Dominant Negative Alleles of SEC10 Reveal Distinct Domains Involved in Secretion and Morphogenesis in Yeast

The accurate targeting of secretory vesicles to distinct sites on the plasma membrane is necessary to achieve polarized growth and to establish specialized domains at the surface of eukaryotic cells. Members of a protein complex required for exocytosis, the exocyst, have been localized to regions of active secretion in the budding yeast Saccharomyces cerevisiae where they may function to specify sites on the plasma membrane for vesicle docking and fusion. In this study we have addressed the function of one member of the exocyst complex, Sec10p. We have identified two functional domains of Sec10p that act in a dominant-negative manner to inhibit cell growth upon overexpression. Phenotypic and biochemical analysis of the dominant-negative mutants points to a bifunctional role for Sec10p. One domain, consisting of the amino-terminal two-thirds of Sec10p directly interacts with Sec15p, another exocyst component. Overexpression of this domain displaces the full-length Sec10 from the exocyst complex, resulting in a block in exocytosis and an accumulation of secretory vesicles. The carboxy-terminal domain of Sec10p does not interact with other members of the exocyst complex and expression of this domain does not cause a secretory defect. Rather, this mutant results in the formation of elongated cells, suggesting that the second domain of Sec10p is required for morphogenesis, perhaps regulating the reorientation of the secretory pathway from the tip of the emerging daughter cell toward the mother–daughter connection during cell cycle progression.

The Yeast Dynactin Complex Is Involved in Partitioning the Mitotic Spindle between Mother and Daughter Cells during Anaphase B

Although vertebrate cytoplasmic dynein can move to the minus ends of microtubules in vitro, its ability to translocate purified vesicles on microtubules depends on the presence of an accessory complex known as dynactin. We have cloned and characterized a novel gene, NIP100, which encodes the yeast homologue of the vertebrate dynactin complex protein p150glued. Like strains lacking the cytoplasmic dynein heavy chain Dyn1p or the centractin homologue Act5p, nip100Δ strains are viable but undergo a significant number of failed mitoses in which the mitotic spindle does not properly partition into the daughter cell. Analysis of spindle dynamics by time-lapse digital microscopy indicates that the precise role of Nip100p during anaphase is to promote the translocation of the partially elongated mitotic spindle through the bud neck. Consistent with the presence of a true dynactin complex in yeast, Nip100p exists in a stable complex with Act5p as well as Jnm1p, another protein required for proper spindle partitioning during anaphase. Moreover, genetic depletion experiments indicate that the binding of Nip100p to Act5p is dependent on the presence of Jnm1p. Finally, we find that a fusion of Nip100p to the green fluorescent protein localizes to the spindle poles throughout the cell cycle. Taken together, these results suggest that the yeast dynactin complex and cytoplasmic dynein together define a physiological pathway that is responsible for spindle translocation late in anaphase.

Farnesyltransferase inhibitors induce dramatic morphological changes of KNRK cells that are blocked by microtubule interfering agents

Farnesyltransferase inhibitors (FTIs) exhibit the remarkable ability to inhibit transformed phenotypes of a variety of human cancer cell lines and to block the growth of cancer cells in a number of animal model systems. In this paper, we report that the addition of FTI to v-K-ras- transformed NRK cells (KNRK) results in dramatic morphological changes. Within 24 h after the addition of FTI, the round morphology of KNRK cells was changed to an elongated (flattened and spread out) morphology resembling those of untransformed NRK cells. No morphological effects were seen when similar concentrations of FTI were added to NRK cells. Phalloidin staining showed that FTI treatment did not restore the disrupted actin cytoskeleton in KNRK cells. In contrast, FTI addition resulted in the appearance of extensive microtubule networks in KNRK cells. The addition of a low concentration (1.2 nM) of vincristine or vinblastine, agents that interfere with microtubule dynamics, blocked the FTI-induced morphological changes in KNRK cells. In contrast, cytochalasin B, which interferes with actin polymerization, did not block the morphological changes. The FTI-induced morphological changes were associated with a decrease in the percentage of cells in S-phase, and the addition of 1.2 nM vincristine did not have additional effects on cell cycle progression. A higher concentration (12 nM) of vincristine caused synergistic effect with FTI to enrich dramatically KNRK cells in G2/M phase. These results suggest that FTI affects cell morphology and that microtubule dynamics are involved in these processes.

Extending the Microtubule/Microfibril Paradigm1 : Cellulose Synthesis Is Required for Normal Cortical Microtubule Alignment in Elongating Cells

The cortical microtubule array provides spatial information to the cellulose-synthesizing machinery within the plasma membrane of elongating cells. Until now data indicated that information is transferred from organized cortical microtubules to the cellulose-synthesizing complex, which results in the deposition of ordered cellulosic walls. How cortical microtubules become aligned is unclear. The literature indicates that biophysical forces, transmitted by the organized cellulose component of the cell wall, provide a spatial cue to orient cortical microtubules. This hypothesis was tested on tobacco (Nicotiana tabacum L.) protoplasts and suspension-cultured cells treated with the cellulose synthesis inhibitor isoxaben. Isoxaben (0.25–2.5 μm) inhibited the synthesis of cellulose microfibrils (detected by staining with 1 μg mL−1 fluorescent dye and polarized birefringence), the cells failed to elongate, and the cortical microtubules failed to become organized. The affects of isoxaben were reversible, and after its removal microtubules reorganized and cells elongated. Isoxaben did not depolymerize microtubules in vivo or inhibit the polymerization of tubulin in vitro. These data are consistent with the hypothesis that cellulose microfibrils, and hence cell elongation, are involved in providing spatial cues for cortical microtubule organization. These results compel us to extend the microtubule/microfibril paradigm to include the bidirectional flow of information.