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.Electron microscopy shows that manipulating kinetochore number alters the number of spindle microtubules: adding extra kinetochores increases the number of spindle microtubules, suggesting kinetochore-based regulation of microtubule number.
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: Does kinetochore inactivation lead to spindle elongation? We used the ndc10-1 mutant to produce cells with no chromosomal attachments and two manipulations to control the progress of the cell cycle: treating cells with a mating pheromone, α-factor, to arrest them in G1, and removing Cdc20, an activator of the anaphase-promoting complex (APC), to arrest cells in metaphase (Hartwell et al., 1973; Hartwell and Smith, 1985). Wild-type and ndc10-1 cells were grown to log phase at 23°C, synchronized in G1 with α-factor, and released into a Cdc20 depletion–induced metaphase arrest at 37°C (Figure 2A). Spindle pole bodies were labeled by fusing a red fluorescent protein (RFP) variant to a spindle pole body component (SPC42-mCherry), and chromosome XV was labeled with green fluorescent protein (GFP) by inserting a Lac operator (LacO) array ∼200 base pairs downstream of CEN15 (see Supplemental Table S1 for exact position; Shonn et al., 2000) and expressing a GFP-Lac repressor fusion (GFP-LACI; Straight et al., 1996). Spindle length was measured as the three-dimensional (3D) distance between the two spindle poles and reported as the average length of measured spindles; error bars are SDs of average spindle length over all trials. Figure 2B shows examples of fluorescent spindles.
Affiliation: Molecular and Cellular Biology Department, Harvard University, Cambridge, MA 02138 FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138.