cut11⁺: A Gene Required for Cell Cycle-dependent Spindle Pole Body Anchoring in the Nuclear Envelope and Bipolar Spindle Formation inSchizosaccharomyces pombe

Abstract

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...

pkl1+andklp2+: Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis

“…This inference is corroborated by a reduction in the percentage of azygotic asci with greater than four spores to near wild type levels (Table 3). Also, microscopy of zygotes formed early in the cross of klp2⌬,dhc1-d1 with itself, with the use of either DAPI to visualize the nuclei or strains carrying Cut11p-GFP to mark the nuclear envelopes (West et al, 1998) revealed that zygotes with a single nucleus were rare in this cross. Most contained two nuclei whose morphology resembled that of diploid nuclei during the "horsetailing" motions of meiotic prophase ( Figure 4C; Chikashige et al, 1994).…”

“…A complete replacement of the klp2 ϩ by ura4 ϩ was made by a singlestep gene replacement protocol (Rothstein, 1991), with the use of a cassette containing two genomic regions flanking the either side of the klp2 ϩ gene and ura4 ϩ as the selectable marker. A 2900-nt PstIHindIII fragment corresponding to the sequence Ϫ2900 to Ϫ68 nt upstream of the klp2 ϩ ORF was subcloned into the PstI-HindIII site of the bacterial cloning vector pSPORT1 (GIBCO BRL), which had been modified to carry the ura4 ϩ gene (pSPORT1-URA4; West et al, 1998). A 1.3-kb BglII-HindIII fragment corresponding to the sequence ϩ83 nt to ϩ1043 nt downstream of the klp2 ϩ ORF was blunt-ended and subcloned into the KpnI site of pSPORT1-URA4 with the aid of KpnI linkers.…”

“…Final images were exported to Adobe Photoshop (San Jose, CA) for figure preparation. To examine nuclear movement in crosses between klp2⌬,dhc1-d1 strains, cut11::GFP (West et al, 1998) was used as a marker for the nuclear envelope.…”

“…Functional Cut11p is required at the onset of mitosis to allow the cell's spindle pole body to enter and attach to an opening that forms in the nuclear envelope; it is required for the formation of a normal mitotic spindle (West et al, 1998). In a cut11-1 background, pkl1⌬ shows defective growth at the semipermissive temperature of 29°C, at which temperature cut11-1 and cut11-1,klp2⌬ grow like wild type (Figure 6).…”

Two Related Kinesins,klp5+andklp6+, Foster Microtubule Disassembly and Are Required for Meiosis in Fission Yeast

“…Experiments with GFP-tagged genes were generally done at 25°C, and others were done at the temperatures noted. Fixed cells were prepared for tubulin and DNA staining with the use of a double aldehyde method (Hagan and Hyams, 1988), and antibodies were applied as previously described (West et al, 1998). DNA staining was also done with cells fixed by adding 1/10 volume of cell culture to methanol at Ϫ20°C for 2-15 min.…”