The fidelity of chromosome segregation in mitosis is essential for the accurate propagation of genetic information to daughter cells, and it is dependent upon timely, coordinated changes in the microtubule (MT) cytoskeleton. In Saccharomyces cerevisiae
, chromosome segregation is achieved in four microtubule-dependent steps. The first step is spindle assembly, which involves the migration of duplicated spindle pole bodies (SPBs) to form a bipolar spindle. This process requires the plus-end-directed activity of the kinesin-like motors of the BimC family, Cin8p and Kip1p, that cross-link and slide antiparallel microtubules of the spindle midzone at the G2
/M transition (27
). The second step is orientation of the mitotic spindle at the site of cytokinesis, which is mediated by cytoplasmic MTs and requires their transient, dynamic interactions with the cell cortex through dynein-dependent (51
) and dynein-independent (50
) pathways. The third step is chromosome movement along kinetochore MTs through their depolymerization in anaphase A (46
), and the fourth step is complete chromosome separation in the process of anaphase B spindle elongation. This last process is powered by Cin8p and Kip1p that cross-link and push apart polar MTs, by polymerization of the same MTs in the midzone driven by a poleward flux of tubulin subunits, and by a pulling force generated by dynein on cytoplasmic MTs (49
These changes in the MT architecture are dependent on tight regulation on different levels. Most directly, motor proteins and MT-associated proteins (MAPs) influence MT polymerization, stability, and dynamics (31
), thereby affecting processes the specific MT state facilitates. For example, the temperature-sensitive double motor cin8
Δ mutant is sensitive to various MT-destabilizing drugs. At elevated temperatures, spindle assembly at G2
/M is compromised (20
). Introduction of the tub2
allele, which hyperstabilizes MTs (28
), suppressed the sensitivities both to elevated temperatures and to MT-destabilizing drugs that are associated with this double mutant (39
), suggesting destabilized MTs in this background.
Another level of regulation is by upstream cyclin/Cdk complexes whose periodic expression drives specific cell cycle events. Late in G1
phase, the Cln3p-Cdc28p protein kinase complex activates two transcription complexes, the MBF complex (MBF for MluI cell cycle box [MCB]-binding factor) and the SBF complex (SBF for Swi4/6 cell cycle box [SCB]-binding factor), and these in turn promote the transcription of a number of genes important for budding and DNA synthesis (10
). At G2
/M, the Clb2p-Cdc28p complex represses the activity of SBF, returning the expression of SBF-regulated genes to low levels (1
). MT regulation by cyclin/cdk complexes may manifest indirectly: Clb2p-Cdc28p, for example, contributes to the stability or localization of motors posttranslationally (8
). It is therefore not surprising that certain Clb/Cdk mutants share the same phenotype exhibited by cin8
double mutants grown at the elevated temperature, being unable to assemble a bipolar spindle due to a failure to segregate duplicated SPBs (20
). These reports reinforce the ties that exist between upstream and downstream MT regulators.
In wild-type cells under normal conditions, phenotypes are often not manifested because other proteins or pathways act redundantly. A general example is KSS1
, a gene whose deletion in wild-type cells has no phenotypic consequence on the mating pathway and therefore was originally thought not to be involved in the process. It was later shown that it can fill in for another protein, Fus3p, which is functionally redundant with Kss1p, when Fus3p is not present (48
). Similarly, in this study we aimed to identify proteins that under normal conditions have no apparent role in MT stability, but when the stability of MTs is compromised, their involvement becomes essential.
We previously conducted a genetic screen to identify proteins that when overexpressed can correct the temperature sensitivity and the MT destabilization phenotype associated with cin8
). Here we describe pathways by which Fcp1p, one of the overexpression suppressors, increases MT stability in this background. The underlying mechanisms were elucidated through the identification of proteins that Fcp1p's effect is dependent upon or that can substitute for it altogether. On the basis of these results, we propose novel pathways that regulate the stability of microtubules.