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At least 577 records · Page 32Linked to original sources
PubMed2004

Feasibility of drug delivery to the posterior pole of the rabbit eye with an episcleral implant.

PURPOSE: To evaluate the feasibility of a nonbiodegradable polymeric episcleral implant as a new controlled intraocular delivery system of betamethasone (BM) to the posterior pole of the eye. METHODS: The episcleral implant, which is composed of a drug-releasing component and a suture tag, released BM through an ethylene vinyl acetate membrane. The implants were placed on the sclera in 12 eyes of 12 Japanese white rabbits so that the drug-releasing surface could attach to the sclera at the posterior pole. BM concentrations in the aqueous humor, vitreous, and retina-choroid (posterior half and anterior half) were determined by high-performance liquid chromatography (HPLC) at weeks 1, 2, and 4 after implantation. In addition, the intraocular tissue distribution of the drug was evaluated by fluorescein microscopy after implantation of the implant loaded with 6-carboxy fluorescein diacetate (6-CFDA) as a drug marker. Retinal toxicity was evaluated by electroretinography and histologic examination. RESULTS: The implant showed zero-order release profiles both in vitro and in vivo for 4 weeks. BM concentrations in the retina-choroid after implantation were maintained above the concentrations effective for suppressing inflammatory reactions for at least 4 weeks. The BM concentration was greater in the posterior half of the retina-choroid than in the vitreous. It was confirmed that 6-CFDA penetrated through the sclera and dispersed into the retina-choroid. Fluorescence from 6-CFDA gradually decreased in intensity with increased distance from the implantation site. Electroretinography and histologic study showed no substantial toxic reactions. CONCLUSIONS: These findings suggest that the episcleral implant may be a useful drug carrier for intraocular delivery of BM, especially for the posterior part of the eye.

Animals↗
PubMedDate not supplied

Catastrophic injuries in pole-vaulters.

Pole vaulting is a unique sport in that athletes often land from heights ranging from 10 to 20 feet. We retrospectively reviewed 32 catastrophic pole-vault injuries that were reported to the National Center for Catastrophic Sports Injury Research between 1982 and 1998. The purpose of this study was to determine the mechanisms of injury so that preventive strategies can be implemented. Information was obtained by means of a telephone interview with someone familiar with the accident. All injuries occurred in male athletes at an average age of 17.5 years; 31 were catastrophic head injuries and 1 was a thoracic spine fracture that resulted in paraplegia. Three common mechanisms were identified: 17 (53%) athletes landed with their body on the landing pad and their head on the surrounding hard ground, 8 (25%) landed in the vault box after being stranded at the height of the jump, and 5 (16%) completely missed the landing pad. The mechanism of injury in the remaining two athletes was unknown. The accident resulted in death in 16 (50%) athletes and in permanent disability in 6 (19%). Increasing the minimum landing pad size and enforcing the rule requiring soft surfaces adjacent to the landing pads are the primary recommendations for preventing injuries. The authors discuss other rule and equipment changes that may help reduce the occurrence of future injuries.

Adolescent↗
PubMed2002

A splicing alteration of 4.1R pre-mRNA generates 2 protein isoforms with distinct assembly to spindle poles in mitotic cells.

The C-terminal region of erythroid cytoskeletal protein 4.1R, encoded by exons 20 and 21, contains a binding site for nuclear mitotic apparatus protein (NuMA), a protein needed for the formation and stabilization of the mitotic spindle. We have previously described a splicing mutation of 4.1R that yields 2 isoforms: One, CO.1, lacks most of exon 20-encoded peptide and carries a missense C-terminal sequence. The other, CO.2, lacks exon 20-encoded C-terminal sequence, but retains the normal exon 21-encoded C-terminal sequence. Knowing that both shortened proteins are expressed in red cells and assemble to the membrane skeleton, we asked whether they would ensure 4.1R mitotic function in dividing cells. We show here that CO.2, but not CO.1, assembles to spindle poles, and colocalizes with NuMA in erythroid and lymphoid mutated cells, but none of these isoforms interact with NuMA in vitro. In microtubule-destabilizing conditions, again only CO.2 localizes to the centrosomes. These data suggest that the stability of 4.1R association with centrosomes requires an intact C-terminal end, either for a proper conformation of the protein, for a direct binding to an unknown centrosome-cytoskeletal network, or for both. We also found that 4.1G, a ubiquitous homolog of 4.1R, is present in mutated as well as control cells and that its C-terminal region binds efficiently to NuMA, suggesting that in fact mitotic spindles host a mixture of the two 4.1 family members. These findings led to the postulate that the coexpression at the spindle poles of 2 related proteins, 4.1R and 4.1G, might reflect a functional redundancy in mitotic cells.

Alternative Splicing↗
PubMed2003

Orb and a long poly(A) tail are required for efficient oskar translation at the posterior pole of the Drosophila oocyte.

During Drosophila oogenesis, the posterior determinant, Oskar, is tightly localized at the posterior pole of the oocyte. The exclusive accumulation of Oskar at this site is ensured by localization-dependent translation of oskar mRNA: translation of oskar mRNA is repressed during transport and activated upon localization at the posterior cortex. Previous studies have suggested that oskar translation is poly(A)-independent. We show that a long poly(A) tail is required for efficient oskar translation, both in vivo and in vitro, but is not sufficient to overcome BRE-mediated repression. Moreover, we show that accumulation of Oskar activity requires the Drosophila homolog of Cytoplasmic Polyadenylation Element Binding protein (CPEB), Orb. As posterior localization of oskar mRNA is an essential prerequisite for its translation, it was critical to identify an allele of orb that does localize oskar mRNA to the posterior pole of the oocyte. We show that flies bearing the weak mutation orb(mel) localize oskar transcripts with a shortened poly(A) that fails to enhance oskar translation, resulting in reduced Oskar levels and posterior patterning defects. We conclude that Orb-mediated cytoplasmic polyadenylation stimulates oskar translation to achieve the high levels of Oskar protein necessary for posterior patterning and germline differentiation.

Animals↗
PubMed2005

POPK-1/Sad-1 kinase is required for the proper translocation of maternal mRNAs and putative germ plasm at the posterior pole of the ascidian embryo.

Maternal mRNAs localized to specific regions in eggs play important roles in the establishment of embryonic axes and germ layers in various species. Type I postplasmic/PEM mRNAs, which are localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, such as the muscle determinant macho-1 mRNA, play key roles in embryonic development. In the present study, we analyzed the function of the postplasmic/PEM RNA Hr-POPK-1, which encodes a kinase of Halocynthia roretzi. When the function of POPK-1 was suppressed by morpholino antisense oligonucleotides, the resulting malformed larvae did not form muscle or mesenchyme, as in macho-1-deficient embryos. Epistatic analysis indicated that POPK-1 acts upstream of macho-1. When POPK-1 was knocked down, localization of every Type I postplasmic/PEM mRNA examined, including macho-1, was perturbed, showing diffuse early distribution and eventual concentration into a smaller area. This is the probable reason for the macho-1 dysfunction. The postplasmic/PEM mRNAs such as macho-1 and Hr-PEM1 are co-localized with the cortical endoplasmic reticulum (cER) and move with it after fertilization. Eventually they become highly concentrated into a subcellular structure, the centrosome-attracting body (CAB), at the posterior pole of the cleaving embryos. The suppression of POPK-1 function reduced the size of the domain of concentrated cER at the posterior pole, indicating that POPK-1 is involved in the movement of postplasmic/PEM RNAs via relocalization of cER. The CAB also shrank. These results suggest that Hr-POPK-1 plays roles in concentration and positioning of the cER, as well as in the concentration of CAB materials, such as putative germ plasm, in the posterior blastomeres.

Animals↗
PubMed1989

l(1)pole hole is required maternally for pattern formation in the terminal regions of the embryo.

Maternal expression of the l(1)pole hole (l(1)ph) gene product is required for the development of the Drosophila embryo. When maternal l(1)ph+ activity is absent, alterations in the embryonic fate map occur as visualized by the expression of segmentation genes fushitarazu and engrailed. If both maternal and zygotic activity is absent, embryos degenerate around 7 h of development. If only maternal activity is missing, embryos complete embryogenesis and show deletions of both anterior and posterior structures. Anteriorly, structures originating from labral and acron head regions are missing. Posteriorly, abdominal segments A8, 9 and 10, the telson and the proctodeum are missing. Similar pattern deletions are observed in embryos derived from the terminal class of female sterile mutations. Thus, the maternal l(1)ph+ gene product is required for the establishment of cell identities at the anterior and posterior poles of the Drosophila embryo.

Animals↗
PubMed1991

Regeneration of the root pole in surgically transected carrot embryos occurs by position-dependent, proximodistal replacement of missing tissues.

Torpedo-stage carrot embryos were surgically transected at various locations along the shoot-root axis and explants of the cotyledon-bearing shoot pole were sectioned and examined in order to provide a more detailed description of root pole regeneration. When excisions occurred at the sites of the future hypocotyl, the future radicle or the future root apical meristem, the regenerating axial tissues exhibited patterns of cellular organization that were nearly identical to those seen in unsevered controls. To accomplish this restoration, new cells, of the type normally found at each cutting site, were produced behind a regeneration dome that formed over the original surgical site. The regeneration dome was displaced by division and expansion-driven extension of the longitudinal axis, and cells in the renewed region quickly acquired individual anatomical traits and collective tissue morphologies that corresponded to those of cells in the analogous locations in intact embryos. Although no clear mechanism is implied, the results of these experiments suggest that interactions between cells near the surgical margin permit them to retain their sense of location within the original structure, and apprise them of the removal of their basipetally positioned neighbors. With varying-length remnants of the shoot serving as the only vestige of the original size and shape of the embryo, cells close to the site of excision were apparently reconfigured to commence ordered divisions that ultimately reconstituted the embryonic axis.

Microscopy, Electron↗
PubMed1992

3' non-translated sequences in Drosophila cyclin B transcripts direct posterior pole accumulation late in oogenesis and peri-nuclear association in syncytial embryos.

We have characterised forms of the Drosophila cyclin B transcript that differ as a result of a splicing event which removes a nucleotide segment from the 3' untranslated region. In oogenesis, both cyclin A RNA and a shorter form of the cyclin B transcript are seen in the cells of the germarium that are undergoing mitosis. The shorter cyclin B transcript alone is then detectable in the presumptive oocyte until stages 7-8 of oogenesis. Both cyclin A RNA and a longer form of the cyclin B RNA are then synthesised in the nurse cells during stages 9-11, to be deposited in the oocyte during stages 11-12. These transcripts become evenly distributed throughout the oocyte cytoplasm but, in addition, those of cyclin B become concentrated at the posterior pole. Examination of the distributions of RNAs transcribed from chimeric cyclin genes indicates that sequences in the 3' untranslated region of the larger cyclin B RNA are required both for it to become concentrated at the posterior pole and to direct those transcripts in the body of the syncytial embryo to their peri-nuclear localisation. These sequences are disrupted by the splicing event which generates smaller cyclin B transcripts.

Animals↗
PubMed1997

Animal and vegetal pole cells of early Xenopus embryos respond differently to maternal dorsal determinants: implications for the patterning of the organiser.

The maternal dorsal determinants required for the specification of the dorsal territories of Xenopus early gastrulae are located at the vegetal pole of unfertilised eggs and are moved towards the prospective dorsal region of the fertilised egg during cortical rotation. While the molecular identity of the determinants is unknown, there are dorsal factors in the vegetal cortical cytoplasm (VCC). Here, we show that the VCC factors, when injected into animal cells activate the zygotic genes Siamois and Xnr3, suggesting that they act along the Wnt/beta-catenin pathway. In addition, Siamois and Xnr3 are activated at the vegetal pole of UV-irradiated embryos, indicating that these two genes are targets of the VCC factors in all embryonic cells. However, the consequences of their activation in cells that occupy different positions along the animal-vegetal axis differ. Dorsal vegetal cells of normal embryos or VCC-treated injected animal cells are able to dorsalise ventral mesoderm in conjugate experiments but UV-treated vegetal caps do not have this property. This difference is unlikely to reflect different levels of activation of FGF or activin-like signal transduction pathways but may reflect the activation of different targets of Siamois. Chordin, a marker of the head and axial mesoderm, is activated by the VCC/Siamois pathway in animal cells but not in vegetal cells whereas cerberus, a marker of the anterior mesendoderm which lacks dorsalising activity, can only be activated by the VCC/Siamois pathway in vegetal cells. We propose that the regionalisation of the organiser during gastrulation proceeds from the differential interpretation along the animal-vegetal axis of the activation of the VCC/beta-catenin/Siamois pathway.

Animals↗
PubMed2000

Animal and vegetal poles of the mouse egg predict the polarity of the embryonic axis, yet are nonessential for development.

Recent studies suggest early (preimplantation) events might be important in the development of polarity in mammalian embryos. We report here lineage tracing experiments with green fluorescent protein showing that cells located either near to or opposite the polar body at the 8-cell stage of the mouse embryo retain their same relative positions in the blastocyst. Thus they come to lie on either end of an axis of symmetry of the blastocyst that has recently been shown to correlate with the anterior-posterior axis of the postimplantation embryo (see R. J. Weber, R. A. Pedersen, F. Wianny, M. J. Evans and M. Zernicka-Goetz (1999). Development 126, 5591-5598). The embryonic axes of the mouse can therefore be related to the position of the polar body at the 8-cell stage, and by implication, to the animal-vegetal axis of the zygote. However, we also show that chimeric embryos constructed from 2-cell stage blastomeres from which the animal or the vegetal poles have been removed can develop into normal blastocysts and become fertile adult mice. This is also true of chimeras composed of animal or vegetal pole cells derived through normal cleavage to the 8-cell stage. We discuss that although polarity of the postimplantation embryo can be traced back to the 8-cell stage and in turn to the organisation of the egg, it is not absolutely fixed by this time.

Animals↗
PubMed2005

Role of the spindle-pole-body protein ApsB and the cortex protein ApsA in microtubule organization and nuclear migration in Aspergillus nidulans.

Nuclear migration and positioning in Aspergillus nidulans depend on microtubules, the microtubule-dependent motor protein dynein, and auxiliary proteins, two of which are ApsA and ApsB. In apsA and apsB mutants nuclei are clustered and show various kinds of nuclear navigation defects, although nuclear migration itself is still possible. We studied the role of several components involved in nuclear migration through in vivo fluorescence microscopy using fluorescent-protein tagging. Because ApsA localizes to the cell cortex and mitotic spindles were immobile in apsA mutants, we suggest that astral microtubule-cortex interactions are necessary for oscillation and movement of mitotic spindles along hyphae, but not for post-mitotic nuclear migration. Mutation of apsA resulted in longer and curved microtubules and displayed synthetic lethality in combination with the conventional kinesin mutation DeltakinA. By contrast, ApsB localized to spindle-pole bodies (the fungal centrosome), to septa and to spots moving rapidly along microtubules. The number of cytoplasmic microtubules was reduced in apsB mutants in comparison to the wild type, indicating that cytoplasmic microtubule nucleation was affected, whereas mitotic spindle formation appeared normal. Mutation of apsB suppressed dynein null mutants, whereas apsA mutation had no effect. We suggest that nuclear positioning defects in the apsA and apsB mutants are due to different effects on microtbule organisation. A model of spindle-pole body led nuclear migration and the roles of dynein and microtubules are discussed.

Aspergillus nidulans↗
PubMed1993

Primary structure and microtubule-interacting domain of the SP-H antigen: a mitotic MAP located at the spindle pole and characterized as a homologous protein to NuMA.

Using a human autoantibody, SP-H, we identified a 200-230 kDa mitotic MAP in a variety of mammalian cell lines which shows affinity for the minus end of microtubules and also becomes associated with the spindle pole during mitosis. To examine the detailed structure and functional organization of the protein, the gene coding for the end-specific MAP was isolated and characterized by screening a human placenta lambda gt11 expression library using SP-H as a probe. Overlapping cDNA clones, which covered the entire length of the coding region of the SP-H antigen, were obtained. Polyclonal antibodies raised against fusion proteins generated from non-overlapping cDNA fragments stained the HeLa SP-H antigen in interphase and mitotic cells, and recognized a single 215 kDa band on immunoblots, as did the original SP-H antibody. Analysis of the nucleotide sequence revealed a 7,091 nucleotide sequence with an open reading frame of 6,345 nucleotides encoding a 2,115 amino acid polypeptide with a calculated molecular mass of 238,376 Da. The predicted amino acid sequence showed the protein to be composed of an alpha-helical domain, flanked by globular domains located at the amino and carboxy termini. The sequence contained five repeats of the hypothetical leucine zipper motif: one is in the N-terminal globular domain, and four are in the central alpha-helical stalk. Comparison with other sequences in the database shows that the SP-H antigen is identical to the NuMA protein reported by Yang et al. (1992) J. Cell Biol. 116, 1303-1317, but there are differences between the SP-H antigen and NuMA sequence reported by Compton et al. (1992) J. Cell Biol. 116, 1395-1408. cDNA inserts of the truncated SP-H antigen were expressed in both insect Sf9 cells and in cultured mammalian cells. The recombinant protein corresponding to the C-terminal half of the protein was restricted to the nucleus, whereas the N-terminal half of the protein was localized in the cytoplasm, suggesting the presence of a nuclear translocation signal(s) in the C-terminal domain. The C-terminal polypeptide expressed in mitotic COS cells was shown to specifically localize at the spindle pole. Microtubule-binding assays using in vitro transcribed/translated polypeptide products from different domains of the SP-H antigen further suggested that the SP-H antigen interacts with microtubules through the globular domain at the C-terminus.

Amino Acid Sequence↗
PubMed1994

Cloning of a cDNA encoding human centrin, an EF-hand protein of centrosomes and mitotic spindle poles.

A human cDNA expression library was screened using anti-centrin antibodies to obtain a cDNA clone encoding human centrin. The cDNA clone contains an open reading frame of 516 base pairs and predicts a product of 172 amino acids with a calculated molecular mass of 19,528 and a pI of 4.61. Sequence analysis demonstrates that human centrin and centrins from higher plants, protozoa, algae, Xenopus and the yeast CDC31 gene product are closely related members of a subfamily of the EF-hand superfamily of calcium-binding proteins. The human centrin sequence has four putative calcium-binding domains as defined by the EF-hand consensus. Immunoprecipitation and western blot studies from HeLa cells confirm that human centrin is a protein of approximately 20,000 M(r) as predicted from the cDNA clone. Indirect immunofluorescence analysis of HeLa cells demonstrates that centrin is localized at the centrosome of interphase cells and that it redistributes to the region of the spindle poles during mitosis. When taken together with earlier genetic studies, these results demonstrate that centrin is a ubiquitous component of centrosomes and mitotic spindle poles of diverse organisms and suggest that centrin plays a role in centrosome separation at the time of mitosis.

Amino Acid Sequence↗
PubMed1994

A calcineurin-like gene ppb1+ in fission yeast: mutant defects in cytokinesis, cell polarity, mating and spindle pole body positioning.

A calcineurin (type 2B)-like protein phosphatase gene designated ppb1+ was isolated from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence was 57% identical to rat PP2B alpha. ppb1 null mutant could form colonies at 33 degrees C but the size of the colonies was small at 22 degrees C. Cytokinesis was greatly delayed at 22 degrees C, and a large number of multi-septate cells were produced. The cell polarity control was impaired, causing branched cells. ppb1 null was virtually sterile. These phenotypes were rescued by a plasmid carrying the ppb1+ gene. Multi-septate cells were also produced in wild type at 22 degrees C by cyclosporin A, an inhibitor of calcineurin. This drug effect was enhanced in stst1 null mutant, which was hypersensitive to various drugs and cations. ppb1 null was not affected by cyclosporin A, consistent with the hypothesis that ppb1 is its target. Double-mutant analysis indicated that ppb1 had a function related to that of two other phosphatases, type 1-like dis2 and 2A-like ppa2.ppb1 null-sts1 null showed the severe multi-septate phenotype in the absence of cyclosporin A. ppb1+ and sts1+ gene functions are related. The double mutant ppb1-sts5 was lethal, indicating that the ppb1+ gene shared an essential function with the sts5+ gene. Overexpression of ppb1+ caused anomalies in cell and nuclear shape, microtubule arrays and spindle pole body positioning in interphase cells. Thus the ppb1+ gene appears to be involved in cytokinesis, mating, transport, nuclear and spindle pole body positioning, and cell shape.

Amino Acid Sequence↗
PubMed1996

Human keratinocytes migrate to the negative pole in direct current electric fields comparable to those measured in mammalian wounds.

Previous measurements of the lateral electric fields near skin wounds in guinea pigs have detected DC fields between 100-200 mV/mm near the edge of the wound. We have studied the translocation response of motile primary human keratinocytes migrating on a collagen substrate while exposed to similar physiological DC electric fields. We find that keratinocytes migrate randomly on collagen in fields of 5 mV/mm or less, but in larger fields they migrate towards the negative pole of the field, exhibiting galvanotaxis. Since these cells have an average cell length of 50 microns, this implies that they are able to detect a voltage gradient as low as 0.5 mV along their length. This cath-odally-directed movement exhibits increased directedness with increasing field strengths between 10 and 100 mV/mm. We observe a maximally directed response at 100 mV/mm with half of the cells responding to the field within 14 minutes. The average speed of migration tended to be greater in fields above 50 mV/mm than in smaller fields. We conclude that human keratinocytes migrate towards the negative pole in DC electric fields that are of the same magnitude as measured in vivo near wounds in mammalian skin.

Animals↗
PubMed1997

A yeast heat shock transcription factor (Hsf1) mutant is defective in both Hsc82/Hsp82 synthesis and spindle pole body duplication.

Cdc28 is a cyclin-dependent protein kinase of Saccharomyces cerevisiae that is required for the G1/S and G2/M transitions of the cell division cycle. All previously described cdc28 mutants aside from cdc28-1N arrest division specifically in the G1 phase. cdc28-1N arrests division in G2/mitosis. We show here that the cdc28-109 mutant exhibits a mixed cell division arrest at 37 degrees C with cells in both the G1 and G2 phases. In order to identify proteins that functionally interact with Cdc28, we isolated mutants that are colethal with cdc28-109 at its permissive temperature. We describe here our phenotypic analysis of two such mutants, hsf1-82 and ydj1-10, that affect the heat shock transcription factor and a yeast dnaj-like protein chaperone, respectively. hsf1-82 and ydj1-10 temperature-sensitive mutants arrest the cell division cycle at several stages. However, one predominant class of cells in both mutants was arrested with a large bud and a single vertex of microtubules. Electron microscopic analysis of such hsf1-82 cells showed that they contained an unduplicated spindle pole body with an enlarged half-bridge. Two-dimensional gel electrophoresis of total cell proteins revealed that the hsf1-82 cells were specifically defective in the expression of the Hsc82 and Hsp82 proteins. Furthermore, the hsf1-82 mutation was suppressed by the HSC82 gene on a multicopy plasmid that restored Hsc82 protein to high levels in these cells. These results show that Hsf1 is required for spindle pole body duplication at 37 degrees C.

Alleles↗
PubMed1997

The checkpoint control for anaphase onset does not monitor excess numbers of spindle poles or bipolar spindle symmetry.

Exit from mitosis in animal cells is substantially delayed when spindle assembly is inhibited, spindle bipolarity is disrupted, or when a monopolar spindle is formed. These observations have led to the proposal that animal cells have a 'spindle assembly' checkpoint for the metaphase-anaphase transition that monitors bipolar spindle organization. However, the existence of such a checkpoint is uncertain because perturbations in spindle organization can produce unattached kinetochores, which by themselves are known to delay anaphase onset. In this study we have tested if cells monitor bipolar spindle organization, independent of kinetochore attachment, by analyzing the duration of mitosis in sea urchin zygotes and vertebrate somatic cells containing multipolar spindles in which all kinetochores are attached to spindle poles. We found that sea urchin zygotes containing tripolar or tetrapolar spindles progressed from nuclear envelope breakdown to anaphase onset with normal timing. We also found that the presence of supernumerary, unpaired spindle poles did not greatly prolong mitosis. Observation of untreated PtK1 cells that formed tripolar or tetrapolar spindles revealed that they progressed through mitosis, on average, at the normal rate. More importantly, the interval between the bipolar attachment of the last monooriented chromosome and anaphase onset was normal. Thus, neither of these cell types can detect the presence of gross aberrations in spindle architecture that inevitably lead to aneuploidy. We conclude that animal cells do not have a checkpoint for the metaphase-anaphase transition that monitors defects in spindle architecture independent of the checkpoint that monitors kinetochore attachment to the spindle. For dividing cells in which spindle microtubule assembly is not experimentally compromised, we propose that the completion of kinetochore attachment is the event which limits the time of the metaphase-anaphase transition.

Anaphase↗
PubMed1978

The role of spindle pole bodies and modified microtubule ends in the initiation of microtubule assembly in Saccharomyces cerevisiae.

The spindle poles of the budding yeast, Saccharomyces cerevisiae, have been removed from mitotic and meiotic cells by osmotic lysis of spheroplasts. The spindle pole bodies (SPBs)--diskoidal structures also termed 'spindle plaques'--have been analysed for their ability to potentiate the polymerization of microtubules in vitro. Free SPBs were completely deprived of any detectable native microtubules by incubation in the absence of added tubulin and were then challenged with chick neurotubulin, which had been rendered partially defective in self-initiation of repolymerization. Electron microscopy revealed that these SPBs served as foci for the initiation of microtubule polymerization in vitro. Because the attached microtubules elongated linearly with time but did not increase in numbers after the first stage of the reaction, it is apparent that there are a limited number of sites for initiation. The initiating potential of the SPBs was found to be inhibited by enzymic hydrolysis of protein but not of DNA. The microtubule end proximal to the site of initiation on the SPB is distinguished by a 'closed' appearance because of a terminal component which is continuous with the microtubule wall, whereas the distal end has the 'open' appearance characteristic of freely repolymerized neurotubules. SPBs which were partially purified on sucrose gradients retained their ability to initiate the assembly of microtubules with the same structural differentiation of their ends. The occurrence of closed proximal ends on native yeast microtubules suggests that closed ends may play a role in the initiation of microtubule polymerization in vivo, as well as in vitro.

Cell Differentiation↗