scholarly journals Eg5 is static in bipolar spindles relative to tubulin

2001 ◽  
Vol 154 (6) ◽  
pp. 1125-1134 ◽  
Author(s):  
Tarun M. Kapoor ◽  
Timothy J. Mitchison
Keyword(s):  
Motor Activity ◽  
Half Life ◽  
Microtubule Dynamics ◽  
Spindle Matrix ◽  
Spindle Poles ◽  
Kinesin Eg5 ◽  
Mitotic Kinesin Eg5 ◽  
Bipolar Spindles

We used fluorescent speckle microscopy to probe the dynamics of the mitotic kinesin Eg5 in Xenopus extract spindles, and compared them to microtubule dynamics. We found significant populations of Eg5 that were static over several seconds while microtubules flux towards spindle poles. Eg5 dynamics are frozen by adenylimidodiphosphate. Bulk turnover experiments showed that Eg5 can exchange between the spindle and the extract with a half life of <55 s. Eg5 distribution in spindles was not perturbed by inhibition of its motor activity with monastrol, but was perturbed by inhibition of dynactin with p50 dynamitin. We interpret these data as revealing the existence of a static spindle matrix that promotes Eg5 targeting to spindles, and transient immobilization of Eg5 within spindles. We discuss alternative interpretations of the Eg5 dynamics we observe, ideas for the biochemical nature of a spindle matrix, and implications for Eg5 function.

scholarly journals A Novel Action of Terpendole E on the Motor Activity of Mitotic Kinesin Eg5

2003 ◽  
Vol 10 (2) ◽  
pp. 131-137 ◽  
Author(s):  
Junko Nakazawa ◽  
Junichiro Yajima ◽  
Takeo Usui ◽  
Masashi Ueki ◽  
Akira Takatsuki ◽  
...  
Keyword(s):  
Motor Activity ◽  
Kinesin Eg5 ◽  
Mitotic Kinesin Eg5

scholarly journals Monastrol Inhibition of the Mitotic Kinesin Eg5

2005 ◽  
Vol 280 (13) ◽  
pp. 12658-12667 ◽  
Author(s):  
Jared C. Cochran ◽  
Joseph E. Gatial ◽  
Tarun M. Kapoor ◽  
Susan P. Gilbert
Keyword(s):  
Kinesin Eg5 ◽  
Mitotic Kinesin Eg5

scholarly journals Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis

2003 ◽  
Vol 160 (5) ◽  
pp. 671-683 ◽  
Author(s):  
Alexey Khodjakov ◽  
Lily Copenagle ◽  
Michael B. Gordon ◽  
Duane A. Compton ◽  
Tarun M. Kapoor
Keyword(s):  
Three Dimensional ◽  
Differential Interference Contrast ◽  
Differential Interference Contrast Microscopy ◽  
Spindle Formation ◽  
Capture Mechanism ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
Kinesin Eg5 ◽  
Mitotic Kinesin Eg5 ◽  
Dependent Mechanism

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living α-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates.

Novel photochromic inhibitor for mitotic kinesin Eg5 which forms multiple isomerization states

2021 ◽  
Author(s):  
Islam Md Alrazi ◽  
Kei Sadakane ◽  
Shinsaku Maruta
Keyword(s):  
Inhibitory Activity ◽  
Molecular Motor ◽  
The Novel ◽  
Precise Control ◽  
Atpase Assay ◽  
Ic50 Value ◽  
Motor Activities ◽  
Ic50 Values ◽  
Kinesin Eg5 ◽  
Mitotic Kinesin Eg5

Abstract The mitotic kinesin Eg5 is a plus-end directed homotetrameric molecular motor essential for the formation of bipolar spindles during cell division. Kinesin Eg5 is overexpressed in cancer cells and hence considered as a target for cancer therapy; the inhibitors specific for Eg5 have been developed as anticancer drugs. In this study, we synthesized a novel functional photoresponsive inhibitor composed of spiropyran and azobenzene derivatives to control Eg5 function with multistage inhibitory activity accompanied by the formation of different isomerization states. The photochromic inhibitor spiropyran-sulfo-azobenzene (SPSAB) exhibited three isomerization states: spiro (SP)-trans, merocyanine (MC)-cis and MC-trans, upon exposure to visible light, ultraviolet and in the dark, respectively. SPSAB-induced reversible changes in the inhibitory activity of ATPase and motor activities correlating with photoisomerization among the three states. Among the three isomerization states of SPSAB, the SP-trans isomer showed potent inhibitory activity at an IC50 value of 30 µM in the basal ATPase assay. MC-trans and MC-cis exhibited less inhibitory activity at IC50 values of 38 and 86 µM, respectively. The results demonstrated that the novel photochromic inhibitor enabled precise control of Eg5 function at three different levels using light irradiation.

Differential pathways of recruitment for centrosomal antigens to the mitotic poles during bipolar spindle formation

1991 ◽  
Vol 100 (3) ◽  
pp. 533-540 ◽  
Author(s):  
T. Maekawa ◽  
R. Kuriyama
Keyword(s):  
Drug Treatment ◽  
Single Cell ◽  
Subcellular Distribution ◽  
Immunofluorescence Staining ◽  
Cell Periphery ◽  
Spindle Formation ◽  
Spindle Poles ◽  
Specific Manner ◽  
Bipolar Spindles ◽  
Antibody Localization

As cells enter mitosis, centrosomes undergo many transformations and become associated with different molecules in a stage-specific manner. We have developed a protocol for immunofluorescence staining with four antibody probes that can help us to follow the interaction of centrosomal components during mitosis. The cells were first stained with a human autoimmune serum (5051); a monoclonal anti-phosphocentrosomal antibody (CHO3); and an antitubulin antibody. Localization of the antibodies was detected using rhodamine-, fluorescein- and AMCA-conjugated second antibodies, respectively. After photographing marked mitotic cells, coverslips were soaked with 0.2 M glycine-HCl at pH 1.0 for 1 h to release all antibodies bound to the structures. The same cells were re-stained with a human autoantibody (SP-H) specific for spindle poles and a fluorescein-conjugated second antibody. This allowed us to compare the subcellular distribution of three kinds of centrosomal antigens in a single cell. Mitotic PtK1 cells treated with either nocodazole or taxol included microtubule-containing cytoplasmic foci and parallel bundles of short microtubules at the cell periphery. All the centrosomal antibodies stained the same one or two dots corresponding to structures labeled by the tubulin antibody. CHO3 also revealed extra cytoplasmic foci, whereas the SP-H antigen was additionally localized at one end of the free microtubule bundles. As the microtubules reorganized into bipolar spindles during the recovery from drug treatment, the CHO3 and SP-H antigens coalesced into the spindle poles where the 5051 antigen was located, suggesting that centrosomal antigens become associated with spindle poles through very different recruitment pathways.

scholarly journals K858, a Novel Inhibitor of Mitotic Kinesin Eg5 and Antitumor Agent, Induces Cell Death in Cancer Cells

2009 ◽  
Vol 69 (9) ◽  
pp. 3901-3909 ◽  
Author(s):  
Ryuichiro Nakai ◽  
Shin-ichi Iida ◽  
Takeshi Takahashi ◽  
Tetsuya Tsujita ◽  
Seiho Okamoto ◽  
...  
Keyword(s):  
Cell Death ◽  
Cancer Cells ◽  
Antitumor Agent ◽  
Kinesin Eg5 ◽  
Mitotic Kinesin Eg5

scholarly journals Kinetochore-mediated outward force promotes spindle pole separation in fission yeast

2019 ◽  
Vol 30 (22) ◽  
pp. 2802-2813 ◽  
Author(s):  
Yutaka Shirasugi ◽  
Masamitsu Sato
Keyword(s):  
Fission Yeast ◽  
Motor Proteins ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Double Knockout ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Bipolar Spindles ◽  
Meiosis I

Bipolar spindles are organized by motor proteins that generate microtubule-­dependent forces to separate the two spindle poles. The fission yeast Cut7 (kinesin-5) is a plus-end-directed motor that generates the outward force to separate the two spindle poles, whereas the minus-end-directed motor Pkl1 (kinesin-14) generates the inward force. Balanced forces by these antagonizing kinesins are essential for bipolar spindle organization in mitosis. Here, we demonstrate that chromosomes generate another outward force that contributes to the bipolar spindle assembly. First, it was noted that the cut7 pkl1 double knockout failed to separate spindle poles in meiosis I, although the mutant is known to succeed it in mitosis. It was assumed that this might be because meiotic kinetochores of bivalent chromosomes joined by cross-overs generate weaker tensions in meiosis I than the strong tensions in mitosis generated by tightly tethered sister kinetochores. In line with this idea, when meiotic mono-oriented kinetochores were artificially converted to a mitotic bioriented layout, the cut7 pkl1 mutant successfully separated spindle poles in meiosis I. Therefore, we propose that spindle pole separation is promoted by outward forces transmitted from kinetochores to spindle poles through microtubules.

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