The cortical protein Num1p is essential for dynein-dependent interactions of microtubules with the cortex.

In budding yeast, the mitotic spindle moves into the neck between the mother and bud via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud. How dynein and microtubules interact with the cortex is unknown. We found that cells lacking Num1p failed to exhibit dynein-dependent microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation. Num1p localized to the bud cortex, and that localization was independent of microtubules, dynein, or dynactin. These data are consistent with Num1p being an essential element of the cortical attachment mechanism for dynein-dependent sliding of microtubules in the bud.

Dynein-dependent movements of the mitotic spindle in Saccharomyces cerevisiae Do not require filamentous actin.

In budding yeast, the mitotic spindle is positioned in the neck between the mother and the bud so that both cells inherit one nucleus. The movement of the mitotic spindle into the neck can be divided into two phases: (1) Kip3p-dependent movement of the nucleus to the neck and alignment of the short spindle, followed by (2) dynein-dependent movement of the spindle into the neck and oscillation of the elongating spindle within the neck. Actin has been hypothesized to be involved in all these movements. To test this hypothesis, we disrupted the actin cytoskeleton with the use of mutations and latrunculin A (latrunculin). We assayed nuclear segregation in synchronized cell populations and observed spindle movements in individual living cells. In synchronized cell populations, no actin cytoskeletal mutant segregated nuclei as poorly as cells lacking dynein function. Furthermore, nuclei segregated efficiently in latrunculin-treated cells. Individual living cell analysis revealed that the preanaphase spindle was mispositioned and misaligned in latrunculin-treated cells and that astral microtubules were misoriented, confirming a role for filamentous actin in the early, Kip3p-dependent phase of spindle positioning. Surprisingly, mispositioned and misaligned mitotic spindles moved into the neck in the absence of filamentous actin, albeit less efficiently. Finally, dynein-dependent sliding of astral microtubules along the cortex and oscillation of the elongating mitotic spindle in the neck occurred in the absence of filamentous actin.

The role of Saccharomyces cerevisiae coronin in the actin and microtubule cytoskeletons.

Coronin was originally identified as a cortical protein associated with the actin cytoskeleton in Dictyostelium [1]. More recent studies have revealed that coronin is involved in actin-based motility, cytokinesis and phagocytosis [2,3]. Here, we describe the identification of a single homolog of coronin in Saccharomyces cerevisiae, which we show localizes to cortical actin patches in an actin-dependent manner. Unlike Dictyostelium mutants that lack coronin, yeast strains lacking coronin had no detectable defects in actin-based processes. This may reflect differences in the functions of the actin cytoskeleton in these two organisms. Previous studies have shown that cortical actin may mediate astral microtubule-based movements of the mitotic spindle in S. cerevisiae [4,5] and that, during mitosis in Dictyostelium, the regions of the cell cortex that overlap with astral microtubules become enriched in actin and coronin [6]. We therefore examined whether yeast lacking coronin had defects in the microtubule cytoskeleton. The mutant strains had increased sensitivity to the microtubule-destabilizing drug benomyl and an increased number of large-budded cells with short spindles. Further examination of microtubule-related processes, including spindle formation, migration of the mitotic spindle to the bud neck, spindle elongation, and translocation of the elongating spindle through the bud neck, failed to reveal any defects in the coronin mutant. Taken together, these results suggest that S. cerevisiae coronin is a component of the actin cytoskeleton that may interact with the microtubule cytoskeleton.

Characterization of changes in F-actin during maturation of starfish oocytes.

Major morphological and mechanical changes occur in the starfish oocyte during maturation. Measurements made by quantitative fluorescence microscopy of fixed specimens stained with saturating levels of rhodamine-phalloidin demonstrated that changes in the level of F-actin in intact oocytes, in the endoplasm, and in the cortex may contribute to these changes. The level of F-actin increased transiently after exposure of oocytes to the maturation inducing hormone, 1-methyladenine (1-MA). This increase correlated with the formation of spikes on the cell surface. The level of F-actin decreased at the time of germinal vesicle breakdown (GVBD), which may account for the decrease in stiffness that occurs at this time. No increase in the level of F-actin was observed during formation of polar bodies, suggesting the existence of a secondary mechanism affecting oocyte stiffness. The changes in the amount of F-actin during oocyte maturation were largest in the cortex. The data also suggested that there are two distinct populations of cortical actin that are regulated both spatially and temporally; these are the F-actin in spikes and nonspike cortical F-actin. Changes in either or both of these populations of cortical actin were induced independently of GVBD by short exposures to 1-MA, induction of maturation with dithiothreitol, and pretreatment of immature oocytes with forskolin before adding 1-MA. Stabilization of F-actin by microinjection of phalloidin had no effect on GVBD. These results suggest that polymerization and depolymerization of actin during maturation are responsible for morphological and mechanical changes in the oocyte. In addition, the data suggest that the regulation of actin polymerization and depolymerization can be dissociated from GVBD.

Relative changes in F-actin during the first cell cycle: evidence for two distinct pools of F-actin in the sea urchin egg.

The cortical actin cytoskeleton undergoes dramatic rearrangements during fertilization of sea urchin eggs. To characterize these changes further, we quantified the relative changes in filamentous actin (F-actin) during fertilization and the first cell cycle in both intact eggs and in isolated cortices by quantitative fluorescence microscopy. The level of F-actin in the intact egg decreased after fertilization and continued to decrease throughout the first cell cycle. By 60 min after fertilization, the level of F-actin had decreased to 50% of the unfertilized sea urchin egg. By cytokinesis, the level of F-actin had decreased to 30% of the unfertilized egg. After completion of cell division, individual blastomeres had 10% of the F-actin in the unfertilized egg. In contrast, there was an increase in cortical F-actin to 370% of the level in the unfertilized egg after fertilization. This increase corresponded to the formation of microvilli. There was little change in the level of cortical F-actin during the first cell cycle. We draw parallels to other systems that increase the amount of F-actin in the Triton-insoluble cytoskeleton by recruiting actin from a Triton-soluble pool of F-actin.