figure 6C). Figure 6D shows an example, indicating the width of spindle pole bodies, microtubules from opposite poles, and the nuclear envelope.
Affiliation: Molecular and Cellular Biology Department, Harvard University, Cambridge, MA 02138 FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138.