Spindle Tubulin Research Articles

Dynamic localization of the Na+-HCO3- co-transporter NBCn1 to the plasma membrane, centrosomes, spindle and primary cilia.

Finely tuned regulation of transport protein localization is vital for epithelial function. The Na+-HCO3- co-transporter NBCn1 (also known as SLC4A7) is a key contributor to epithelial pH homeostasis, yet the regulation of its subcellular localization is not understood. Here, we show that a predicted N-terminal β-sheet and short C-terminal α-helical motif are essential for NBCn1 plasma membrane localization in epithelial cells. This localization was abolished by cell-cell contact disruption, and co-immunoprecipitation (co-IP) and proximity ligation (PLA) revealed NBCn1 interaction with E-cadherin and DLG1, linking it to adherens junctions and the Scribble complex. NBCn1 also interacted with RhoA and localized to lamellipodia and filopodia in migrating cells. Finally, analysis of native and GFP-tagged NBCn1 localization, subcellular fractionation, co-IP with Arl13B and CEP164, and PLA of NBCn1 and tubulin in mitotic spindles led to the surprising conclusion that NBCn1 additionally localizes to centrosomes and primary cilia in non-dividing, polarized epithelial cells, and to the spindle, centrosomes and midbodies during mitosis. We propose that NBCn1 traffics between lateral junctions, the leading edge and cell division machinery in Rab11 endosomes, adding new insight to the role of NBCn1 in cell cycle progression.

SETD2 Regulates Nuclear Division to Achieve Polyploidization in Megakaryocyte

Megakaryocytes (MKs) undergo a fascinating endomitosis process to become naturally large multilobulated, polyploid nuclei cells that give rise to platelets. Two key steps involving in this process are the controlled failure in cytokinesis (cytoplasm division) and karyokinesis (nuclear division). The cytokinesis defect has been reported to be regulated by factors related to abnormal actomyosin ring and cleavage furrow formation such as RhoA, MYH9 and Rac1. In contrast, it remains largely unknown how nuclear division defect is regulated during MK endomitosis. Using a mouse model and an in vitro differentiation system, we found SETD2 expression markedly declined when MKs underwent polyploidization. Knockdown of SETD2 increased the high ploidy of primary bone marrow (BM) murine MKs, as well as MKs derived from cultured human cord-blood CD34+ cells and MEG-01 cells. Furthermore, the higher ploidy of MKs was accompanied by the increase of platelet formation both in the mouse model and the in vitro differentiation system. With forced overexpression of SETD2, the proportion of high ploidy MKs (≥8N) was remarkably decreased. These data suggest that SETD2, an α-tubulin methyltransferase, negatively regulates polyploidization during megakaryopoiesis. To investigate how SETD2 regulates endomitosis, we employed live-cell imaging to monitor the whole process of endomitosis at the single-cell level by observing the spindle geometry. We found SETD2 deficient MKs were capable of completing the endomitotic cycle, allowing cells to reach a higher ploidy in a significantly shorter time compared to control cells (2.9 hours vs. 4.9 hours). In comparison, overexpression of SETD2 significantly increased endomitosis cycle time compared to control conditions (5.2 hours vs. 4.3 hours). These results show that SETD2 deficiency accelerates the endomitotic cell cycle. Moreover, loss of SETD2 increased the percentage of cells within the presence of nucleoplasmic bridges (NPBs), a thin DNA link referred as a characteristic structure for karyokinesis defect. These results suggest that SETD2 may regulate endomitosis through facilitating the defect of karyokinesis. During karyokinesis, microtubules localized near spindle poles are need to be depolymerized as force-generating apparatus to separate nuclei. Thus, we measured the amount of polymerized and depolymerized microtubule forms in the presence and absence of SETD2. We found that loss of SETD2 decreased the proportion of the depolymerized form of tubulin in spindles, while SETD2 overexpression increased depolymerized microtubule forms. These data suggest that loss of SETD2 promotes the defect of karyokinesis by reducing depolymerized microtubule state. Mechanistically, as SETD2 could trimethylate α-tubulin at lysine 40 (α-tubK40me3), we found that the levels of α-TubK40me3 near spindle poles were inversely correlated with the degree of karyokinesis defect and MK polyploidization. Moreover, SETD2-deficiency resulted in an absence of α-TubK40me3 with an increase of the MK ploidy. And this ploidy increase was largely reversed by overexpressing a mutant tubulin protein that mimics the α-tubK40me3. Together, these data indicate that SETD2-mediated α-TubK40me3 regulates MK polyploidization by influencing microtubule dynamics. In summary, our study reveals that SETD2 is an important, novel regulator of MK endomitosis by regulating α-TubK40me3 to influence microtubule dynamics. These findings help fill current knowledge gaps in the mechanism of the karyokinesis defect during MK endomitosis. Targeting SETD2 may improve high ploidy MKs and platelet production that could promote the regeneration of platelets in vitro and help treat megakaryocytic dysplasia diseases with abnormal polyploidization in the future.

O-171 Altered meiotic spindle morphology and composition in in vitro matured oocytes

Abstract Study question How does the meiotic spindle tubulin PTMs of MII oocytes matured in vitro compare to that of MII oocytes matured in vivo? Summary answer MII cultured in vitro present detyrosinated tubulin in the spindle microtubules, while MII oocytes matured in vivo do not. What is known already A functional spindle is required for chromosomal segregation during meiosis, but the role of tubulin post-translational modifications (PTMs) in spindle meiotic dynamics remains poorly characterized. In contrast with GVs matured in vitro within the cumulus oophorous, in vitro maturation of denuded GVs to the MII stage (GV-MII) is associated with spindle abnormalities, chromosome misalignment and compromised developmental potential. Although aneuploidy rates in GV-MII are not higher than in vivo matured MII, disorganized chromosomes may contribute to compromised developmental potential. However, to date, spindle PTMs morphology of GV-MII has not been compared to that of in vivo cultured MII oocytes. Study design, size, duration GV (n = 125), and MII oocytes (n = 24) were retrieved from hormonally stimulated women, aged 20 to 35 years old. GVs were matured to the MII stage in vitro in G-2 PLUS medium for 30h; the maturation rate was 68,2%; the 46 GV-MII oocytes obtained were vitrified, stored, and warmed before fixing and subjecting to immunofluorescent analysis. In vivo matured MII oocytes donated to research were used as controls. Participants/materials, setting, methods Women were stimulated using a GnRH antagonist protocol, with GnRH agonist trigger. Trigger criterion was ≥2 follicles ≥18mm; oocytes were harvested 36h later. Spindle microtubules were incubated with antibodies against alpha tubulin and tubulin PTMs (acetylation, tyrosination, polyglutamylation, Δ2-tubulin, and detyrosination); chromosomes were stained with Hoechst 33342 and samples subjected to confocal immunofluorescence microscopy (ZEISS LSM780), with ImageJ software analysis. Differences in spindle morphometric parameters were assessed by non-parametric Kruskal–Wallis and Fisher’s exact tests. Main results and the role of chance Qualitatively, Δ2-tubulin, tyrosination and polyglutamylation were similar for both groups. Acetylation was also present in both groups, albeit in different patterns: while in vivo matured MII oocytes showed acetylation at the poles, GV-MII showed a symmetrical distribution of signal intensity, but discontinuous signal on individual microtubule tracts, suggesting apparent islands of acetylation. In contrast, detyrosination was detected in in vivo matured MII oocytes but was absent from GV-MII. Regarding spindle pole morphology, of the four possible phenotypes described in the literature (double flattened and double focused; flattened-focused, focused-flattened, with the first word characterizing the cortex side of the spindle), we observed double flat shaped spindle poles in 86% of GV-MII oocytes (25/29) as opposed to 40.5% (15/37) for the in vivo matured MII oocytes (p = 0.0004, Fisher’s exact test). Further morphometric analysis of the spindle size (maximum projection, major and minor axis length) and the metaphase plate position (proximal to distal ratio, angle) revealed decreased spindle size in GV-MII oocytes (p = 0.019, non parametric Kruskal- Wallis test). Limitations, reasons for caution Oocytes retrieved from hyperstimulation cycles could be intrinsically impaired since they failed to mature in vivo. Our conclusions should not be extrapolated to IVM in non-stimulated cycles, as in this model, the cumulus oophorus is a major factor in oocyte maturation and correlation with spindle dynamics has been inferred. Wider implications of the findings The metaphase II spindle stability compared to the mitotic or metaphase I meiotic one justifies the presence of PTMs such as acetylation and glutamylation, which are found in stable, long-lived microtubules. The significance of the absence of detyrosinated microtubules in the MII-GV group remains to be determined Trial registration number not applicable

Spindle tubulin and MTOC asymmetries may explain meiotic drive in oocytes

In the first meiotic division (MI) of oocytes, the cortically positioned spindle causes bivalent segregation in which only the centre-facing homologue pairs are retained. ‘Selfish’ chromosomes are known to exist, which bias their spindle orientation and hence retention in the egg, a process known as ‘meiotic drive’. Here we report on this phenomenon in oocytes from F1 hybrid mice, where parental strain differences in centromere size allows distinction of the two homologue pairs of a bivalent. Bivalents with centromere and kinetochore asymmetry show meiotic drive by rotating during prometaphase, in a process dependent on aurora kinase activity. Cortically positioned homologue pairs appear to be under greater stretch than their centre-facing partners. Additionally the cortex spindle-half contain a greater density of tubulin and microtubule organising centres. A model is presented in which meiotic drive is explained by the impact of microtubule force asymmetry on chromosomes with different sized centromeres and kinetochores.

Combination of spindle and first polar body chromosome images for the enhanced prediction of developmental potency of mouse metaphase II oocytes.

The objective of this study was to classify spindle and first polar body (PB1) chromosome images in ovulated mouse oocytes over time to predict the developmental competence of metaphase II (MII) oocytes. Oocytes were collected at 12, 15, 20, and 25 h after human chorionic gonadotropin (hCG) injection, and stained for spindle tubulin, chromosomes, and PB1 chromosomes. MII spindle morphology was classified as tapered type or barrel type and PB1 chromosomes were categorized as aggregated, separated, dot, or collapsed. To determine whether differences in spindle and PB1 images in MII oocytes are associated with fertilization success, we performed in vitro fertilization (IVF) at various times after hCG injection. Barrel-type spindles and aggregate-type PB1 were dominant at 12 h after hCG injection. Oocyte spindles collected 1 h after injection were tapered, and PB1 chromosomes were separated. At 20 and 25 h after treatment, spindle and PB1 images were classified as collapsed. The rate of development to 2-cell embryos after IVF did not differ between the 12 h and 15 h treatments; however, it was significantly lower for the 25 h treatment than for other treatments. The rates of development to blastocysts at 12, 15, 20, and 25 h after hCG injection were 61, 46, 42, and 9%, respectively. MII oocytes with barrel-type spindles and aggregate-type PB1 had high rates of fertilization and blastocyst development, and spindle and PB1 characteristics were correlated with the outcomes of IVF and embryo culture. These results suggested that images of spindles combined with those of PB1 chromosomes enable the prediction of oocytic and/or embryonic quality.

Overexpression of spindlin1 induces metaphase arrest and chromosomal instability

Spin/Ssty gene family is high conserved and very abundant transcript involved in gametogenesis, which was repeatedly detected in early embryo. Nevertheless, the biologic roles of the members are still largely unknown. Previously we have identified human gene spindlin1 as a homologue of the family from ovarian cancer cells, and reported that stable overexpression of spindlin1 could transform NIH3T3 cells and induce tumorigenesis in nude mouse. Here, we showed that spindlin1, as a nuclear protein, was relocated during mitosis. A fraction of spindlin1 proteins was dynamic distributed along mitotic spindle tubulin and enriched at midzone following anaphase entering. We also showed that transient overexpression of spindlin1 induced cell cycle delay in metaphase, caused mitotic spindle defects, and resulted in chromosome instability, micronucleus and multinuclear giant cells formation. Moreover, time-lapse microscopy revealed that these cells arrested at metaphase for more than 3 h with chromosome nondisjunction or missegregation. Furthermore, Mad2 up-regulation in these cells suggested that overexpression of spindlin1 may affect the bipolar spindle correctly attachment to chromosomes and activate spindle checkpoint. Taken together, these data demonstrated that excess spindlin1 protein may be detrimental for spindle microtubule organization, chromosomal stability and can potentially contribute to the development of cancer.

Safety and Efficacy of Marqibo (Vincristine Sulfate Liposomes Injection, OPTISOME™) in for the Treatment of Adults with Relapsed or Refractory Acute Lymphoblastic Leukemia (ALL).

Introduction: Vincristine sulfate (VCR) is a lipophilic, cell-cycle specific, antineoplastic agent that inhibits cell division by specifically binding to tubulin in mitotic spindles. The activity of VCR is dose and time-dependent, but central and peripheral neuropthy prohibits its use beyond doses of 1.4 mg/m2 (capped at 2 mg). Marqibo is a formulation of VCR encapsulated in a sphingomyelin/cholesterol liposome (OPTISOME) with a longer half-life than VCR. In murine models using L1210 or P388 lymphoid leukemia cell lines, Marqibo demonstrated greater anti-tumor activity compared with VCR. Marqibo was therefore an appropriate agent to study in relapsed or refractory ALL.Methods: Two clinical trials have been completed. First, a phase II trial of single agent Marqibo given at 2 mg/m2 (no dose capping) every 2 weeks enrolled 16 patients (pts) [Thomas et al, Cancer 106:120, 2006]. A multicenter dose escalation phase I trial using weekly Marqibo (1.5, 1.825, 2.0, 2.25, 2.4 mg/m2) in combination with pulse dexamethasone followed. There were no restrictions on the number of prior therapies. Subjects with grade 2 or greater central or peripheral neuropathy were ineligible.Results: In total, 52 pts with relapsed or refractory ALL were treated in the two studies combined. Median age was 34 years (range, 19–77), 31 males/21 females, with a median number of prior salvage regimens of 2 (range, 1–3). Nine pts had confirmed Philadelphia positive disease (8 in the pre-imatinib era, 1 resistant to tyrosine kinase inhibitor therapy). All pts had previously received conventional VCR therapy. There were 8 complete remissions and 3 partial remissions for an overall response rate of 21% [95% CI, 11, 35]. An additional 12 pts (23%) achieved hematological improvements (e.g., clearance of marrow blasts, platelet transfusion independence). Five responders were able to undergo allogeneic stem cell transplantation following therapy with Marqibo. The maximum tolerated dose in the phase I trial was 2.25 mg/m2 owing to grade 3 motor neuropathy, grade 4 seizure, and grade 4 hepatotoxicity observed in 1 patient each at the 2.4 mg/m2 dose level. Grade 1–2 peripheral neuropathy was manageable with dose modifications and anti-neuralgia agents such as gabapentin. Commonly observed toxicities included febrile neutropenia, myelosuppression, abdominal pain, nausea, constipation, diarrhea, fatigue, and infusion-related pyrexia.Conclusions: In reviewing the clinical experience to date, Marqibo with or without pulse dexamethasone has provided clinically meaningful activity in heavily pre-treated adults with ALL. A multicenter trial of single agent Marqibo in the setting of second salvage therapy for adults with relapsed ALL is underway. A phase III multicenter trial of Marqibo in combination with standard chemotherapy for de novo elderly ALL is in the planning phase.

Marqibo (Vincristine Sulfate Liposomes Injection, OPTISOME™) Concentrates Vincristine in Tumor Tissue and Lymphoid Malignancy Oriented Tissues in Tumor-Bearing Mice.

Background: Vincristine sulfate (VCR) is a cell-cycle specific, lipophilic, anti-cancer drug that inhibits cell division by binding to tubulin in mitotic spindles. Marqibo is a proprietary sphingomyelin/cholesterol liposome (OPTISOME) encapsulated formulation of VCR with an extended circulating half-life and the potential for preferential malignancy targeting, exposure, and anti-cancer activity. This report evaluates the concentration and accumulation of VCR in solid tumor tissue as well as tissues frequently involved with lymphoid malignancies in tumor-bearing mice following equivalent doses of Marqibo or conventional VCR.Methods: Mice were implanted subcutaneously with MX-1 human breast tumor tissue. When the tumor volume reached 162–485 mm3, mice received a single intravenous dose of Marqibo, containing 1.5 mg/kg of [14C]-VCR, or 1.5 mg/kg of non-liposomal [14C]-VCR. The total radioactivity from parent compound and metabolites in tissue, VCReq, was analyzed by Quantitative Whole Body Autoradioluminography (QWBA) and tissue digestion (TD). QWBA analysis of bone marrow (BM), tumor tissue, lymph nodes, liver, and spleen required 1 flash-frozen mouse per group at various time intervals. Sagittal sections were examined histologically for radioactivity. For TD, all tissues except bone marrow were obtained from 3 mice per group prior to infusion and at selected time points. The samples were chemically digested and analyzed for radioactivity by liquid scintillation counting.Results: QWBA analysis revealed at least two-fold higher VCReq tissue concentrations in the Marqibo-injected mice compared to the conventional VCR-injected mice.VCReq Tissue Concentration (mcg/g) at 48 hours Post-InjectionBMTumorLymphLiverSpleenMarqibo0.991.351.980.476.08VCR0.360.31--0.130.76Marqibo/VCR ratio2.754.35--3.618The TD results are consistent with the QWBA results shown in the table. Marqibo-injected mice demonstrated a minimum 5-fold increase in VCR exposure over a 96 hour period (VCReq AUC analysis) compared to VCR-injected mice over a 48 hour period, as defined in the parameters of the study, in the implanted tumor (123 vs. 21 hr*μg/g), lymph nodes (121 vs. 16.5 hr*μg/g), liver (68.8 vs. 12 hr*μg/g), and spleen (512 vs. 68.9 hr*μg/g). Though the parameters for the AUC values may favor Marqibo, the Tmax of all specified tissues are at least 16 times greater in Marqibo-injected mice than in VCR-injected mice (16 hrs vs. 1hr in tumor, spleen, and lymph; 4 hrs vs. 15 min. in liver; 16 hrs. vs. 15 min. in the kidney). The greatest increase in VCR exposure produced by Marqibo was observed in the spleen and lymph nodes (7.4- and 7.3-fold).Conclusions: Marqibo resulted in targeted delivery of VCR; concentration of VCR in tumor tissue, BM, lymph nodes, liver, and spleen; and maintenance of significant tissue drug concentrations for an extended period of time compared to conventional VCR. The ability of Marqibo to target drug to these tissues and organs makes it particularly attractive as a treatment for hematologic malignancies like myeloma, lymphoma, and acute lymphoblastic leukemia (ALL). Late-stage clinical development of Marqibo in adult ALL is underway.

Modulation of vinblastine cytotoxicity by dilantin (phenytoin) or the protein phosphatase inhibitor okadaic acid involves the potentiation of anti-mitotic effects and induction of apoptosis in human tumour cells

Cellular insensitivity to vinca alkaloids is suggested to be primarily due to drug efflux by P-glycoprotein (P-gp). The anti-epileptic phenytoin (DPH), which does not bind to P-gp, can selectively enhance vincristine (VCR) cytotoxicity in wild-type (WT) or multidrug-resistant (MDR) cells. We now demonstrate that the protein phosphatase inhibitor okadaic acid (OKA) can mimic the effect of DPH by selectively enhancing cytotoxicity of vinblastine (VBL), but not taxol and doxorubicin, in human leukaemia HL-60 cells. Both DPH and OKA potentiate the anti-mitotic effects of VBL by enhanced damage to the mitotic spindle, resulting in prolonged growth arrest. Also, unlike VBL alone, in human leukaemia or non-small-cell lung carcinoma cells treated with VBL plus DPH, recovery from damage to the mitotic spindle is compromised in drug-free medium and cell death by apoptosis in interphase ensues. Since protein phosphatases are involved with the regulation of metaphase to anaphase transit of cells during the mitotic cycle, enhanced VBL cytotoxicity in the presence of DPH or OKA may involve effects during metaphase on the mitotic spindle tubulin leading to growth arrest and apoptosis in interphase. These novel results suggest that DPH or OKA could be powerful tools to study cellular effects of vinca alkaloids and possibly for the development of novel therapeutic strategies.

Physiological evidence for involvement of a kinesin-related protein during anaphase spindle elongation in diatom central spindles.

We have developed a new model system for studying spindle elongation in vitro using the pennate, marine diatom Cylindrotheca fusiformis. C. fusiformis can be grown in bulk to high densities while in log phase growth and synchronized by a simple light/dark regime. Isolated spindles can be attained in quantities sufficient for biochemical analysis and spindle tubulin is approximately 5% of the total protein present. The spindle isolation procedure results in a 10-fold enrichment of diatom tubulin and a calculated 40-fold increase in spindle protein. Isolated spindles or spindles in permeabilized cells can elongate in vitro by the same mechanism and with the same pharmacological sensitivities as described for other anaphase B models (Cande and McDonald, 1986; Masuda et al., 1990). Using this model, in vitro spindle elongation rate profiles were developed for a battery of nucleotide triphosphates and ATP analogs. The relative rates of spindle elongation produced by various nucleotide triphosphates parallel relative rates seen for kinesin-based motility in microtubule gliding assays. Likewise ATP analogs that allow discrimination between myosin-, dynein-, and kinesin-mediated motility produce relative spindle elongation rates characteristic of kinesin motility. Also, isolated spindle fractions are enriched for a kinesin related protein as identified by a peptide antibody against a conserved region of the kinesin superfamily. These data suggest that kinesin-like motility contributes to spindle elongation during anaphase B of mitosis.

Incorporation and turnover of labeled exogenous tubulin in the mitotic spindles of Chaetopterus oocytes and HeLa cells.

The incorporation of tubulin into mitotic spindles in situ was studied by incubating permeabilized mitotic cells in solutions containing [3H]GTP-labeled or dichlorotriazinylamino fluorescein (DTAF)-labeled tubulin. Metaphase HeLa cells or spindle-containing "minicells" from Chaetopterus oocytes were lysed in a microtubule-assembly buffer plus 0.5% Nonidet P-40, 1 mg/ml 120,000g supernatant mammalian brain tubulin, and [3H]GTP. After different periods of incubation, mitotic spindles were isolated in 2 M-glycerol-containing assembly buffer and separated from unbound counts by centrifugation through a 4 M-glycerol cushion; 3H counts per mg protein increase linearly for 8-12 min and then reach a plateau or steady state in both Chaetopterus oocytes and HeLa cells. Addition of 4 mM CaCl2 blocks or reverses incorporation. Little or no [3H]GTP is incorporated if exogenous tubulin or lysed cells are omitted from the assembly mixture. To measure the loss rate of [3H]GTP-tubulin from mitotic spindles, cells were incubated in tubulin plus [3H]GTP for 30 min, and a 20-fold excess of cold GTP (2 mM) was added. Samples were removed after incubation for different periods, and spindles were isolated as described above and counted for 3H content. [3H]GTP is lost from spindles at a rate of about 16%/min until a new steady state is reached in about 8 min. These results are consistent with an incorporation and turnover of [3H]GTP-tubulin in spindle microtubules of these lysed-cell models. The location of this newly incorporated tubulin in the spindle was investigated by incorporating fluorescent DTAF-tubulin into mitotic spindles of these lysed cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

Tubulin composition and microtubule nucleation of a griseofulvin-resistant Chinese hamster ovary cell mutant with abnormal spindles

A griseofulvin-resistant Chinese hamster ovary (CHO) mutant (Grs-2) which has an altered β-tubulin subunit as well as wild-type β-tubulin is temperature-sensitive (ts) for growth at 40.5 °C. This growth defect appears to result from the formation of abnormal mitotic spindles at the non-permissive temperature (Abraham, I et al., J cell biol 97 (1983) 1055) [19]. Light microscopy of spindles isolated from mutant cells cultured at the permissive temperature showed a typical bipolar morphology, whereas spindles isolated at the non-permissive temperature were multipolar. In order to study the role of tubulin in spindle formation, we analyzed the tubulin composition of the multipolar spindles. Two-dimensional gels and immunoblotting analysis of one-dimensional electrophoretic gels stained with monoclonal anti-Chinese hamster brain β-tubulin antibody revealed that both mutant and wild-type β-tubulins were present in similar proportions in both bipolar spindles at 37 °C and multipolar spindles at 40.5 °C. The ratio between wild-type and mutant tubulin in spindles was also found to be the same as in the cytoplasmic microtubule network in interphase cells, providing evidence that the mutant β-tubulin appeared to be incorporated in a similar manner into both interphase and mitotic microtubule structures. In vitro microtubule polymerization onto centrosomes prepared from mutant Grs-2 demonstrated that 80% of the sites for microtubule nucleation were without centrioles, suggesting fragmentation of pericentriolar material away from centrioles. This may be one of the causes of multipolar spindle formation in the mutant cells. These results, therefore, suggest that abnormal formation of spindles in mutant cells is due not to the presence of the mutant tubulin per se, but to the abnormal behavior of this mutant tubulin in the cellular environment during mitosis or abnormal interaction with other components in the spindle at 40.5 °C.

Aprotic polar solvents inducing chromosomal malsegregation in yeast interfere with the assembly of porcine brain tubulin in vitro

A number of aprotic solvents which had previously been found to induce mitotic aneuploidy in yeast were tested for their effects on re-assembly of twice recycled tubulin from pig brain. Some of the solvents which were strong aneuploidy-inducing mutagens in yeast slowed down tubulin assembly in vitro at concentrations lower than those required for aneuploidy induction. Ethyl acetate, methyl acetate, diethyl ketone and acetonitrile fell into this category. Other strong aneuploidy-inducing agents like acetone and 2-methoxyethyl acetate accelerated tubulin assembly. Non-genetically active methyl isopropyl ketone and isopropyl acetate both accelerated assembly, whereas methyl n-propyl ketone and n-propyl acetate were weak inducers of aneuploidy and slowed down the rate and extent of assembly. Those chemicals which slowed down the assembly rate also reduced the extent of assembly. Most chemicals which accelerated assembly also led to an increased extent of assembly, with the exception of isopropyl acetate. At the higher concentrations, however, a maximum assembly rate was reached which was followed by a slow decline. Although a perfect correlation between effects on the induction of chromosomal malsegregation and the interference with tubulin assembly in vitro was not seen, the experiments with tubulin were carried out using this class of chemicals because some of them strongly induced mitotic aneuploidy under conditions which suggested tubulin to be the prime target. The lack of a perfect coincidence might be due to species differences between the porcine brain and the yeast spindle tubulin, or the test for aneuploidy induction may have been negative because the concentrations required for an effect on yeast tubulin may be greater than the general lethal toxicity limit. Bearing this reservation in mind, the results suggest that the yeast aneuploidy test has a considerable predictive value for mammalian mutagenicity.

Programmed expression of beta-tubulin genes during development and differentiation of the chicken.

We have previously demonstrated that the chicken genome contains at least four different, functional beta-tubulin genes. By using gene specific probes we have now analyzed the relative levels of expression of the four encoded messenger RNA (mRNA) transcripts as a function of chicken development and differentiation. We have found that the RNA transcript from the beta 2 gene is present in large amounts in embryonic chick brain and is also preferentially expressed in spinal cord neurons, indicating that this transcript encodes the dominant neuronal beta-tubulin polypeptide. The beta 3 mRNA is present in overwhelming amounts in RNA from chicken testis suggesting that this gene encodes a flagellar or meiotic spindle tubulin. However, both of these genes are transcribed to varying, but lesser, degrees in a number of additional cell and tissue types indicating that they are not neuronal or testis specific, respectively. Beta 4' transcripts are present at moderate levels in all cell and tissue types examined, suggesting that this mRNA encodes a constitutive beta-tubulin polypeptide that is involved in an essential or housekeeping microtubule function. Transcripts from the beta 1 gene are a minor component of the beta-tubulin mRNA populations in all cells and tissues tested. Overall, we conclude that specific beta-tubulin mRNA species are expressed in markedly different ratios in different tissues in the chicken. Such developmental regulation may reflect the function(s) of the individual beta-tubulin polypeptides or, alternatively, may be required for precise control of tubulin gene expression in cells that utilize microtubules for divergent purposes.

Purification and characterization of oocyte cytoplasmic tubulin and meiotic spindle tubulin of the surf clam Spisula solidissima.

Assembly-competent tubulin was purified from the cytoplasm of unfertilized and parthogenetically activated oocytes, and from isolated meiotic spindles of the surf clam, Spisula solidissima. At 22 degrees C or 37 degrees C, Spisula tubulin assembled into 48-51-nm macrotubules during the first cycle of polymerization and 25-nm microtubules during the third and subsequent cycles of assembly. Macrotubules were formed from sheets of 26-27 protofilaments helically arranged at a 36 degree angle relative to the long axis of the polymer and were composed of alpha and beta tubulins and several other proteins ranging in molecular weight from 30,000 to 270,000. Third cycle microtubules contained 14-15 protofilaments in cross-section and were composed of greater than 95% alpha and beta tubulins. After three cycles of polymerization at 37 degrees C, unfertilized and activated oocyte tubulin self-assembled into microtubules at a critical concentration (Ccr) of 0.09 mg/ml. At the physiological temperature of 22 degrees C, unfertilized oocyte tubulin assembled into microtubules at a Ccr of 0.36 mg/ml, activated oocyte tubulin assembled at a Ccr of 0.42 mg/ml, and isolated meiotic spindle tubulin assembled at a Ccr of 0.33 mg/ml. The isoelectric points of tubulin from both unfertilized oocytes and isolated meiotic spindles were 5.8 for alpha tubulin and 5.6 for beta tubulin. In addition, one dimensional peptide maps of oocyte and spindle alpha and beta tubulins were very similar, if not identical. These results indicate that unfertilized oocyte tubulin and tubulin isolated from the first meiotic spindle are indistinguishable on the basis of assembly properties, isoelectric focusing, and one dimensional peptide mapping. These results suggest that the transition of tubulin from the quiescent oocyte state to that competent to form spindle microtubules in vivo does not require special modification of tubulin but may involve changes in the availability of microtubule organizing centers or assembly-promoting microtubule-associated proteins.

Strongylocentrotus purpuratus spindle tubulin. I. Characteristics of its polymerization and depolymerization in vitro.

Tubulin was extracted from spindles isolated from embryos of the sea urchin Strongylocentrotus purpuratus, repolymerized in vitro, and purified through three cycles of temperature-dependent assembly and disassembly. In addition to the tubulin, these preparations contain a protein of 80 kdaltons and a small but variable amount of actin. At 37 degrees C, the tubulin polymerizes with a critical concentration of 0.15-0.2 mg/ml into smooth-walled polymers which contain predominantly 14 protofilaments. Removal of the 80 kdalton protein and the actin by DEAE-chromatography does not change the critical concentration for polymerization. At 15 degrees C, which is within the range of physiological temperatures for S. purpuratus embryos, the spindle tubulin will self-assemble, but the rate of total polymer formation is very slow, requiring hours in the test tube. This rate can be increased by shearing the polymerizing microtubules, creating more ends for assembly, indicating that the slow rate of polymer formation is due to a slow rate of self-initiation. If spindle tubulin is polymerized at 37 degrees C and then lowered to 15 degrees C, some polymer will be retained, the percentage of which depends on the protein concentration. These results demonstrate that spindle tubulin from S. purpuratus will assemble at 37 degrees C with a low critical concentration for polymerization in the absence of detectable MAPs and will self-assemble and maintain steady state levels of polymer at physiological temperatures.

Identification of microtubule-associated proteins in the meiotic spindle of surf clam oocytes.

Meiotic spindles isolated from surf clam oocytes to morphological purity are biochemically complex, consisting of many polypeptides. These proteins fall into two classes: (a) polypeptides that are apparently cytoplasmic proteins and are not specifically associated with the spindle; and (b) polypeptides that are specifically associated with the spindle. A subset of the spindle-associated proteins, including a 250,000 mol wt component, remain with spindle tubulin through cycles of cold depolymerization and warm polymerization, showing that they are microtubule-associated proteins.

THE MECHANISM OF SOMATIC ASSOCIATION IN COMMON WHEAT, TRITICUM AESTIVUM L. IV. FURTHER EVIDENCE FOR MODIFICATION OF SPINDLE TUBULIN THROUGH THE SOMATIC-ASSOCIATION GENES AS MEASURED BY VINBLASTINE BINDING

Treatment with the antitubulin vinblastine was found to disrupt the spindle system in dividing root-tip cells of common wheat, Triticum aestivum L. Genotypes lacking the somatic association suppressor gene on 5B(L), or containing the somatic-association promoter on 5B(S), were found to be more sensitive to the treatment. In genetic lines carrying the somatic association suppressor, sensitivity to vinblastine was lower and there was a direct correlation between dosage of the suppressor gene (0, 2, and 4) and the decrease in spindle disruption on exposure to various concentrations of vinblastine. It is concluded that the somatic association genes affect binding ability of spindle tubulin to vinblastine. Since the same genes affect binding of colchicine to tubulin and since the two alkaloids attach to different sites it is assumed that the somatic association suppressor gene has a broad effect on the tubulin molecules which is not confined to a single site. The relevance of genetic control of antitubulin binding to somatic association is discussed.