Abstract. Mitotic spindles of Schizosaccharomyces pombe have been studied by EM, using serial cross sections to reconstruct 12 spindles from cells that were ultrarapidly frozen and fixed by freeze substitution. The resulting distributions of microtubules (NITs) have been analyzed by computer. Short spindles contain two kinds of MTS: continuous ones that run from pole to pole and MTS that originate at one pole and end in the body of the spindle. Among the latter there are three pairs of MT bundles that end on fibrous, darkly staining structures that we interpret as kinetochores. The number of MTs ending at each putative kinetochore ranges from two to four; all kinetochore-associated MTS disappear as the spindle elongates from 3-6/~m. At this and greater spindle lengths, there are no continuous MTS, only polar MTs that interdigitate at the spindle midzone, but the spindle continues to elongate. An analysis of the density of neighboring MTs at the midzone of long spindles shows that their most common spacing is ~40 nm, center to center, and that there is a preferred angular separation of 90 ~ . Only hints of such square-packing are found at the midzone of short spindles, and near the poles there is no apparent order at any mitotic stage. Our data suggest that the kinetochore Mrs (KNITs) do not interact directly with nonkinetochore MTs, but that interdigitating MTS from the two spindle poles do interact to form a mechanically stable bundle that connects the poles. As the spindle elongates, the number of MTs decreases while the mean length of the Mrs that remain increases. We conclude that the chromosomes of S. pombe become attached to the spindle by kinetochore MTs, that these MTs disappear as the chromosomes segregate, that increased separation of daughter nuclei is accompanied by a sliding apart of anti-parallel MTS, and that the mitotic processes of S. pombe are much like those in other eukaryotic cells.

Abstract, Spindle microtubules (MTs) in PtK~ cells, fixed at stages from metaphase to telophase, have been reconstructed using serial sections, electron microscopy, and computer image processing. We have studied the class of MTs that form an interdigitating system connecting the two spindle poles (interpolar MTs or ipMTs) and their relationship to the spindle MTs that attach to kinetochores (kMTs). Viewed in cross section, the ipMTs cluster with antiparallel near neighbors throughout mitosis; this bundling becomes much more pronounced as anaphase proceeds. While the minus ends of most kMTs are near the poles, those of the ipMTs are spread over half of the spindle length, with at least 50% lying >1.5 #m from the poles. Longitudinal views of the ipMT bundles demonstrate a major rearrangement of their plus ends between midand late anaphase B. However, the minus ends of these MTs do not move appreciably farther from the spindle midplane, suggesting that sliding of these MTs contributes little to anaphase B. The minus ends of ipMTs are markedly clustered in the bundles of kMTs throughout anaphase A. These ends lie close to kMTs much more frequently than would be expected by chance, suggesting a specific interaction. As sister kinetochores separate and kMTs shorten, the minus ends of the kMTs remain associated with the spindle poles, but the minus ends of many ipMTs are released from the kMT bundles, allowing the spindle pole and the kMTs to move away from the ipMTs as the spindle elongates.

The cycle of spindle pole body (SPB) duplication, differentiation, and segregation in Schizosaccharomyces pombe is different from that in some other yeasts. Like the centrosome of vertebrate cells, the SPB of S. pombe spends most of interphase in the cytoplasm, immediately next to the nuclear envelope. Some y-tubulin is localized on the SPB, suggesting that it plays a role in the organization of interphase microtubules (MTs), and serial sections demonstrate that some interphase MTs end on or very near to the SPB. y-Tubulin is also found on osmiophilic material that lies near the inner surface of the nuclear envelope, immediately adjacent to the SPB, even though there are no MTs in the interphase nucleus. Apparently, the MT initiation activities of y-tubulin in S. pombe are regulated. The SPB duplicates in the cytoplasm during late G2 phase, and the two resulting structures are connected by a darkly staining bridge until the mitotic spindle forms. As the cell enters mitosis, the nuclear envelope invaginates beside the SPB, forming a pocket of cytoplasm that accumulates dark amorphous material. The nuclear envelope then opens to form a fenestra, and the duplicated SPB settles into it. Each part of the SPB initiates intranuclear MTs, and then the two structures separate to lie in distinct fenestrae as a bipolar spindle forms. Through metaphase, the SPBs remain in their fenestrae, bound to the polar ends of spindle MTs; at about this time, a small bundle of cytoplasmic MTs forms in association with each SPB. These MTs are situated with one end near to, but not on, the SPBs, and they project into the cytoplasm at an orientation that is oblique to the spindle axis. As anaphase proceeds, the nuclear fenestrae close, and the SPBs are extruded back into the cytoplasm. These observations define new fields of enquiry about the control of SPB duplication and the dynamics of the nuclear envelope.

The "cut" mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality. Analysis of temperature-sensitive alleles of cut11 ϩ suggests that this gene is required for the formation of a functional bipolar spindle. Defective spindle structure was revealed with fluorescent probes for tubulin and DNA. Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm. Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase. This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope. Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes. Cloning and sequencing showed that cut11 ϩ encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1. These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis. INTRODUCTIONAccurate chromosome segregation requires proper assembly and function of a mitotic spindle. The spindle is constructed from microtubules (MT) 1 whose polymerization is nucleated by the centrosome (reviewed in Kellogg et al., 1994), which is known in fungi as the spindle pole body (SPB) (reviewed in Snyder, 1994). Although these two organelles are structurally distinct, genetic and biochemical approaches have identified several common components of centrosomes and SPBs, including ␥-tubulin (reviewed in Kellogg et al., 1994), CDC31/centrin (reviewed in Schiebel and Bornes, 1995), and p34 cdc2 (Bailly et al., 1989; Raibowol et al., 1989). Thus, analyses of SPBs have been informative about centrosomes in general.Recent work has demonstrated that the SPB of Schizosaccharomyces pombe is a dynamic organelle, undergoing significant changes in morphology and cellular localization as cells progress through their growth and division cycle (Ding et al., 1997). The nature of these changes distinguishes the fission yeast centrosome from that of other organisms. For example, the SPBs of the budding yeast Saccharomyces cer- evisiae duplicate in G 1 and remain in the nuclear envelope through the entire cell cycle (Byers, 1981;. The fission yeast SPB, on the other hand, resides in the cytoplasm through most of interphase, where it duplicates during late G 2 . As the cell enters mitosis, the nuclear envelope invaginates beneath the SPB and forms an opening, or fenestra, into which the duplicated SPB settles. Each part of the double SPB initiates intranuclear MTs; then the two parts separate to lie in distinct fenestrae, bound to the polar ends of the spindle MTs. As anaphas...

Cell-cell interactions are central to vascular development. We have developed an in vitro system in which endothelial cells (EC) are co-cultured with 10T1/2 cells as smooth muscle cell (SMC)/pericyte precursors. 10T1/2 cells, in contact with EC, differentiate to SMC in a process mediated, at least in part, by a transforming growth factor-beta (TGF-beta)-mediated event. Co-culture with EC or TGF-beta treatment induced expression of SM22alpha, with co-culture inducing a significantly greater response. To dissect the molecular mechanisms of SMC/pericyte differentiation, reporter constructs containing the promoter for SM22alpha, a SMC-specific gene, were stably transfected into 10T1/2 cells and response to EC-co-culture and TGFbeta were compared. Co-culture with EC or TGFbeta treatment stimulated activity of a 441-bp SM22-alpha promoter to about the same extent, whereas co-culture induced the activity of a 3.7-kb promoter to about twice that of TGBbeta. Neutralization of TGFbeta in EC-10T1/2 co-cultures partially reduced the 3.7-kb SM22alpha promoter activity in 10T1/2 cells. Previously unidentified CArG and TCE elements near the 5' end of the promoter are responsible for full promoter activity. EC-mesenchymal contact appears to be required for full promoter activity of the SM22alpha gene in 10T1/2 and requires upstream CArG and TCE elements. The 3.7-kb SM22alpha promoter can direct expression of lacZ in vivo to SMC of the large vessels and the smaller intersomitic vessels. We have identified the expression of SM22alpha in pericytes of the retinal microvasculature in developing and remodeling vessels.