Dynamin 1 is a neuron-specific guanosine triphosphatase thought to be critically required for the fission reaction of synaptic vesicle endocytosis. Unexpectedly, mice lacking dynamin 1 were able to form functional synapses, even though their postnatal viability was limited. However, during spontaneous network activity, branched, tubular plasma membrane invaginations accumulated, capped by clathrin-coated pits, in synapses of dynamin 1-knockout mice. Synaptic vesicle endocytosis was severely impaired during strong exogenous stimulation but resumed efficiently when the stimulus was terminated. Thus, dynamin 1-independent mechanisms can support limited synaptic vesicle endocytosis, but dynamin 1 is needed during high levels of neuronal activity.

Centrioles are necessary for flagella and cilia formation, cytokinesis, cell-cycle control and centrosome organization/spindle assembly. They duplicate once per cell cycle, but the mechanisms underlying their duplication remain unclear. Here we show using electron tomography of staged C. elegans one-cell embryos that daughter centriole assembly begins with the formation and elongation of a central tube followed by the peripheral assembly of nine singlet microtubules. Tube formation and elongation is dependent on the SAS-5 and SAS-6 proteins, whereas the assembly of singlet microtubules onto the central tube depends on SAS-4. We further show that centriole assembly is triggered by an upstream signal mediated by SPD-2 and ZYG-1. These results define a structural pathway for the assembly of a daughter centriole and should have general relevance for future studies on centriole assembly in other organisms.

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...

Endophilin is a membrane binding protein with curvature-generating/sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin, and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles but not of clathrin-coated pits at synapses. The absence of endophilin impairs, but does not abolish synaptic transmission and results in perinatal lethality, while partial endophilin absence causes severe neurological defects, including epilepsy, and neurodegeneration. Our data supports a model in which endophilin recruitment to coated pit necks, due to its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.

Polyploidy, increased sets of chromosomes, occurs during development, cellular stress, disease and evolution. Despite its prevalence, little is known about the physiological alterations that accompany polyploidy. We previously described 'ploidy-specific lethality', where a gene deletion that is not lethal in haploid or diploid budding yeast causes lethality in triploids or tetraploids. Here we report a genome-wide screen to identify ploidy-specific lethal functions. Only 39 out of 3,740 mutations screened exhibited ploidy-specific lethality. Almost all of these mutations affect genomic stability by impairing homologous recombination, sister chromatid cohesion, or mitotic spindle function. We uncovered defects in wild-type tetraploids predicted by the screen, and identified mechanisms by which tetraploidization affects genomic stability. We show that tetraploids have a high incidence of syntelic/monopolar kinetochore attachments to the spindle pole. We suggest that this defect can be explained by mismatches in the ability to scale the size of the spindle pole body, spindle and kinetochores. Thus, geometric constraints may have profound effects on genome stability; the phenomenon described here may be relevant in a variety of biological contexts, including disease states such as cancer.

SummaryCilia/flagella are highly conserved organelles that play diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia/flagella often result in primary ciliary dyskinesia (PCD). However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a novel gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in PCD patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment.

The spindle pole body (SPB) is the major microtubule-organizing center of budding yeast and is the functional equivalent of the centrosome in higher eukaryotic cells. We used fast-frozen, freeze-substituted cells in conjunction with high-voltage electron tomography to study the fine structure of the SPB and the events of early spindle formation. Individual structures were imaged at 5-10 nm resolution in three dimensions, significantly better than can be achieved by serial section electron microscopy. The SPB is organized in distinct but coupled layers, two of which show ordered two-dimensional packing. The SPB central plaque is anchored in the nuclear envelope with hook-like structures. The minus ends of nuclear microtubules (MTs) are capped and are tethered to the SPB inner plaque, whereas the majority of MT plus ends show a distinct flaring. Unbudded cells containing a single SPB retain 16 MTs, enough to attach to each of the expected 16 chromosomes. Their median length is ϳ150 nm. MTs growing from duplicated but not separated SPBs have a median length of ϳ130 nm and interdigitate over the bridge that connects the SPBs. As a bipolar spindle is formed, the median MT length increases to ϳ300 nm and then decreases to ϳ30 nm in late anaphase. Three-dimensional models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complement and extend what is known about the three-dimensional structure of the yeast mitotic spindle and further our understanding of the organization of the SPB in intact cells. INTRODUCTIONMicrotubule-organizing centers (MTOCs) nucleate and anchor microtubules (MTs) during cell differentiation and division (for review, see Brinkley, 1985;Rose et al., 1993;Kellogg et al., 1994;Pereira and Schiebel, 1997). Although MTOCs vary widely in structure, their functions are largely conserved. In metazoa, the principal MTOC is the centrosome, which organizes both the interphase MTs and those of the mitotic spindle. In the yeast Saccharomyces cerevisiae, this role is served by the spindle pole body (SPB), a complex and dynamic organelle that undergoes significant structural changes during the yeast cell cycle (for review, see Winey and Byers, 1993;Kilmartin, 1994;Snyder, 1994). Nuclear MTs, which form the meiotic or mitotic spindles, attach to an inner plaque of the SPB, whereas cytoplasmic MTs, important for nuclear movements and position, are attached to an outer plaque (Moens and Rapport, 1971;Byers and Goetsch, 1974, 1975;Rabinow and Marak, 1966). Recent studies of isolated SPBs by cryomicroscopy and electron tomography have shown that SPBs are organized from six major layers, including a central crystalline core that contains the SPC42 gene product (Bullitt et al., 1997).Duplication of the SPB during the cell cycle begins with the formation of a "satellite" on the cytoplasmic face of the half-bridge, a specialized region of the nuclear envelope that lies immediately adjacent to the existing SPB. By processes not yet described in detail, the satellite develops into a...