A centrosomal antigen localized on intermediate filaments and mitotic spindle poles

1990 ◽  
Vol 97 (2) ◽  
pp. 259-271
Author(s):  
B. Buendia ◽  
C. Antony ◽  
F. Verde ◽  
M. Bornens ◽  
E. Karsenti
Keyword(s):  
Monoclonal Antibody ◽  
Intermediate Filaments ◽  
Mitotic Spindle ◽  
Mdck Cells ◽  
Large Fraction ◽  
Human Lymphocytes ◽  
Spindle Poles ◽  
Pericentriolar Material ◽  
Xenopus Egg

A monoclonal antibody (CTR2611) raised against centrosomes isolated from human lymphocytes (KE37) stains the pericentriolar material and intermediate filaments in the same cells. In MDCK cells, where most of the microtubules do not originate from the pericentriolar region during interphase, the antigen is distributed along intermediate filaments. At the onset of mitosis, a large fraction of the CTR2611 antigen associates with the minus-end domain of the microtubules of the mitotic spindle but not with the pericentriolar region itself. Treatment of mitotic MDCK cells with taxol leads to the assembly of many microtubule asters in the cytoplasm at the expense of the mitotic spindle. The CTR2611 antigen is present in the center of each of these asters. Similar asters can also be produced in vitro by adding taxol to concentrated Xenopus egg mitotic cytoplasm. Again, the antigen is found close to the center of the asters. These results suggest that CTR2611 antigen is associated with a material involved in microtubule nucleation or microtubule minus-end stabilization. The monoclonal antibody recognizes a 74 × 10(3) Mr polypeptide and other polypeptides at 120 × 10(3) Mr and 170 × 10(3) Mr. The 74 × 10(3) Mr polypeptide is found in all species examined so far, suggesting that it contains a highly conserved epitope.

scholarly journals Nudel/NudE and Lis1 promote dynein and dynactin interaction in the context of spindle morphogenesis

2013 ◽  
Vol 24 (22) ◽  
pp. 3522-3533 ◽  
Author(s):  
Shusheng Wang ◽  
Stephanie A. Ketcham ◽  
Arne Schön ◽  
Benjamin Goodman ◽  
Yueju Wang ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Cytoplasmic Dynein ◽  
Spindle Poles ◽  
Interesting Possibility ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Lis1, Nudel/NudE, and dynactin are regulators of cytoplasmic dynein, a minus end–directed, microtubule (MT)-based motor required for proper spindle assembly and orientation. In vitro studies have shown that dynactin promotes processive movement of dynein on MTs, whereas Lis1 causes dynein to enter a persistent force-generating state (referred to here as dynein stall). Yet how the activities of Lis1, Nudel/NudE, and dynactin are coordinated to regulate dynein remains poorly understood in vivo. Working in Xenopus egg extracts, we show that Nudel/NudE facilitates the binding of Lis1 to dynein, which enhances the recruitment of dynactin to dynein. We further report a novel Lis1-dependent dynein–dynactin interaction that is essential for the organization of mitotic spindle poles. Finally, using assays for MT gliding and spindle assembly, we demonstrate an antagonistic relationship between Lis1 and dynactin that allows dynactin to relieve Lis1-induced dynein stall on MTs. Our findings suggest the interesting possibility that Lis1 and dynactin could alternately engage with dynein to allow the motor to promote spindle assembly.

scholarly journals The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound.

1995 ◽  
Vol 6 (2) ◽  
pp. 227-236 ◽  
Author(s):  
J Rosenblatt ◽  
P Peluso ◽  
T J Mitchison
Keyword(s):  
Actin Polymerization ◽  
Critical Concentration ◽  
Large Fraction ◽  
Nucleotide Exchange ◽  
Chromatography Analysis ◽  
Thymosin Beta 4 ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Non-muscle cells contain 15-500 microM actin, a large fraction of which is unpolymerized. Thus, the concentration of unpolymerized actin is well above the critical concentration for polymerization in vitro (0.2 microM). This fraction of actin could be prevented from polymerization by being ADP bound (therefore less favored to polymerize) or by being ATP bound and sequestered by a protein such as thymosin beta 4, or both. We isolated the unpolymerized actin from Xenopus egg extracts using immobilized DNase 1 and assayed the bound nucleotide. High-pressure liquid chromatography analysis showed that the bulk of soluble actin is ATP bound. Analysis of actin-bound nucleotide exchange rates suggested the existence of two pools of unpolymerized actin, one of which exchanges nucleotide relatively rapidly and another that apparently does not exchange. Native gel electrophoresis of Xenopus egg extracts demonstrated that most of the soluble actin exists in complexes with other proteins, one of which might be thymosin beta 4. These results are consistent with actin polymerization being controlled by the sequestration and release of ATP-bound actin, and argue against nucleotide exchange playing a major role in regulating actin polymerization.

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly

1998 ◽  
Vol 111 (5) ◽  
pp. 557-572 ◽  
Author(s):  
C. Roghi ◽  
R. Giet ◽  
R. Uzbekov ◽  
N. Morin ◽  
I. Chartrain ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Pericentriolar Material ◽  
Spindle Microtubules ◽  
Cycle Dependent

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 ‘invades’ the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

scholarly journals Stu1p Is Physically Associated with β-Tubulin and Is Required for Structural Integrity of the Mitotic Spindle

2002 ◽  
Vol 13 (6) ◽  
pp. 1881-1892 ◽  
Author(s):  
Hongwei Yin ◽  
Liru You ◽  
Danielle Pasqualone ◽  
Kristen M. Kopski ◽  
Tim C. Huffaker
Keyword(s):  
Mitotic Spindle ◽  
Structural Integrity ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spindle Poles ◽  
Balance Of Forces ◽  
Related Proteins ◽  
Β Tubulin

Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Here, we provide evidence that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1–5mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1–5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin–related protein. Stu1p was observed to be a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and we have examined the nature of this interaction. We show that Stu1p interacts specifically with β-tubulin and identify the domains required for this interaction on both Stu1p and β-tubulin. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles.

scholarly journals Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum

2015 ◽  
Vol 26 (7) ◽  
pp. 1286-1295 ◽  
Author(s):  
Francisco Lázaro-Diéguez ◽  
Iaroslav Ispolatov ◽  
Anne Müsch
Keyword(s):  
Mitotic Spindle ◽  
Cell Shape ◽  
Spindle Assembly Checkpoint ◽  
Mdck Cells ◽  
Spindle Pole ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Spindle Positioning ◽  
Spindle Poles ◽  
Spindle Position

All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow β angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell's height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule–mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning.

A human monoclonal antibody produced by in Vitro sensitization of human lymphocytes with an antigen from urine of a sarcoma patient

1987 ◽  
Vol 42 (6) ◽  
pp. 591-596 ◽  
Author(s):  
James F. Huth ◽  
Romaine E. Saxton ◽  
Donald L. Morton ◽  
Reiko F. Irie
Keyword(s):  
Monoclonal Antibody ◽  
Human Lymphocytes ◽  
Human Monoclonal Antibody ◽  
Sarcoma Patient

Two domains of p80 katanin regulate microtubule severing and spindle pole targeting by p60 katanin

2000 ◽  
Vol 113 (9) ◽  
pp. 1623-1633 ◽  
Author(s):  
K.P. McNally ◽  
O.A. Bazirgan ◽  
F.J. McNally
Keyword(s):  
Mitotic Spindle ◽  
Binding Proteins ◽  
Spindle Pole ◽  
Negative Regulator ◽  
Microtubule Binding ◽  
Microtubule Severing ◽  
Spindle Poles ◽  
Wd40 Domain

The assembly and function of the mitotic spindle requires the activity of a number of microtubule-binding proteins. Some microtubule-binding proteins bind microtubules in vitro but do not co-localize with microtubules in interphase cells. Instead these proteins associate with specific subregions of the mitotic spindle. Katanin, a heterodimeric microtubule-severing ATPase, is found localized at mitotic spindle poles. In this paper we demonstrate that human p60 katanin and the C-terminal domain of human p80 katanin both bind microtubules in vitro. Association of these two proteins results in an increased microtubule affinity and increased microtubule-severing activity in vitro. Association of these subunits in transfected HeLa cells increases microtubule disassembly activity and targeting to spindle poles. The N-terminal WD40 domain of p80 katanin acts as a negative regulator of microtubule disassembly activity and is also required for spindle pole localization, possibly through interactions with another spindle-pole protein. These results support a model in which katanin is targeted to spindle poles through a combination of direct microtubule binding by the p60 subunit and through interactions between the WD40 domain and an unknown protein. We propose that both domains of p80 are essential in precisely regulating katanin's activity in vivo.

A microtubule-interacting protein involved in coalignment of vimentin intermediate filaments with microtubules

1993 ◽  
Vol 106 (4) ◽  
pp. 1263-1273 ◽  
Author(s):  
E. Draberova ◽  
P. Draber
Keyword(s):  
Monoclonal Antibody ◽  
Intermediate Filaments ◽  
3T3 Cells ◽  
Double Immunofluorescence ◽  
Interacting Protein ◽  
Cytoplasmic Microtubules ◽  
Microtubular Structures ◽  
Vimentin Filaments

A protein of M(r) 210,000 was identified in 3T3 cells by immunoblotting and by immunoprecipitation with a monoclonal antibody MA-01. The protein was thermolabile and was located on 3T3 microtubules prepared by taxol-driven polymerization in vitro. On fixed cells the MA-01 antigen was located on interphase and mitotic microtubular structures, vinblastine paracrystals, taxol bundles and colcemid-resistant microtubules. Microinjection experiments with purified MA-01 antibody followed by double immunofluorescence have shown that the injection of antibody led to disruption of vimentin filaments, whereas the distribution of cytoplasmic microtubules was unchanged. The collapse of vimentin filaments started 30 minutes after injecting the antibody at immunoglobulin concentrations 2 mg ml-1 or higher and reached its maximum 3–6 hours after the injection. Within 20 hours after the injection vimentin filaments became reconstituted. Microinjection of the antibody into cells pre-treated with vinblastine resulted in localization of the MA-01 antigen on vinblastine paracrystals as well as on coiled vimentin filaments. The data presented suggest that the MA-01 antigen is a new microtubule-interacting protein that mediates, directly or indirectly, an interaction between microtubules and vimentin intermediate filaments.

scholarly journals mini spindles

1999 ◽  
Vol 146 (5) ◽  
pp. 1005-1018 ◽  
Author(s):  
C. Fiona Cullen ◽  
Peter Deák ◽  
David M. Glover ◽  
Hiroyuki Ohkura
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Structural Integrity ◽  
Sequence Similarity ◽  
High Similarity ◽  
Microtubule Associated Proteins ◽  
Spindle Poles ◽  
Associated Proteins ◽  
Diploid Cells

We describe a new Drosophila gene, mini spindles (msps) identified in a cytological screen for mitotic mutant. Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells. Nucleation of microtubules from centrosomes, metaphase alignment of chromosomes, or the focusing of spindle poles appears much less affected. The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast. Consistent with their sequence similarity, Msps protein also associates with microtubules in vitro. In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase. The absence of centrosomal staining in interphase of the cellularized embryos suggests that the interactions between Msps protein and microtubules or centrosomes may be regulated during the cell cycle.

scholarly journals A Potential Role for Human Cohesin in Mitotic Spindle Aster Assembly

2001 ◽  
Vol 276 (50) ◽  
pp. 47575-47582 ◽  
Author(s):  
Heather C. Gregson ◽  
John A. Schmiesing ◽  
Jong-Soo Kim ◽  
Toshiki Kobayashi ◽  
Sharleen Zhou ◽  
...  
Keyword(s):  
Sister Chromatid ◽  
Mitotic Spindle ◽  
Potential Role ◽  
Metaphase Plate ◽  
Spindle Pole ◽  
Sister Chromatid Cohesion ◽  
Cycle Arrest ◽  
Spindle Poles ◽  
Chromatid Cohesion

The cohesin multiprotein complex containing SMC1, SMC3, Scc3 (SA), and Scc1 (Rad21) is required for sister chromatid cohesion in eukaryotes. Although metazoan cohesin associates with chromosomes and was shown to function in the establishment of sister chromatid cohesion during interphase, the majority of cohesin was found to be off chromosomes and reside in the cytoplasm in metaphase. Despite its dissociation from chromosomes, however, microinjection of an antibody against human SMC1 led to disorganization of the metaphase plate and cell cycle arrest, indicating that human cohesin still plays an important role in metaphase. To address the mitotic function of human cohesin, the subcellular localization of cohesin components was reexamined in human cells. Interestingly, we found that cohesin localizes to the spindle poles during mitosis and interacts with NuMA, a spindle pole-associated factor required for mitotic spindle organization. The interaction with NuMA persists during interphase. Similar to NuMA, a significant amount of cohesin was found to associate with the nuclear matrix. Furthermore, in the absence of cohesin, mitotic spindle asters failed to formin vitro. Our results raise the intriguing possibility that in addition to its well demonstrated function in sister chromatid cohesion, cohesin may be involved in spindle assembly during mitosis.

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