Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae.

During mitosis in budding yeast the nucleus first moves to the mother-bud neck and then into the neck. Both movements depend on interactions of cytoplasmic microtubules with the cortex. We investigated the mechanism of these movements in living cells using video analysis of GFP-labeled microtubules in wild-type cells and in EB1 and Arp1 mutants, which are defective in the first and second steps, respectively. We found that nuclear movement to the neck is largely mediated by the capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization. Efficient microtubule interactions with the capture site require that microtubules be sufficiently long and dynamic to probe the cortex. In contrast, spindle movement into the neck is mediated by microtubule sliding along the bud cortex, which requires dynein and dynactin. Free microtubules can also slide along the cortex of both bud and mother. Capture/shrinkage of microtubule ends also contributes to nuclear movement into the neck and can serve as a backup mechanism to move the nucleus into the neck when microtubule sliding is impaired. Conversely, microtubule sliding can move the nucleus into the neck even when capture/shrinkage is impaired.

The surveillance mechanism of the spindle position checkpoint in yeast.

The spindle position checkpoint in Saccharomyces cerevisiae delays mitotic exit until the spindle has moved into the mother-bud neck, ensuring that each daughter cell inherits a nucleus. The small G protein Tem1p is critical in promoting mitotic exit and is concentrated at the spindle pole destined for the bud. The presumed nucleotide exchange factor for Tem1p, Lte1p, is concentrated in the bud. These findings suggested the hypothesis that movement of the spindle pole through the neck allows Tem1p to interact with Lte1p, promoting GTP loading of Tem1p and mitotic exit. However, we report that deletion of LTE1 had little effect on the timing of mitotic exit. We also examined several mutants in which some cells inappropriately exit mitosis even though the spindle is within the mother. In some of these cells, the spindle pole body did not interact with the bud or the neck before mitotic exit. Thus, some alternative mechanism must exist to coordinate mitotic exit with spindle position. In both wild-type and mutant cells, mitotic exit was preceded by loss of cytoplasmic microtubules from the neck. Thus, the spindle position checkpoint may monitor such interactions.

Role of yeast insulin-degrading enzyme homologs in propheromone processing and bud site selection.

The Saccharomyces cerevisiae AXL1 gene product Axl1p shares homology with the insulin-degrading enzyme family of endoproteases. Yeast axl1 mutants showed a defect in a-factor pheromone secretion, and a probable site of processing by Axl1p was identified within the a-factor precursor. In addition, Axl1p appears to function as a morphogenetic determinant for axial bud site selection. Amino acid substitutions within the presumptive active site of Axl1p caused defects in propheromone processing but failed to perturb bud site selection. Thus, Axl1p has been shown to participate in the dual regulation of distinct signaling pathways, and a member of the insulinase family has been implicated in propeptide processing.

Bni1p, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis.

The Saccharomyces cerevisiae BNI1 gene product (Bni1p) is a member of the formin family of proteins, which participate in cell polarization, cytokinesis, and vertebrate limb formation. During mating pheromone response, bni1 mutants showed defects both in polarized morphogenesis and in reorganization of the underlying actin cytoskeleton. In two-hybrid experiments, Bni1p formed complexes with the activated form of the Rho-related guanosine triphosphatase Cdc42p, with actin, and with two actin-associated proteins, profilin and Bud6p (Aip3p). Both Bni1p and Bud6p (like Cdc42p and actin) localized to the tips of mating projections. Bni1p may function as a Cdc42p target that links the pheromone response pathway to the actin cytoskeleton.

TOR signaling regulates microtubule structure and function.

The functional diversity and structural heterogeneity of microtubules are largely determined by microtubule-associated proteins (MAPs) [1] [2]. Bik1p (bilateral karyogamy defect protein) is one of the MAPs required for microtubule assembly, stability and function in cell processes such as karyogamy and nuclear migration and positioning in the yeast Saccharomyces cerevisiae [3]. The macrocyclic immunosuppressive antibiotic rapamycin, complexed with its binding protein FKBP12, binds to and inhibits the target of rapamycin protein (TOR) in yeast [4] [5]. We report here that TOR physically interacts with Bik1p, the yeast homolog of human CLIP-170/Restin [6] [7]. Inhibition of TOR by rapamycin significantly affects microtubule assembly, elongation and stability. This function of TOR is independent of new protein synthesis. Rapamycin also causes defects in spindle orientation, nuclear movement and positioning, karyogamy and chromosomal stability, defects also found in the bikDelta mutant. Our data suggest a role for TOR signaling in regulating microtubule stability and function, possibly through Bik1p.

Regulation of expression of the cyclic nucleotide phosphodiesterase gene in phosphodiesterase inhibitor-negative mutants of Dictyostelium discoideum.

During early development of Dictyostelium discoideum, the enzyme cyclic nucleotide phosphodiesterase (PD) is produced at a low rate during the period its specific inhibitor (PDI) is being synthesized. In addition, PD gene expression is derepressed in the aggregation-deficient (Agg-), Pdi- mutant HC35. These observations suggest that the PDI might function to regulate PD gene expression, as well as modulate its activity. To explore this idea further, five new Agg-, Pdi- mutants were isolated and analyzed. All of the mutants produced high PD activity and overexpressed PD mRNA; four exhibited elevated levels of the 2400-nucleotide aggregation transcript and one overproduced the 1900-nucleotide vegetative transcript. In contrast, PD transcripts were not elevated in two Agg-, Pdi+ mutants. To determine if PDI production regulates PD expression, HC35 cells were transformed with plasmids carrying the PDI structural gene under the control of either the vegetative or aggregative PD promoter. Neither expression of PDI by the transformants nor addition of partially purified PDI to HC35 cells affected PD transcription. These results suggest that PD overexpression in the Pdi- mutants is not a direct consequence of the inability of these cells to produce inhibitor.

Involvement of intracellular calcium in protein secretion in Dictyostelium discoideum.

We reported previously that Ca2+ depletion of Dictyostelium discoideum cells severely inhibits extracellular cyclic nucleotide phosphodiesterase (PD) synthesis at a post-transcriptional step. In this study, further experiments were performed to learn more about the nature of this phenomenon. Examination of the polysomal distribution of PD transcripts in control cells and in cells depleted of Ca2+ by incubation with EGTA and A23187 (EA) suggested that inhibition of PD production does not involve translational control. Kinetic analysis of this inhibitory process revealed that soluble, intracellular PD activity, synthesized from either the 2.4 or 1.9 kb PD mRNA, decreased very rapidly upon addition of EA. Furthermore, this decrease in activity was accompanied by the preferential loss of PD-related polypeptides, indicating a proteolytic event. EA-induced PD degradation required cellular energy and concomitant protein synthesis but was unaffected by most of the lysosomotropic agents tested. Therefore, PD proteolysis might not occur in the lysosome. In cell fractionation experiments, the EA-sensitive, intracellular PD activity comigrated with a rough ER marker in Percoll/KCl gradients. In addition to its effect on the PD, EA were also observed to inhibit production and rapidly lower the intracellular levels of another secreted glycoprotein, the PD inhibitor. Together, these results suggest that depletion of some intracellular Ca2+ store(s) in Dictyostelium, possibly the ER, disrupts the normal function of the secretory pathway, resulting in selective degradation of certain proteins.

A cytokinesis checkpoint requiring the yeast homologue of an APC-binding protein.

Checkpoint controls ensure that events of the cell-division cycle are completed with fidelity and in the correct order. In budding yeast with a mutation in the motor protein dynein, the mitotic spindle is often misaligned and therefore slow to enter the neck between mother cell and budding daughter cell. When this occurs, cytokinesis (division of the cytoplasm into two) is delayed until the spindle is properly positioned. Here we describe mutations that abolish this delay, indicating the existence of a new checkpoint mechanism. One mutation lies in the gene encoding the yeast homologue of EB1, a human protein that binds the adenomatous polyposis coli (APC) protein, a tumour suppressor. EB1 is located on microtubules of the mitotic spindle and is important in spindle assembly. EB1 may therefore, by associating with microtubules, contribute to the sensor mechanism that activates the checkpoint. Another mutation affects Stt4, a phosphatidylinositol-4-OH kinase. Cold temperature is an environmental stimulus that causes misalignment of the mitotic spindle in yeast and appears to activate this checkpoint mechanism.