Abstract. The three dimensional organization of microtubules in mitotic spindles of the yeast Saccharomyces cerevisiae has been determined by computer-aided reconstruction from electron micrographs of serially cross-sectioned spindles. Fifteen spindles ranging in length from 0.6-9.4 Ixm have been analyzed. Ordered microtubule packing is absent in spindles up to 0.8 Ixm, but the total number of microtubules is sufficient to allow one microtubule per kinetochore with a few additional microtubules that may form an interpolar spindle. An obvious bundle of about eight interpolar microtubules was found in spindles 1.3-1.6 p~m long, and we suggest that the ~32 remaining microtubules act as kinetochore fibers. The relative lengths of the microtubules in these spindles suggest that they may be in an early stage of anaphase, even though these spindles are all situated in the mother cell, not in the isthmus between mother and bud. None of the reconstructed spindles exhibited the uniform populations of kinetochore microtubules characteristic of metaphase. Long spindles (2.7-9.4 p,m), presumably in anaphase B, contained short remnants of a few presumed kinetochore microtubules clustered near the poles and a few long microtubules extending from each pole toward the spindle midplane, where they interdigitated with their counterparts from the other pole. Interpretation of these reconstructed spindles offers some insights into the mechanisms of mitosis in this yeast. THE structure of the mitotic spindle and its function have been studied in a wide variety of organisms, yielding a few global observations about spindle structure. In general, spindles are organized from two spindle poles, each of which nucleates several classes of microtubules. These classes include astral, kinetochore, and interpolar spindle microtubules. The astral microtubules are not part of the spindle per se, but can be involved in the orientation, and perhaps the elongation of the spindle. Kinetochore microtubules connect the chromosomes to the spindle poles and are involved in chromosome movements. The interpolar spindle microtubules extend from each spindle pole, interdigitate with their counterparts from the other pole, and are involved in separating the poles with their attached chromosomes during anaphase B. This generic view of spindle organization is thought by many to hold true for most spindles, including

Nucleus-vacuole (NV) junctions in Saccharomyces cerevisiae are formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p in the nuclear envelope. Herein, we report that NV junctions in yeast promote piecemeal microautophagy of the nucleus (PMN). During PMN, teardrop-like blebs are pinched from the nucleus, released into the vacuole lumen, and degraded by soluble hydrolases. PMN occurs in rapidly dividing cells but is induced to higher levels by carbon and nitrogen starvation and is under the control of the Tor kinase nutrient-sensing pathway. Confocal and biochemical assays demonstrate that Nvj1p is degraded in a PMN-dependent manner. PMN occurs normally in apg7-⌬ cells and is, therefore, not dependent on macroautophagy. Transmission electron microscopy reveals that portions of the granular nucleolus are often sequestered into PMN structures. These results introduce a novel mode of selective microautophagy that targets nonessential components of the yeast nucleus for degradation and recycling in the vacuole. INTRODUCTIONAutophagy functions in dividing cells to recycle the cytoplasm and is essential for cell viability during extended periods of starvation (Klionsky and Ohsumi, 1999). Autophagy in yeast and mammals occurs by various modes, including morphologically distinct macro-and microautophagic pathways. Macroautophagy in Saccharomyces cerevisiae is induced by starvation and involves the formation of double membrane autophagosomes around bulk cytoplasm and organelles (Takeshige et al., 1992;Baba et al., 1994). Vesicular targeting factors mediate the fusion of the outer autophagosomal membrane with the vacuole (Darsow et al., 1997;Sato et al., 1998), and an autophagic body is subsequently released into the vacuole lumen (Baba et al., 1994) where it is degraded by acid hydrolases (Jones et al., 1997). Most vacuolar hydrolases are synthesized as inactive proenzymes, which are activated in the vacuole by Pep4p and Prb1p proteinases. Thus, autophagic bodies accumulate in the vacuoles of pep4 or prb1 mutant cells (Takeshige et al., 1992;Woolford et al., 1993;Baba et al., 1994;Jones et al., 1997) due to their slower degradation rates (Jones et al., 1982;Zubenko et al., 1983).Many of the factors necessary for the formation of autophagosomes are used in the cytosol-to-vacuole targeting (Cvt) of proaminopeptidase I to the vacuole lumen (Scott et al., 1996;Teter and Klionsky, 2000). APG/AUT/CVT genes, which are required for the formation of Cvt vesicles and their conversion into larger autophagosomes (Abeliovich et al., 2000;Kim et al., 2001a), also comprise components of a novel system of ubiquitin-like conjugation reactions (Klionsky and Ohsumi, 1999). Common to these reactions is Apg7p, a conserved E1-like enzyme (Mizushima et al., 1998a,b) that is required both for the conjugation of Apg12p to Apg5p and of Aut7p/Apg8p to phosphatidylethanolamine (Ichimura et al., 2000). Recently, it was shown that some Apg proteins, including Apg5p and Aut7p/Apg8p, are required for early steps in the fo...

Abstract. It is crucial to the eucaryotic cell cycle that the centrosome undergo precise duplication to generate the two poles of the mitotic spindle . In the budding yeast Saccharomyces cerevisiae, centrosomal functions are provided by the spindle pole body (SPB), which is duplicated at the time of bud emergence in GI of the cell cycle. Genetic control of this process has previously been revealed by the characterization of mutants in CDC31 and KARL, which prevent SPB duplication and lead to formation of a monopolar spindle . Newly isolated mutations described here (mpsl and mps2, for monopolar spindle) similarly

The spindle assembly checkpoint keeps cells with defective spindles from initiating chromosome segregation. The protein kinase Mps1 phosphorylates the yeast protein Mad1p when this checkpoint is activated, and the overexpression of Mps1p induces modification of Mad1p and arrests wild-type yeast cells in mitosis with morphologically normal spindles. Spindle assembly checkpoint mutants overexpressing Mps1p pass through mitosis without delay and can produce viable progeny, which demonstrates that the arrest of wild-type cells results from inappropriate activation of the checkpoint in cells whose spindle is fully functional. Ectopic activation of cell-cycle checkpoints might be used to exploit the differences in checkpoint status between normal and tumor cells and thus improve the selectivity of chemotherapy.

Nucleation of microtubules by eukaryotic microtubule organizing centers (MTOCs) is required for a variety of functions, including chromosome segregation during mitosis and meiosis, cytokinesis, fertilization, cellular morphogenesis, cell motility, and intracellular trafficking. Analysis of MTOCs from different organisms shows that the structure of these organelles is widely varied even though they all share the function of microtubule nucleation. Despite their morphological diversity, many components and regulators of MTOCs, as well as principles in their assembly, seem to be conserved. This review focuses on one of the best-characterized MTOCs, the budding yeast spindle pole body (SPB). We review what is known about its structure, protein composition, duplication, regulation, and functions. In addition, we discuss how studies of the yeast SPB have aided investigation of other MTOCs, most notably the centrosome of animal cells.