Multipolar Spindle Formation Research Articles

HTPX2 Is Required for Normal Spindle Morphology and Centrosome Integrity during Vertebrate Cell Division

Bipolar spindle formation is essential for the accurate segregation of genetic material during cell division. Although centrosomes influence the number of spindle poles during mitosis, motor and non-motor microtubule-associated proteins (MAPs) also play key roles in determining spindle morphology [1]. TPX2 is a novel MAP also characterized in Xenopus cell-free extracts [2, 3]. To examine hTPX2 (human TPX2) function in human cells, we used siRNA to knock-down its expression and found that cells lacking hTPX2 arrest in mitosis with multipolar spindles. NuMA, γ-tubulin, and centrin localize to each pole, and nocodazole treatment of cells lacking hTPX2 demonstrates that the localization of γ-tubulin to multiple spindle poles requires intact microtubules. Furthermore, we show that the formation of monopolar microtubule arrays in human cell extracts does not require hTPX2, demonstrating that the mechanism by which hTPX2 promotes spindle bipolarity is independent of activities focusing microtubule minus ends at spindle poles. Finally, inhibition of the kinesin Eg5 in hTPX2-depleted cells leads to monopolar spindles, indicating that Eg5 function is necessary for multipolar spindle formation in the absence of hTPX2. Our observations reveal a structural role for hTPX2 in spindles and provide evidence for a balance between microtubule-based motor forces and structural spindle components.

Centrosome replication, genomic instability and cancer.

Karyotypic alterations, including whole chromosome loss or gain, ploidy changes, and a variety of chromosome aberrations are common in cancer cells. If proliferating cells fail to coordinate centrosome duplication with DNA replication, this will inevitably lead to a change in ploidy, and the formation of monopolar or multipolar spindles will generally provoke abnormal segregation of chromosomes. Indeed, it has long been recognized that errors in the centrosome duplication cycle may be an important cause of aneuploidy and thus contribute to cancer formation. This view has recently received fresh impetus with the description of supernumerary centrosomes in almost all solid human tumors. As the primary microtubule organizing center of most eukaryotic cells, the centrosome assures symmetry and bipolarity of the cell division process, a function that is essential for accurate chromosome segregation. In addition, a growing body of evidence indicates that centrosomes might be important for initiating S phase and completing cytokinesis. Centrosomes undergo duplication precisely once before cell division. Recent reports have revealed that this process is linked to the cell division cycle via cyclin-dependent kinase (cdk) 2 activity that couples centriole duplication to the onset of DNA replication at the G(1)/S phase transition. Alterations in G(1)/S phase regulating proteins like the retinoblastoma protein, cyclins D and E, cdk4 and 6, cdk inhibitors p16(INK4A) and p15(INK4B), and p53 are among the most frequent aberrations observed in human malignancies. These alterations might not only lead to unrestrained proliferation, but also cause karyotypic instability by uncontrolled centrosome replication. Since several excellent reports on cell cycle regulation and cancer have been published, this review will focus on the role of centrosomes in cell cycle progression, as well as causes and consequences of aberrant centrosome replication in human neoplasias.

Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function

Survivin is a member of the inhibitor of apoptosis gene family that has been implicated in both apoptosis inhibition and regulation of mitosis. However, the subcellular distribution of survivin has been controversial and variously described as a microtubule-associated protein or chromosomal passenger protein. Here, we show that antibodies directed to the survivin sequence Ala(3)-Ile(19) exclusively recognized a nuclear pool of survivin that segregated with nucleoplasmic proteins, but not with outer nuclear matrix or nuclear matrix proteins. By immunofluorescence, nuclear survivin localized to kinetochores of metaphase chromosomes, and to the central spindle midzone at anaphase. However, antibodies to Cys(57)-Trp(67) identified a cytosolic pool of survivin, which associated with interphase microtubules, centrosomes, spindle poles and mitotic spindle microtubules at metaphase and anaphase. Polyclonal antibodies recognizing survivin epitopes Ala(3)-Ile(19), Met(38)-Thr(48), Pro(47)-Phe(58) and Cys(57)-Trp(67) identified both survivin pools within the same mitotic cell. A ratio of approximately 1:6 for nuclear versus cytosolic survivin was obtained by quantitative subcellular fractionation. In synchronized cultures, cytosolic survivin abruptly increased at mitosis, physically associated with p34(cdc2), and was phosphorylated by p34(cdc2) on Thr(34), in vivo. By contrast, nuclear survivin began to accumulate in S phase, was not complexed with p34(cdc2) and was not phosphorylated on Thr(34). Intracellular loading of a polyclonal antibody to survivin caused microtubule defects and resulted in formation of multipolar mitotic spindles, but did not interfere with cytokinesis. These data demonstrate that although both reported localizations of survivin exist in mitotic cells, the preponderant survivin pool is associated with microtubules and participates in the assembly of a bipolar mitotic spindle.

Calcium, Calmodulin, and CaMKII Requirement for Initiation of Centrosome Duplication in Xenopus Egg Extracts

Aberrant centrosome duplication is observed in many tumor cells and may contribute to genomic instability through the formation of multipolar mitotic spindles. Cyclin-dependent kinase 2 (Cdk2) is required for multiple rounds of centrosome duplication in Xenopus egg extracts but not for the initial round of replication. Egg extracts undergo periodic oscillations in the level of free calcium. We show here that chelation of calcium in egg extracts or specific inactivation of calcium/calmodulin-dependent protein kinase II (CaMKII) blocks even initial centrosome duplication, whereas inactivation of Cdk2 does not. Duplication can be restored to inhibited extracts by addition of CaMKII and calmodulin. These results indicate that calcium, calmodulin, and CaMKII are required for an essential step in initiation of centrosome duplication. Our data suggest that calcium oscillations in the cell cycle may be linked to centrosome duplication.

HSP70 overexpression increases resistance of V79 cells to cytotoxicity of airborne pollutants, but does not protect the mitotic spindle against damage caused by airborne toxins

Exposure of Chinese hamster V79 cells to extracts of airborne pollutants induced formation of multipolar or incomplete mitotic spindles. To find out whether overexpression of the HSP70 chaperone protein could protect spindles against airborne toxins we constructed V79 cells stably transfected with an expression vector containing rat heat-inducible hsp70.1 gene under the control of a constitutive CMV promoter. When cells were incubated with extracts of airborne pollutants (5–20 μg/ml) no protective effect of the HSP70 protein against mitotic spindle damage was observed. Moreover, at 20 μg/ml of extracts of airborne toxins the frequency of mitotic malformations was even higher in HSP70-overexpressing cells than in control ones. Extracts of airborne pollutants of 50 μg/ml blocked the formation of mitotic figures both in control and HSP70-overexpressing cells and led to destruction of cell nuclei. However, the HSP70-overproducing cells exhibited higher survival rates when exposed to heat shock and airborne toxins than the control ones, as determined by MTT assay. This suggests that HSP70 overexpression—a frequent feature of cancer cells—should be considered as a factor facilitating survival of cells with damaged mitotic spindles and aberrantly segregated chromosomes.

Effect of serum depletion on centrosome overduplication and death of human pancreatic cancer cells after exposure to radiation

The tumor microenvironment is one of the key factors affecting the cellular response to radiation; however, the influence of serum concentration on tumor radiosensitivity remains poorly understood. We recently discovered that γ-irradiation of tumor cells causes centrosome overduplication, which may lead to lethal nuclear fragmentation through the establishment of multipolar mitotic spindles. In the present study, we investigated the effect of serum depletion on radiation-induced cell death in relation to the centrosome dynamics in human pancreatic cancer cells. Exposure of Capan-1 cells to γ-irradiation resulted in a time-dependent increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death. Treatment of irradiated cells with serum depletion drastically accelerated centrosome overduplication and the formation of multipolar spindles, resulting in increased nuclear fragmentation and cell death. Cell cycle analysis of irradiated cultures revealed that the reduced serum level increased the population of cells arrested in the G2/M phase, which might be responsible for the abnormal centrosome accumulation. These findings suggest that serum concentration can influence radiation-induced cell killing through modulating cell cycle progression and possibly centrosome overduplication.

Centrosome abnormalities in human carcinomas of the gallbladder and intrahepatic and extrahepatic bile ducts.

During mitosis, 2 centrosomes ensure accurate assembly of bipolar spindles and fidelity of the chromosomal segregation. The presence of more than 2 copies of centrosomes during mitosis can result in the formation of multipolar spindles, unbalanced chromosome segregation, and aneuploidy. Recent studies have provided evidence that centrosome hyperamplification plays a pivotal role in carcinogenesis. Using immunofluorescence analysis with gamma-tubulin and pericentrin antibodies, paraffin-embedded sections from 40 malignant biliary diseases including gallbladder cancers (GC; n = 13), intrahepatic cholangiocellular carcinoma (CCC; n = 19), and extrahepatic bile duct cancers (BDC; n = 8) were examined. Thirty-seven benign biliary diseases including chronic cholecystitis, gallbladder adenoma, hepatolithiasis, and choledochal cyst were included as benign controls. The frequencies of the centrosome abnormalities were 70% for GC, 58% for CCC, and 50% for BDC, respectively. The frequencies of centrosome abnormalities in malignant biliary diseases were significantly higher than in their benign counterparts (GC, CCC, BDC; P =.001,.002, and.001, respectively). The results of current study also indicated that biliary malignancy in the advanced stage (III-IV) displayed a higher frequency of centrosome abnormalities than in the early stage (I-II) (P <.001). We conclude that abnormalities in size, number, and shape of the centrosome are frequently observed in biliary tract malignancy. Centrosome abnormalities started to occur in the early stage of biliary malignancy and became very frequent in the advanced stage. This implies that centrosome abnormality might relate to the transition from early to advanced malignancy in biliary malignancy.

Differential regulation of maternal vs. paternal centrosomes.

Centrosomes are the main microtubule-organizing centers in animal cells. During meiosis and mitosis, two centrosomes form the poles that direct the assembly of a bipolar spindle, thus ensuring the accurate segregation of chromosomes. Cells cannot tolerate the presence of more than two active centrosomes during meiosis or mitosis because doing so results in the formation of multipolar spindles, infidelity in chromosome segregation, and aneuploidy. Here, we show that fertilization of Spisula solidissima oocytes results in cells that contain three active centrosomes, two maternal and one paternal. During meiosis I, the paternal centrosome's ability to nucleate microtubules is selectively shut off while maternal centrosomes remain competent to nucleate microtubules and assemble asters in the same cytoplasm. We propose that embryos can identify paternal vs. maternal centrosomes and can control them differentially.

Requirement of Cdk2-cyclin E activity for repeated centrosome reproduction in Xenopus egg extracts.

The abnormally high number of centrosomes found in many human tumor cells can lead directly to aneuploidy and genomic instability through the formation of multipolar mitotic spindles. To facilitate investigation of the mechanisms that control centrosome reproduction, a frog egg extract arrested in S phase of the cell cycle that supported repeated assembly of daughter centrosomes was developed. Multiple rounds of centrosome reproduction were blocked by selective inactivation of cyclin-dependent kinase 2-cyclin E (Cdk2-E) and were restored by addition of purified Cdk2-E. Confocal immunomicroscopy revealed that cyclin E was localized at the centrosome. These results demonstrate that Cdk2-E activity is required for centrosome duplication during S phase and suggest a mechanism that could coordinate centrosome reproduction with cycles of DNA synthesis and mitosis.

The effect of taxol onTriticum preprophase root cells: preprophase microtubule band organization seems to depend on new microtubule assembly

To examine whether preprophase microtubule band (PPB) organization occurs by rearrangement of pre-existing, or by assembly of new microtubules (Mts), we treated root cells ofTriticum turgidum with taxol, which stabilizes pre-existing Mts by slowing their depolymerization. With taxol early preprophase cells failed to form a normal PPB and PPB narrowing was prevented in cells that had already formed a wide one. The PPB became persistent in prometaphase cells and the formation of multipolar prophase-prometaphase spindles was induced. These data favour the suggestion that PPB formation and narrowing, as well as prophase spindle development, are dynamic processes depending on continuous Mt assembly at the PPB site and in the perinuclear cytoplasm.

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.

The effect of microtubule- and microfilament-disrupting drugs on preimplantation mouse embryos

The sensitivity of early preimplantation mouse embryos to drugs which disrupt microfilament function (cytochalasin B-CB and cytochalasin D-CD and microtubule assembly (colchicine, colcemid, vinblastine and griseofulvin) was examined. CD inhibited cleavage at a concentration 35-fold lower than CB (3 X 10-7 M v. 1 X 10-5 M). Treatment of 2-cell embryos for 6 h with 1 X 10-5 M CB or 1 X 10-6 M CD or continuous exposure to lower concentrations of CB or CD did not affect development to the blastocyst stage in vitro. Vinblastine inhibited cleavage at a concentration tenfold lower than colcemid or colchicine (1 X 10-8 M v. 1 X 10-7 M). The continuous presence of colcemid at 10-8 M did not affect the development of 2-cell embryos to the blastocyst stage, but development was reduced with vinblastine at 1 X 10-9 M and completely inhibited with colchicine at 1 X 10-8 M. The drugs produced similar responses when 2-cell embryos were treated for 6 h with concentration that inhibited cleavage. Complete inhibition of cleavage was obtained after only a 2 h exposure to 2 X 10-7 M colchicine. A similar concentration of lumicolchicine did not affect cleavage or blastocyst formation. Embryos were less sensitive to griseofulvin; the first cleavage division was unaffected by concentrations as high as 3 X 10-4 M and only 50% of 2-cell embryos failed to cleave in 1 X 10-4 and 3 X 10-4 M griseofulvin. At these concentrations a small proportion of 1-cell embryos and the majority of the 2-cell embryos showed unequal cytoplasmic division probably caused by the formation of multipolar spindles. The continuous exposure of 2-cell embryos to 3 X 10-5 M griseofulvin did not affect blastocyst formation.

Chromosome analysis with special reference to centromeric heterochromatin and ploidy variation in mouse sarcoma-180 cells.

Chromosome analysis of an ascitic form of mouse sarcoma 180 has been made both by conventional Giemsa staining as well as by C-banding. The modal number has been found as 75 with one biarmed marker in the present ascitic tumour. C-banding analysis reveals that this submetacentric marker is actually a dicentric chromosome with two closely situated C-positive heterochromatic zones. In addition to this marker, the cell line also possesses another stable and transmissible dicentric chromosome with two distinctly terminal centromeres, and an another unusual chromosome with an extended centromeric heterochromatin.Spontaneous chromosome elimination during anaphase separation, endoreduplication, multipolar spindle formation, etc. have been recorded in a high frequency which are assumed to be responsible for the characteristic ploidal variation in this cell line.

Chromosome condensation activity in Rana pipiens eggs matured in vivo and in blastomeres arrested by cytostatic factor (CSF)

The ability of brain nuclei to give rise to condensed chromosomes was studied in Rana pipiens eggs which had undergone meiotic maturation in vivo, in blastomeres of two-cell embryos which had been arrested at metaphase by the injection of cytostatic factor (CSF) from mature eggs, and in immature fully grown ovarian oocytes with and without prior CSF injection. Chromosomes from brain nuclei were found to condense within 4 h in mature eggs and this chromosome condensation activity was enhanced by the chelation of free Ca 2+ in the nuclear isolation medium. Chromosomes also condensed in CSF-arrested blastomeres whether they were placed in the blastomere 30 min before the CSF injection or as long as 22 h after the CSF. Both the Ca 2+-sensitive CSF, 1 ∘CSF, and the Ca 2+-insensitive CSF, 2 ∘CSF, resulted in chromosome condensation within arrested blastomeres. The condensation was accompanied by the formation of multipolar spindles and asters. However, it was found that cytoplasm in CSF-arrested blastomeres does not arrest mitosis at metaphase when transferred into a cleaving blastomere. Other experiments demonstrated that chromosome condensation does not occur in ovarian oocytes even when supplied with CSF. The results are interpreted as indicating that CSF does not directly bring about chromosome condensation, but arrests the cell cycle at metaphase and stabilizes the cytoplasmic conditions of metaphase which, in turn, induce chromosome condensation in foreign nuclei as well as spindle and aster formation.

Cytological Studies in Lopezieae (Onagraceae)

Information is presented on chromosome number and pairing in 23 of the 25 recognized taxa of the primarily Mexican genus Lopezia (Onagraceae), based on the examination of 193 individuals from 99 different populations. Of these, 5 have n = 11, the presumed original basic chromosome number for the genus and for the family; 4 have n = 10; 6 have n = 9; 2 have n = 8, of which one also includes populations with n = 7; and 1, n = 7 only. Three additional entities are polyploids, 1 a very distinct species with n = 22; the other 2, with n = 20, are closely related to entities with n = 10. Both the annual habit and self-pollination are correlated with reduced chromosome numbers in Lopezia. Multipolar spindle formation is frequent and thought to play a role in the production of aneuploids. Distributional evidence indicates the probability of saltational speciation in semiarid marginal habitats, as in Clarkia, especially among annuals.