scholarly journals Mitotic Regulation of Protein 4.1R Involves Phosphorylation by cdc2 Kinase

2005 ◽  
Vol 16 (1) ◽  
pp. 117-127 ◽  
Author(s):  
Shu-Ching Huang ◽  
Eva S. Liu ◽  
Siu-Hong Chan ◽  
Indira D. Munagala ◽  
Heidi T. Cho ◽  
...  
Keyword(s):  
Hela Cells ◽  
Mitotic Spindle ◽  
Mitotic Apparatus ◽  
Spindle Poles ◽  
Associated Proteins ◽  
Dynamic Structures ◽  
Protein 4.1R ◽  
Mitotic Regulation

The nonerythrocyte isoform of the cytoskeletal protein 4.1R (4.1R) is associated with morphologically dynamic structures during cell division and has been implicated in mitotic spindle function. In this study, we define important 4.1R isoforms expressed in interphase and mitotic cells by RT-PCR and mini-cDNA library construction. Moreover, we show that 4.1R is phosphorylated by p34cdc2kinase on residues Thr60 and Ser679 in a mitosis-specific manner. Phosphorylated 4.1R135isoform(s) associate with tubulin and Nuclear Mitotic Apparatus protein (NuMA) in intact HeLa cells in vivo as well as with the microtubule-associated proteins in mitotic asters assembled in vitro. Recombinant 4.1R135is readily phosphorylated in mitotic extracts and reconstitutes mitotic aster assemblies in 4.1R-immunodepleted extracts in vitro. Furthermore, phosphorylation of these residues appears to be essential for the targeting of 4.1R to the spindle poles and for mitotic microtubule aster assembly in vitro. Phosphorylation of 4.1R also enhances its association with NuMA and tubulin. Finally, we used siRNA inhibition to deplete 4.1R from HeLa cells and provide the first direct genetic evidence that 4.1R is required to efficiently focus mitotic spindle poles. Thus, we suggest that 4.1R is a member of the suite of direct cdc2 substrates that are required for the establishment of a bipolar spindle.

scholarly journals A microtubule-associated protein antigen unique to mitotic spindle microtubules in PtK1 cells.

1983 ◽  
Vol 96 (2) ◽  
pp. 424-434 ◽  
Author(s):  
J G Izant ◽  
J A Weatherbee ◽  
J R McIntosh
Keyword(s):  
Mitotic Spindle ◽  
Microtubule Network ◽  
Mitotic Apparatus ◽  
Microtubule Associated Proteins ◽  
Immunoglobulin Preparations ◽  
Rapid Dissolution ◽  
Associated Proteins ◽  
Cultured Human Cells

Microtubule-associated proteins (MAPs) that copurify with tubulin through multiple cycles of in vitro assembly have been implicated as regulatory factors and effectors in the in vivo activity of microtubules. As an approach to the analysis of the functions of these molecules, a collection of lymphocyte hybridoma monoclonal antibodies has been generated using MAPs from HeLa cell microtubule protein as antigen. Two of the hybridoma clones secrete IgGs that bind to distinct sites on what appears to be a 200,000-dalton polypeptide. Both immunoglobulin preparations stain interphase and mitotic apparatus microtubules in cultured human cells. One of the clones (N-3B4.3.10) secretes antibody that reacts only with cells of human origin, while antibody from the other hybridoma (N-2B5.11.2) cross-reacts with BSC and PtK1 cells, but not with 3T3 cells. In PtK1 cells the N-2B5 antigen is associated with the microtubules of the mitotic apparatus, but there is no staining of the interphase microtubule array; rather, the antibody stains an ill-defined juxtanuclear structure. Further, neither antibody stains vinblastine crystals in either human or marsupial cells at any stage of the cell cycle. N-2B5 antibody microinjected into living PtK1 cells binds to the mitotic spindle, but does not cause a rapid dissolution of either mitotic or interphase microtubule structures. When injected before the onset of anaphase, however, the N-2B5 antibody inhibits proper chromosome partition in mitotic PtK1 cells. N-2B5 antibody injected into interphase cells causes a redistribution of MAP antigen onto the microtubule network.

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.

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 Interaction of NuMA protein with the kinesin Eg5: its possible role in bipolar spindle assembly and chromosome alignment

2013 ◽  
Vol 451 (2) ◽  
pp. 195-204 ◽  
Author(s):  
Yuko Iwakiri ◽  
Sachiko Kamakura ◽  
Junya Hayase ◽  
Hideki Sumimoto
Keyword(s):  
Spindle Assembly ◽  
Cytoplasmic Dynein ◽  
Mitotic Apparatus ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Interphase Cells ◽  
Correct Alignment ◽  
Spindle Microtubules

Bipolar spindle assembly in mitotic cells is a prerequisite to ensure correct alignment of chromosomes for their segregation to each daughter cell; spindle microtubules are tethered at plus ends to chromosomes and focused at minus ends to either of the two spindle poles. NuMA (nuclear mitotic apparatus protein) is present solely in the nucleus in interphase cells, but relocalizes during mitosis to the spindle poles to play a crucial role in spindle assembly via focusing spindle microtubules to each pole. In the present study we show that the kinesin-5 family motor Eg5 is a protein that directly interacts with NuMA, using a proteomics approach and various binding assays both in vivo and in vitro. During mitosis Eg5 appears to interact with NuMA in the vicinity of the spindle poles, whereas the interaction does not occur in interphase cells, where Eg5 is distributed throughout the cytoplasm but NuMA exclusively localizes to the nucleus. Slight, but significant, depletion of Eg5 in HeLa cells by RNA interference results in formation of less-focused spindle poles with misaligned chromosomes in metaphase; these phenotypes are similar to those induced by depletion of NuMA. Since NuMA is less accumulated at the spindle poles in Eg5-depleted cells, Eg5 probably contributes to spindle assembly via regulating NuMA localization. Furthermore, depletion of cytoplasmic dynein induces mislocalization of NuMA and phenotypes similar to those observed in NuMA-depleted cells, without affecting Eg5 localization to the spindles. Thus dynein appears to control NuMA function in conjunction with Eg5.

scholarly journals Microtubule associated proteins and motors required for ectopic microtubule array formation in S. cerevisiae

2021 ◽  
Author(s):  
Brianna R. King ◽  
Janet B. Meehl ◽  
Tamira Vojnar ◽  
Mark Winey ◽  
Eric G. Muller ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Polymerase Activity ◽  
Nucleation Site ◽  
Microtubule Nucleation ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
A Subunit ◽  
Nucleation Activity

AbstractThe mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the γ-tubulin complex, nucleation and elongation of microtubules, and the organization of microtubules relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Further, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.

scholarly journals Immunofluorescence localization of HeLa cell microtubule-associated proteins on microtubules in vitro and in vivo.

1980 ◽  
Vol 87 (3) ◽  
pp. 792-801 ◽  
Author(s):  
J C Bulinski ◽  
G G Borisy
Keyword(s):  
Hela Cells ◽  
Cross Reactivity ◽  
Fiber Network ◽  
Reactive Material ◽  
Microtubule Associated Proteins ◽  
Molecular Weights ◽  
Associated Proteins ◽  
A Minor

Rabbit antisera were prepared against the two major groups of microtubule-associated proteins (MAPs) from HeLa cells, proteins of approximately 210,000 molecular weight (210k MAPs), and 125,000 mol wt (125k MAPs). These antisera were characterized by a sensitive antigen detection technique that employs immunofluorescence to localize cross-reactive material in polyacrylamide gels. Antisera prepared against the 210k MAPs showed no cross-reactivity with extract proteins of other molecular weights or with bran MAPs, but did react with proteins of 210,000 mol wt and with a minor HeLa MAP of approximately 255,000 mol wt. Antibodies prepared against the 125k HeLa MAPs, likewise, reacted specifically with proteins of 125,000 mol wt, showing no cross-reactivity with other HeLa extract proteins or porcine brain MAPs. Immunofluorescence with the 210k and 125k MAP antisera was used to demonstrate the association of each of the MAPs with fixed HeLa microtubules in vitro. In addition, immunofluorescence with these antisera revealed a physical association of 210k and 125k MAPs with a Colcemid-sensitive fiber network in fixed interphase and mitotic HeLa cells. Thus, using specific, well-characterized antisera to the two major groups of HeLa MAPs, we have shown that these proteins are components of microtubules in HeLa cells.

scholarly journals Microtubule associated proteins and motors required for ectopic microtubule array formation in S. cerevisiae

Genetics ◽  
2021 ◽  
Author(s):  
Brianna R King ◽  
Janet B Meehl ◽  
Tamira Vojnar ◽  
Mark Winey ◽  
Eric G Muller ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Polymerase Activity ◽  
Nucleation Site ◽  
Microtubule Nucleation ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
A Subunit ◽  
Nucleation Activity

Abstract The mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the γ-tubulin complex, nucleation and elongation of microtubules, and the organization of microtubules relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Further, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.

scholarly journals Poleward microtubule flux mitotic spindles assembled in vitro.

1991 ◽  
Vol 112 (5) ◽  
pp. 941-954 ◽  
Author(s):  
K E Sawin ◽  
T J Mitchison
Keyword(s):  
Mitotic Spindle ◽  
Preceding Paper ◽  
Mitotic Spindles ◽  
Microtubule Motor ◽  
Mitotic Spindle Assembly ◽  
Spindle Elongation ◽  
Dynamic Structures ◽  
Poleward Flux

In the preceding paper we described pathways of mitotic spindle assembly in cell-free extracts prepared from eggs of Xenopus laevis. Here we demonstrate the poleward flux of microtubules in spindles assembled in vitro, using a photoactivatable fluorescein covalently coupled to tubulin and multi-channel fluorescence videomicroscopy. After local photoactivation of fluorescence by UV microbeam, we observed poleward movement of fluorescein-marked microtubules at a rate of 3 microns/min, similar to rates of chromosome movement and spindle elongation during prometaphase and anaphase. This movement could be blocked by the addition of millimolar AMP-PNP but was not affected by concentrations of vanadate up to 150 microM, suggesting that poleward flux may be driven by a microtubule motor similar to kinesin. In contrast to previous results obtained in vivo (Mitchison, T. J. 1989. J. Cell Biol. 109:637-652), poleward flux in vitro appears to occur independently of kinetochores or kinetochore microtubules, and therefore may be a general property of relatively stable microtubules within the spindle. We find that microtubules moving towards poles are dynamic structures, and we have estimated the average half-life of fluxing microtubules in vitro to be between approximately 75 and 100 s. We discuss these results with regard to the function of poleward flux in spindle movements in anaphase and prometaphase.

The nucleolar phosphoprotein B23 redistributes in part to the spindle poles during mitosis

1999 ◽  
Vol 112 (4) ◽  
pp. 455-466 ◽  
Author(s):  
O.V. Zatsepina ◽  
A. Rousselet ◽  
P.K. Chan ◽  
M.O. Olson ◽  
E.G. Jordan ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Mitotic Cell ◽  
Cell Extract ◽  
In Vitro Assay ◽  
Animal Cells ◽  
Mitotic Apparatus ◽  
Vitro Assay ◽  
Spindle Poles ◽  
Hyperphosphorylated Form

B23 is a major phosphoprotein in the interphasic nucleolus where it is involved in the assembly of pre-ribosomes. Using several cultured animal cells, we report that, in addition to the known redistribution of the protein during mitosis, B23 also becomes associated with mitotic spindle poles starting from early prometaphase onwards. Colocalization of B23 with the protein NuMA (Nuclear Mitotic Apparatus protein) was studied in mitotic cells and taxol-arrested cells. During the onset of mitosis, we observed that a fraction of B23 associates with, and dissociates from, the poles later than NuMA. At metaphase, both proteins are colocalized at the poles. The polar redistribution of both B23 and NuMA is mediated by microtubules. In taxol-treated cells, B23 is associated with the microtubule minus ends in the center of mitotic asters together with NuMA. Association of B23 with microtubule minus ends of mitotic asters was further confirmed with an in vitro assay, where B23 was found by western blotting to co-sediment with taxol-induced microtubule asters formed in a mitotic cell extract. Immunolabeling demonstrated that B23 and NuMA were both present at the center of the asters. Furthermore, an additional hyperphosphorylated form of B23 appeared when microtubule asters formed and associated with the asters. Immunodepletion of B23 from the mitotic extract revealed that taxol-induced microtubule asters were still observed in B23-immunodepleted mitotic extract, indicating that the presence of B23 at the poles is unlikely to be essential for spindle formation or stabilisation.

scholarly journals A Nonerythroid Isoform of Protein 4.1R Interacts with the Nuclear Mitotic Apparatus (NuMA) Protein

1999 ◽  
Vol 145 (1) ◽  
pp. 29-43 ◽  
Author(s):  
Subhendra N. Mattagajasingh ◽  
Shu-Ching Huang ◽  
Julia S. Hartenstein ◽  
Michael Snyder ◽  
Vincent T. Marchesi ◽  
...  
Keyword(s):  
Interphase Nucleus ◽  
Mdck Cells ◽  
Single Gene ◽  
Spindle Pole ◽  
Cell Protein ◽  
Mitotic Apparatus ◽  
Spindle Poles ◽  
Nuclear Mitotic Apparatus ◽  
Protein 4.1R

Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788–1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22–24-kD domain.

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