In budding yeast, spindle disassembly is achieved by arrest of spindle elongation, ipMT depolymerization, and disengagement of spindle halves; these subprocesses are largely governed by the Aurora B kinase, kinesin-8, and the APC. This study reveals, surprisingly, that these subprocesses are not essential for spindle breakdown, mitotic exit, and cell division, but rather for spindle assembly in the subsequent cell cycle of the daughter cells. In this paper, we propose that complete spindle disassembly is required to regenerate the pool of tubulin that is to be incorporated into growing MTs to permit efficient spindle assembly in daughter cells.
Previously we showed that cytokinetic ring contraction can break the spindle and that cells exit mitosis and continue with cell division when the normal mechanisms of spindle disassembly are impaired (Woodruff et al., 2010
). However, in the subsequent cell cycle of the daughter cells, the spindle assembles poorly and often collapses or falls apart under these conditions. Four observations suggest these spindles are not completely dysfunctional, but simply lack the necessary pool of assembly-competent tubulin to sustain spindle MT growth. First, the total amount of tubulin incorporated into spindles was ~2.5-fold lower in td-kip3 doc1Δ
cells as compared with wild-type cells. In addition, we found that aMTs were ~1.8-fold shorter in td-kip3 doc1Δ
cells versus wild-type cells (Figure S4), suggesting the overall pool of assembly-competent tubulin had been reduced after a round of defective spindle disassembly. Second, during the assembly of these spindles, duplication and initial separation of the spindle MT-nucleating centers, the SPBs, proceeded normally, suggesting SPB biogenesis was not affected. Third, spindles in td-kip3 doc1Δ
cells recruited key midzone-stabilizing proteins (e.g., Cin8, Ase1). Fourth, malformed spindles displayed the capacity to form stable bipolar structures if provided with free tubulin dimers expressed exogenously from a plasmid source. In light of these observations, the fact that a nocodazole pulse rescues spindle assembly in td-kip3 doc1Δ
daughter cells following dysfunctional spindle disassembly suggests MTs are not being efficiently depolymerized to generate assembly-competent tubulin dimers, or any assembly-competent oligomeric complex of tubulin, when spindle-disassembly mechanisms are inhibited. The appearance of small, persistent spindle remnants after a defective round of spindle disassembly supports this conclusion. Although the frequency of spindle remnants (8% of cells) did not match the frequency of malformed spindles (40% of cells), a substantial population of small spindle remnants may have escaped our detection due to resolution limitations of light microscopy. However, the fact that the nocodazole-pulse and ectopic tubulin expression experiments did not completely reduce the frequency of malformed spindles to wild-type levels means we cannot conclude that the availability of free tubulin is the sole solution to the problem. There may be other mechanism(s) we have yet to discover, such as improper modification of the pool of free tubulin to make it assembly competent, for example. Moreover, we cannot exclude the possibility that a minority of the spindle defects seen in td-kip3 doc1Δ
cells resulted from problems unrelated to defective spindle disassembly. Considering that td-kip3 doc1Δ
cells incubated at the permissive temperature displayed a low frequency of malformed spindles (~9%) and chromosome missegregation (1.5-fold higher than wild-type), it is possible that misexpression of genes vital for spindle assembly may have contributed to the malformed spindle phenotype.
In addition to addressing the importance of spindle disassembly for cell proliferation, our work touches on whether multimeric tubulin assemblies or only tubulin dimers can incorporate into the growing MT polymer. Both in vitro and in vivo studies suggest that actin filaments can grow by incorporating both actin monomers and oligomers (Kawamura and Maruyama, 1970
; Murphy et al., 1988
; Okreglak and Drubin, 2010
). However, it is unclear whether microtubules share this property. It has been demonstrated in vitro that tubulin oligomers can nucleate MTs (Caudron et al., 2002
), but whether these oligomers can incorporate into a growing MT sheet remains to be seen. Whether tubulin oligomers per se influence MT nucleation or dynamics in vivo is also unclear. Our work suggests that, in vivo, growth of spindle MTs is sensitive to the oligomeric state of tubulin. It is possible that spindle MTs favor incorporation of tubulin dimers, rather than more complex multimeric assemblies. This proposal seems logical when considering that the growing end of the MT is a curved sheet that eventually zips up to generate a tube (Simon and Salmon, 1990
; Chretien et al., 1995
). This three-dimensional architecture might favor incorporation of smaller tubulin assemblies (e.g., dimers) and restrict incorporation of higher-order tubulin assemblies that, most likely, do not match the geometry of the growing sheet.
In all eukaryotes, spindle assembly depends on many MT-stabilizing proteins and the availability of assembly-competent tubulin to permit MT polymerization (Goshima et al., 2005
; Srayko et al., 2005
). Similar to what we and others (Lacefield et al., 2006
) have observed for budding yeast, RNA interference–mediated knockdown of the tubulin chaperone prefoldin (PFD-3) decreased α-tubulin levels in the Caenorhabditis elegans
embryo, resulting in decreased MT polymerization and short metaphase spindles (Lundin et al., 2008
). In the future, it will be important to test whether spindle disassembly serves an essential role in tubulin dimer regeneration and/or spindle assembly in C. elegans
and other metazoans.