Role of chromosomes in assembly of meiotic and mitotic spindles

1997 ◽  
pp. 271-284 ◽  
Author(s):  
Régis Tournebize ◽  
Rebecca Heald ◽  
Anthony Hyman
Keyword(s):  
Mitotic Spindles

scholarly journals Interaction between Poly(ADP-ribose) and NuMA Contributes to Mitotic Spindle Pole Assembly

2009 ◽  
Vol 20 (21) ◽  
pp. 4575-4585 ◽  
Author(s):  
Paul Chang ◽  
Margaret Coughlin ◽  
Timothy J. Mitchison
Keyword(s):  
Binding Proteins ◽  
Magnetic Beads ◽  
Spindle Pole ◽  
Covalent Modification ◽  
Mitotic Spindles ◽  
Spindle Poles ◽  
Tankyrase 1 ◽  
Mitotic Spindle Pole

Poly(ADP-ribose) (pADPr), made by PARP-5a/tankyrase-1, localizes to the poles of mitotic spindles and is required for bipolar spindle assembly, but its molecular function in the spindle is poorly understood. To investigate this, we localized pADPr at spindle poles by immuno-EM. We then developed a concentrated mitotic lysate system from HeLa cells to probe spindle pole assembly in vitro. Microtubule asters assembled in response to centrosomes and Ran-GTP in this system. Magnetic beads coated with pADPr, extended from PARP-5a, also triggered aster assembly, suggesting a functional role of the pADPr in spindle pole assembly. We found that PARP-5a is much more active in mitosis than interphase. We used mitotic PARP-5a, self-modified with pADPr chains, to capture mitosis-specific pADPr-binding proteins. Candidate binding proteins included the spindle pole protein NuMA previously shown to bind to PARP-5a directly. The rod domain of NuMA, expressed in bacteria, bound directly to pADPr. We propose that pADPr provides a dynamic cross-linking function at spindle poles by extending from covalent modification sites on PARP-5a and NuMA and binding noncovalently to NuMA and that this function helps promote assembly of exactly two poles.

scholarly journals Centrosomes Enhance the Fidelity of Cytokinesis in Vertebrates and Are Required for Cell Cycle Progression

2001 ◽  
Vol 153 (1) ◽  
pp. 237-242 ◽  
Author(s):  
Alexey Khodjakov ◽  
Conly L. Rieder
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Somatic Cells ◽  
Spindle Pole ◽  
Primary Role ◽  
Mitotic Spindles ◽  
Cycle Progression ◽  
Daughter Cells ◽  
Cell Changes

When centrosomes are destroyed during prophase by laser microsurgery, vertebrate somatic cells form bipolar acentrosomal mitotic spindles (Khodjakov, A., R.W. Cole, B.R. Oakley, and C.L. Rieder. 2000. Curr. Biol. 10:59–67), but the fate of these cells is unknown. Here, we show that, although these cells lack the radial arrays of astral microtubules normally associated with each spindle pole, they undergo a normal anaphase and usually produce two acentrosomal daughter cells. Relative to controls, however, these cells exhibit a significantly higher (30–50%) failure rate in cytokinesis. This failure correlates with the inability of the spindle to properly reposition itself as the cell changes shape. Also, we destroyed just one centrosome during metaphase and followed the fate of the resultant acentrosomal and centrosomal daughter cells. Within 72 h, 100% of the centrosome-containing cells had either entered DNA synthesis or divided. By contrast, during this period, none of the acentrosomal cells had entered S phase. These data reveal that the primary role of the centrosome in somatic cells is not to form the spindle but instead to ensure cytokinesis and subsequent cell cycle progression.

scholarly journals SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown

2014 ◽  
Vol 204 (7) ◽  
pp. 1099-1109 ◽  
Author(s):  
Yagmur Turgay ◽  
Lysie Champion ◽  
Csaba Balazs ◽  
Michael Held ◽  
Alberto Toso ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Dominant Negative ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Microtubule Depolymerization ◽  
Nuclear Envelope Breakdown ◽  
And Migration ◽  
Delayed Removal

SUN proteins reside in the inner nuclear membrane and form complexes with KASH proteins of the outer nuclear membrane that connect the nuclear envelope (NE) to the cytoskeleton. These complexes have well-established functions in nuclear anchorage and migration in interphase, but little is known about their involvement in mitotic processes. Our analysis demonstrates that simultaneous depletion of human SUN1 and SUN2 delayed removal of membranes from chromatin during NE breakdown (NEBD) and impaired the formation of prophase NE invaginations (PNEIs), similar to microtubule depolymerization or down-regulation of the dynein cofactors NudE/EL. In addition, overexpression of dominant-negative SUN and KASH constructs reduced the occurrence of PNEI, indicating a requirement for functional SUN–KASH complexes in NE remodeling. Codepletion of SUN1/2 slowed cell proliferation and resulted in an accumulation of morphologically defective and disoriented mitotic spindles. Quantification of mitotic timing revealed a delay between NEBD and chromatin separation, indicating a role of SUN proteins in bipolar spindle assembly and mitotic progression.

Katanin, the microtubule-severing ATPase, is concentrated at centrosomes

1996 ◽  
Vol 109 (3) ◽  
pp. 561-567 ◽  
Author(s):  
F.J. McNally ◽  
K. Okawa ◽  
A. Iwamatsu ◽  
R.D. Vale
Keyword(s):  
Mitotic Spindle ◽  
Dynamic Properties ◽  
Mitotic Spindles ◽  
Microtubule Severing ◽  
Spindle Microtubules ◽  
Gamma Tubulin ◽  
And Function ◽  
Poleward Flux

The assembly and function of the mitotic spindle involve specific changes in the dynamic properties of microtubules. One such change results in the poleward flux of tubulin in which spindle microtubules polymerize at their kinetochore-attached plus ends while they shorten at their centrosome-attached minus ends. Since free microtubule minus ends do not depolymerize in vivo, the poleward flux of tubulin suggests that spindle microtubules are actively disassembled at or near their centrosomal attachment points. The microtubule-severing ATPase, katanin, has the ability actively to sever and disassemble microtubules and is thus a candidate for the role of a protein mediating the poleward flux of tubulin. Here we determine the subcellular localization of katanin by immunofluorescence as a preliminary step in determining whether katanin mediates the poleward flux of tubulin. We find that katanin is highly concentrated at centrosomes throughout the cell cycle. Katanin's localization is different from that of gamma-tubulin in that microtubules are required to maintain the centrosomal localization of katanin. Direct comparison of the localization of katanin and gamma-tubulin reveals that katanin is localized in a region surrounding the gamma-tubulin-containing pericentriolar region in detergent-extracted mitotic spindles. The centrosomal localization of katanin is consistent with the hypothesis that katanin mediates the disassembly of microtubule minus ends during poleward flux.

scholarly journals Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

2016 ◽  
Vol 27 (22) ◽  
pp. 3490-3514 ◽  
Author(s):  
Zachary R. Gergely ◽  
Ammon Crapo ◽  
Loren E. Hough ◽  
J. Richard McIntosh ◽  
Meredith D. Betterton
Keyword(s):  
Fission Yeast ◽  
Microtubule Dynamics ◽  
Chromosome Loss ◽  
Mitotic Spindles ◽  
Aberrant Chromosome ◽  
Large Fluctuations ◽  
Spindle Function ◽  
Chromosome Movements ◽  
Sliding Force

Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen.

scholarly journals A polo-like kinase modulates cytokinesis and flagella biogenesis in Giardia lamblia

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Eun-Ah Park ◽  
Juri Kim ◽  
Mee Young Shin ◽  
Soon-Jung Park
Keyword(s):  
Cell Division ◽  
Giardia Lamblia ◽  
Specific Inhibitor ◽  
Site Directed Mutagenesis ◽  
Kinase Assay ◽  
Basal Bodies ◽  
Putative Open Reading Frame ◽  
Mitotic Spindles ◽  
Dividing Cells

Abstract Background Polo-like kinases (PLKs) are conserved serine/threonine kinases that regulate the cell cycle. To date, the role of Giardia lamblia PLK (GlPLK) in cells has not been studied. Here, we report our investigation on the function of GlPLK to provide insight into the role of this PKL in Giardia cell division, especially during cytokinesis and flagella formation. Methods To assess the function of GIPLK, Giardia trophozoites were treated with the PLK-specific inhibitor GW843286X (GW). Using a putative open reading frame for the PLK identified in the Giardia genomic database, we generated a transgenic Giardia expressing hemagglutinin (HA)-tagged GlPLK and used this transgenic for immunofluorescence assays (IFAs). GlPLK expression was knocked down using an anti-glplk morpholino to observe its effect on the number of nuclei number and length of flagella. Giardia cells ectopically expressing truncated GlPLKs, kinase domain + linker (GlPLK-KDL) or polo-box domains (GlPLK-PBD) were constructed for IFAs. Mutant GlPLKs at Lys51, Thr179 and Thr183 were generated by site-directed mutagenesis and then used for the kinase assay. To elucidate the role of phosphorylated GlPLK, the phosphorylation residues were mutated and expressed in Giardia trophozoites Results After incubating trophozoites with 5 μM GW, the percentage of cells with > 4 nuclei and longer caudal and anterior flagella increased. IFAs indicated that GlPLK was localized to basal bodies and flagella and was present at mitotic spindles in dividing cells. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells. In contrast to Giardia expressing GlPLK-PBD, Giardia expressing GlPLK-KDL was defective in terms of GIPLK localization to mitotic spindles and had altered localization of the basal bodies in dividing cells. Kinase assays using mutant recombinant GlPLKs indicated that mutation at Lys51 or at both Thr179 and Thr183 resulted in loss of kinase activity. Giardia expressing these mutant GlPLKs also demonstrated defects in cell growth, cytokinesis and flagella formation. Conclusions These data indicate that GlPLK plays a role in Giardia cell division, especially during cytokinesis, and that it is also involved in flagella formation.

scholarly journals A novel mitosis-specific Cep215 domain interacts with Cep192 and phosphorylated Aurora A for organization of spindle poles

2020 ◽  
Vol 133 (24) ◽  
pp. jcs240267
Author(s):  
Ryoko Kuriyama ◽  
Cody R. Fisher
Keyword(s):  
Structural Integrity ◽  
Spindle Pole ◽  
Minor Role ◽  
Aurora A ◽  
Mitotic Spindles ◽  
Binding Domains ◽  
Spindle Poles ◽  
Pericentriolar Material

ABSTRACTThe centrosome, which consists of centrioles and pericentriolar material (PCM), becomes mature and assembles mitotic spindles by increasing the number of microtubules (MTs) emanating from the PCM. Among the molecules involved in centrosome maturation, Cep192 and Aurora A (AurA, also known as AURKA) are primarily responsible for recruitment of γ-tubulin and MT nucleators, whereas pericentrin (PCNT) is required for PCM organization. However, the role of Cep215 (also known as CDK5RAP2) in centrosome maturation remains elusive. Cep215 possesses binding domains for γ-tubulin, PCNT and MT motors that transport acentrosomal MTs towards the centrosome. We identify a mitosis-specific centrosome-targeting domain of Cep215 (215N) that interacts with Cep192 and phosphorylated AurA (pAurA). Cep192 is essential for targeting 215N to centrosomes, and centrosomal localization of 215N and pAurA is mutually dependent. Cep215 has a relatively minor role in γ-tubulin recruitment to the mitotic centrosome. However, it has been shown previously that this protein is important for connecting mitotic centrosomes to spindle poles. Based on the results of rescue experiments using versions of Cep215 with different domain deletions, we conclude that Cep215 plays a role in maintaining the structural integrity of the spindle pole by providing a platform for the molecules involved in centrosome maturation.

scholarly journals Expansion microsopy reveals Plasmodium falciparum blood-stage parasites undergo anaphase with a chromatin bridge in the absence of mini-chromosome maintenance complex binding protein

2021 ◽  
Author(s):  
Benjamin Liffner ◽  
Sabrina Absalon
Keyword(s):  
Plasmodium Falciparum ◽  
Nuclear Envelope ◽  
Malaria Parasite ◽  
Binding Protein ◽  
Nuclear Morphology ◽  
Mitotic Spindles ◽  
Human Host ◽  
The Individual ◽  
Chromatin Bridges

ABSTRACTMitosis in the malaria parasite Plasmodium falciparum undergoes closed mitosis, which occurs within an intact nuclear envelope, and differs significantly from its human host. Mitosis is underpinned by the dynamics of microtubules and the nuclear envelope. To date, our ability to study P. falciparum mitosis by microscopy has been hindered by the small size of P. falciparum nuclei. Ultrastructure expansion microscopy (U-ExM) has recently been developed for P. falciparum, allowing visualization of mitosis at the individual nucleus level. Using U-ExM, three intranuclear microtubule structures are observed: hemispindles, mitotic spindles and interpolar spindles. A previous study demonstrated that the mini-chromosome maintenance complex binding-protein (MCMBP) depletion caused abnormal nuclear morphology and microtubule defects. To investigate the role of microtubules following MCMBP depletion and study the nuclear envelope in these parasites, we developed the first nuclear stain enabled by U-ExM in P. falciparum. MCMBP deficient parasites show aberrant hemispindles and mitotic spindles. Moreover, anaphase chromatin bridges, and individual nuclei containing multiple microtubule structures were observed following MCMBP knockdown. Collectively, this study refines our model for the phenotype of MCMBP-deficient parasites and highlights the utility of U-ExM coupled with a nuclear envelope stain for studying mitosis in P. falciparum.

Growth and development of pattern in the cranial neural epithelium of rat embryos during neurulation

Development ◽  
1981 ◽  
Vol 65 (Supplement) ◽  
pp. 225-241
Author(s):  
Gillian M. Morriss-Kay
Keyword(s):  
Neural Crest ◽  
Neural Crest Cell ◽  
Cell Number ◽  
Culture Technique ◽  
Mitotic Spindles ◽  
Rat Embryos ◽  
Constant Area ◽  
High Level ◽  
Crest Cell

The pattern of growth and morphogenesis of the cranial neural epithelium of rat embryos during neurulation is described. Transverse sections of the midbrain/hindbrain neural epithelium at different stages (0–14 somites) show a constant area and cell number throughout neurulation, even though there is a high level of mitosis. Mitotic spindles are orientated parallel to the long axis of the embryo, so that increase in cell number occurs in this direction only. Growth is expressed only as an increase in size of the forebrain, which projects rostrad to the tip of the notochord. In the midbrain/upper hindbrain regions, cellular organization of the neural epithelium changes from columnar to cuboidal to pseudostratified, while its shape changes from flat to biconvex to V shaped. Closure is immediately preceded by neural crest cell emigration from the lateral edges. Throughout neurulation the cranial notochord develops an increasingly convex curvature in the rostrocaudal plane. The attached neural epithelium curves with the notochord (forming the primary cranial flexure) so that as its lateral edges move dorsomedially they form a more distant concentric arc with that of the notochord, and are hence stretched during the final closure period. The whole rat embryo culture technique was used to investigate the morphogenetic role of proteoglycans during neurulation, neural crest cell emigration and other events in the lateral edge region prior to closure, and the importance of microfilament contraction during concave curvature of the neural epithelium.

Sex reversing non-disjunction of the Y chromosome produces exceptionally low sex ratio (% males) and hermaphrodites in the progeny of male BALB/cBm mice: the roles of the maternal genotype and the Y chromosome

1991 ◽  
Vol 3 (3) ◽  
pp. 255 ◽  
Author(s):  
WK Whitten ◽  
SC Carter ◽  
WG Beamer
Keyword(s):  
Sex Ratio ◽  
Y Chromosome ◽  
Dominant Gene ◽  
Chromosome 1 ◽  
F1 Hybrids ◽  
Mitotic Spindles ◽  
Maternal Genome ◽  
Maternal Genotype ◽  
The Many

When females of 21 strains and hybrids were mated to BALB/cBm males to determine the role of the maternal genome in the sex reversing non-disjunction of the Y chromosome, (1) BALB/cBm and BALB/cBy and SJL/J females produced 39.5-41.5% males and 2.4-2.8% hermaphrodites; (2) SWR/J, A/HeJ, DBA/2J and C3HeB/FeJ produced 44.8-49.1% males and 0.2-0.7% hermaphrodites; (3) C3H/HeJ and three strains of C57BL produced normal sex ratios and no hermaphrodites; (4) four F1 hybrids produced 44.5-49.2% males and 0.3-1.9% hermaphrodites; (5) the seven CXB RI strains produced perplexing sets of data: 26.5%-52.0% males and 0.2-3.2% hermaphrodites. These results indicate that a partly dominant gene favouring non-disjunction occurs in the female genomes of BALB/c and SJL/J strains, an enhancing gene occurs in C57BL/6By and there may be others. Heterosis appears to favour normal mitosis. CXBH females produced 26.5% males and 3.2% hermaphrodites, indicating that non-disjunction may have occurred in every male zygote, thus providing models for the generation of Turner's syndrome, hermaphroditism and a predictable non-disjunction. Reciprocal crosses were made between SJL/J and BALB/cBm, followed by 20 backcrosses to the maternal strains, to exchange the Ys and produce two new consomic strains. Males from SJL-BALB/cBm-Y strain, when mated to CXBH females, sired 34.3% males and 4.3% hermaphrodites, whereas BALB/cBm-SJL-Y sired no hermaphrodites and the sex ratio of the offspring was normal. This shows that the non-disjunction involves only the BALB/cBm Y chromosome and is completely independent of genes on the X or autosomal chromosomes. These results indicate that the BALB/cBm Y chromosome is unable to interact normally with the mitotic spindles of some genotypes, particularly CXBH, BALB/c and SJL. The simplest hypothesis is that a primary non-disjunction occurs at first cleavage. This can produce an array of mosaics determined by chance in the many sampling events that take place during development and by the relative vigour and stability of the two original clones.

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