Kinetic analysis of mitotic spindle elongation in vitro

1990 ◽  
Vol 97 (1) ◽  
pp. 79-89
Author(s):  
T.I. Baskin ◽  
W.Z. Cande
Keyword(s):  
Mitotic Spindle ◽  
Average Rate ◽  
Polarized Light ◽  
Total Elongation ◽  
Force Transducer ◽  
Bovine Brain ◽  
Spindle Elongation ◽  
Anaphase B

Studies of mitotic spindle elongation in vitro using populations of diatom spindles visualized with immunofluorescence microscopy have shown that the two interdigitating half-spindles are driven apart by an ATP-dependent process that generates force in the zone of overlap between half-spindles. To characterize further the system responsible for spindle elongation, we observed spindle elongation directly with polarized light or phase-contrast video-microscopy. We report that the kinetics of spindle elongation versus time are linear. A constant rate of spindle elongation occurs despite the continuous decrease in length of the zone of overlap between half-spindles. The average rate of spindle elongation varies as a function of treatment, and rates measured match spindle elongation rates measured in vivo. When spindles elongated in the presence of polymerizing tubulin (from bovine brain), the extent of elongation was greater than the original zone of half-spindle overlap, but the rate of elongation was constant. No component of force due to tubulin polymerization was found. The total elongation observed in the presence of added tubulin could exceed a doubling of original spindle length, matching the elongation in the intact diatom. The linear rate of spindle elongation in vitro suggests that the force transducer for anaphase B is a mechanochemical ATPase, analogous to dynein or myosin, and that the force for spindle elongation does not arise from stored energy, e.g. in an elastic matrix in the midzone. Additionally, on the basis of observations described here, we conclude that the force-transduction system for spindle elongation must be able to remain in the zone of microtubule overlap during the sliding apart of half-spindles, and that the transducer can generate force between microtubules that are not strictly antiparallel.

scholarly journals Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Lara Katharina Krüger ◽  
Matthieu Gélin ◽  
Liang Ji ◽  
Carlos Kikuti ◽  
Anne Houdusse ◽  
...  
Keyword(s):  
Dual Function ◽  
Tubulin Subunit ◽  
Microtubule Growth ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Spindle Stability ◽  
Spindle Function ◽  
Anaphase B

Mitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation in S. pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, creating a link between the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.

The kinesin-like protein CENP-E is kinetochore-associated throughout poleward chromosome segregation during anaphase-A

1996 ◽  
Vol 109 (5) ◽  
pp. 961-969 ◽  
Author(s):  
K.D. Brown ◽  
K.W. Wood ◽  
D.W. Cleveland
Keyword(s):  
Chromosome Segregation ◽  
Chromosome Movement ◽  
Initial Alignment ◽  
Late Prophase ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset ◽  
Spindle Elongation ◽  
Anaphase B

The kinesin-like protein CENP-E transiently associates with kinetochores following nuclear envelope breakdown in late prophase, remains bound throughout metaphase, but sometime after anaphase onset it releases and by telophase becomes bound to interzonal microtubules of the mitotic spindle. Inhibition of poleward chromosome movement in vitro by CENP-E antibodies and association of CENP-E with minus-end directed microtubule motility in vitro have combined to suggest a key role for CENP-E as an anaphase chromosome motor. For this to be plausible in vivo depends on whether CENP-E remains kinetochore associated during anaphase. Using Indian muntjac cells whose seven chromosomes have large, easily tracked kinetochores, we now show that CENP-E is kinetochore-associated throughout the entirety of anaphase-A (poleward chromosome movement), relocating gradually during spindle elongation (anaphase-B) to the interzonal microtubules. These observations support roles for CENP-E not only in the initial alignment of chromosomes at metaphase and in spindle elongation in anaphase-B, but also in poleward chromosome movement in anaphase-A.

scholarly journals Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth

2021 ◽  
Author(s):  
Lara K. Krüger ◽  
Matthieu Gélin ◽  
Liang Ji ◽  
Carlos Kikuti ◽  
Anne Houdusse ◽  
...  
Keyword(s):  
Dual Function ◽  
Tubulin Subunit ◽  
Microtubule Growth ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Spindle Stability ◽  
Spindle Function ◽  
Anaphase B

AbstractMitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation in S. pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, to link the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.

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.

Measurement of axial forces during emptying from the human stomach

1992 ◽  
Vol 263 (2) ◽  
pp. G230-G239 ◽  
Author(s):  
M. J. Vassallo ◽  
M. Camilleri ◽  
C. M. Prather ◽  
R. B. Hanson ◽  
G. M. Thomforde
Keyword(s):  
Axial Force ◽  
Longitudinal Axis ◽  
Force Transducer ◽  
Human Stomach ◽  
In Vitro Studies ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
Axial Forces

Our aim was to measure axial forces in the stomach and to evaluate their relation to circumferential contractions of the gastric walls and the emptying of gastric content. We used a combination of simultaneous radioscintigraphy, gastroduodenal manometry, and an axial force transducer with an inflatable 2-ml balloon fluoroscopically placed in the antrum. In vitro studies demonstrated that the axial force transducer records only antegrade forces along the longitudinal axis of this probe in an intensity-dependent manner. In vivo studies were performed in five healthy subjects for at least 3 h after ingestion of radiolabeled meals. When administered separately, the emptying of liquids or solids from the stomach is associated with generation of antral axial forces and coincident phasic pressure activity; however, almost 20% (average) of gastric axial forces during emptying of liquids or solids are unassociated with proximal or distal antral pressure activity ("isolated" forces). High amplitude antral axial forces and pressures occur during both lag and postlag emptying phases. During emptying of liquids, there is a trend for axial forces to be coincident more often with proximal than with distal antral pressure activity and vice versa for the emptying of solids (P = 0.015). These data suggest that when placed in the antrum, the transducer can semiquantitatively record axial forces during gastric emptying. By combining these observations with the data from in vitro studies, it appears that axial forces predominantly result from traction on the balloon by the longitudinal vector resulting from circumferential gastric contractions. The combination of radioscintigraphy and measurement of antral axial forces is a promising method to evaluate mechanical forces involved in the emptying of the human stomach.

scholarly journals Evidence that myosin does not contribute to force production in chromosome movement.

1982 ◽  
Vol 94 (1) ◽  
pp. 165-178 ◽  
Author(s):  
D P Kiehart ◽  
I Mabuchi ◽  
S Inoué
Keyword(s):  
Gamma Globulin ◽  
Meiotic Chromosome ◽  
Force Production ◽  
Chromosome Movement ◽  
Actomyosin Interaction ◽  
Dividing Cells ◽  
Spindle Elongation ◽  
Chromosome Movements

Antibody against cytoplasmic myosin, when microinjected into actively dividing cells, provides a physiological test for the role of actin and myosin in chromosome movement. Anti-Asterias egg myosin, characterized by Mabuchi and Okuno (1977, J. Cell Biol., 74:251), completely and specifically inhibits the actin activated Mg++ -ATPase of myosin in vitro and, when microinjected, inhibits cytokinesis in vivo. Here, we demonstrate that microinjected antibody has no observable effect on the rate or extent of anaphase chromosome movements. Neither central spindle elongation nor chromosomal fiber shortening is affected by doses up to eightfold higher than those require to uniformly inhibit cytokinesis in all injected cells. We calculate that such doses are sufficient to completely inhibit myosin ATPase activity in these cells. Cells injected with buffer alone, with myosin-absorbed antibody, or with nonimmune gamma-globulin, proceed normally through both mitosis and cytokinesis. Control gamma-globulin, labeled with fluorescein, diffuses to homogeneity throughout the cytoplasm in 2-4 min and remains uniformly distributed. Antibody is not excluded from the spindle region. Prometaphase chromosome movements, fertilization, pronuclear migration, and pronuclear fusion are also unaffected by microinjected antimyosin. These experiments demonstrate that antimyosin blocks the actomyosin interaction thought to be responsible for force production in cytokinesis but has no effect on mitotic or meiotic chromosome motion. They provide direct physiological evidence that myosin is not involved in force production for chromosome movement.

scholarly journals ULTRASTRUCTURAL ANALYSIS OF MITOTIC SPINDLE ELONGATION IN MAMMALIAN CELLS IN VITRO

1971 ◽  
Vol 50 (2) ◽  
pp. 416-431 ◽  
Author(s):  
B. R. Brinkley ◽  
Joiner Cartwright
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Spindle Axis ◽  
Hamster Strain ◽  
Early Anaphase ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Elongation Process

The mitotic spindle of many mammalian cells undergoes an abrupt elongation at anaphase. In both cultured rat kangaroo (strain PtK1) and Chinese hamster (strain Don-C) fibroblasts, the distance from pole to pole at metaphase doubles during anaphase and telophase. In order to determine the organization and distribution of spindle microtubules during the elongation process, cells were fixed and flat embedded in Epon 812. Selected cells were photographed with the phase-contrast microscope and then serially sectioned perpendicular to the major spindle axis. Microtubule profiles were counted in selected sections, and the number was plotted with respect to position along the spindle axis. Interpretation of the distribution profiles indicated that not all interpolar microtubules extended from pole to pole. It is estimated that 55–70% of the interpolar microtubules are overlapped at the cell equator while 30–45% extend across the equator into both half spindles. This arrangement appeared to persist from early anaphase (before elongation) until telophase after the elongation process. Although sliding or shearing of microtubules may occur in the spindle, such appears not to be the mechanism by which the spindle elongates in anaphase. Instead, our data support the hypothesis that spindle elongation occurs by growth of prepositioned microtubules which "push" the poles apart.

The Novel Kinesin Spindle Protein Inhibitor SB-743921 Exhibits Marked Activity In In Vivo and In Vitro In Models of Aggressive Diffuse Large B-Cell Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 118-118
Author(s):  
Danielle C Bongero ◽  
Luca Paoluzzi ◽  
Enrica Marchi ◽  
Neisa Roberto ◽  
Rafael Escandon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Line ◽  
Germinal Center ◽  
Mitotic Spindle ◽  
Tumor Volume ◽  
P Value ◽  
Kinesin Spindle Protein

Abstract Abstract 118 A mitotic spindle target that has emerged as unique and potentially restricted to the mitotic spindle is Eg5, also known as the kinesin spindle protein (KSP). SB-743921 induces mitotic spindle dysfunction and cell cycle arrest by inhibiting Eg5. Preliminary Phase 1 studies of SB-743921 have demonstrated that this compound is not associated with any neuropathy like other anti-mitotic agents. These studies have also demonstrated a potential signal in patients with relapsed and refractory lymphoma. We investigated the efficacy of SB-743921 in aggressive B-cell lymphomas to evaluate effectiveness and tolerability in germinal center (GCB) and post germinal center (ABC) diffuse large B-cell lymphomas (DLBCL). For cytotoxicity assays, luminescent cell viability was performed using CellTiter-Glo™ followed by acquisition with Biotek Synergy HT. The IC50s were calculated using the Calcusyn software (Biosoft). Cell Cycle was assessed by staining with Vybrant DyeCycle Green (Invitrogen) followed by FACSCalibur acquisition. Whole cell lysate proteins were extracted and quantified according to Bradford assay. After electrophoresis on a gradient 4–20% SDS-PAGE gels the proteins were transferred to nitrocellulose membrane. After blocking and incubation with the primary and the secondary antibodies, the chemiluminescent agent was added and the x-ray films were exposed to the membranes. In vivo experiments were performed with five to 7-week-old severe combined immunodeficiency (SCID) beige mice (Taconic Laboratories, Germantown, NY) injected with 1 × 107 Ly1-DLBCL cells on the flank via a subcutaneous (SQ) route. When tumor volumes approached 80 mm3, mice were separated into cohorts of ten mice each. Tumors were assessed using the two largest perpendicular axes (l, length; w, width) as measured with standard calipers. Tumor volume was calculated using the formula 4/3 r3, where r=(l + w) / 4. Tumor-bearing mice were assessed for weight loss and tumor volume at least twice weekly. The IC50 values for SB-743921 across a panel of different DLBCL lines are listed in table 1. Cell cycle analysis showed that compared to the untreated group, after treatment with 100nM of SB 743921 the percentage of GCB cells in G2/M phase increased from 17.6% to 40.3% (+129%) in Ly7, 23.9% to 40.7 % (+70%) in Sudhl6 and from 17.55% to 32.4% (+85%) in Ly1. In comparison, the percent increase of cells in G2/M for the ABC lines was statistically less (p-value 0.001). For example, Ly10 increased from 15% to 27.6% (+45%), Riva from 29.3% to 36.95% (+26%) and Sudhl2 from 22.6% to 27.6% (+22%). Immunoblot analysis of DLBCL cells treated with SB-743921 probed for Eg5, CyclinB1, and phosphorylated BubR1 revealed that although all cells demonstrated a measurable increase in Eg5, the total Eg5 present varied from cell line to cell line. The In vivo xenograft experiment was conducted with the GCB Ly1 cell line and consisted of 4 cohorts; one control and 3 treatments with doses of 2.5 mg/kg, 5 mg/kg and 10 mg/kg. SB-743921 was administered by the intraperitoneal route on days 1, 5, and 9 on a 23 day cycle for 2 cycles. The graph below displays the inhibition of tumor growth in the cohorts after treatment with SB-74321. All 3 cohorts had a p-value of <0.001 relative to the control. In conclusion, SB-743921 is promising as a single agent for treatment of DLBCL. Future studies exploring the specific cell cycle features of different cell lines with respect to their check-point control will afford new opportunities to better understand the mechanisms of increased resistance in ABC compared to GCB. The data suggests SB 743921 overall is effective in the treatment of DLBCL both in vitro and in vivo. Further studies exploring potential synergistic interactions with conventional chemotherapeutic agents as well as establishing the most effective treatment schedules for the agent may provide a new approach to treating these diseases. Disclosures: Escandon: Cytokinetics: Employment. Wood:Cytokinetics: Employment. O'Connor:Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C.

1996 ◽  
Vol 16 (7) ◽  
pp. 3576-3586 ◽  
Author(s):  
C H Yang ◽  
J Tomkiel ◽  
H Saitoh ◽  
D H Johnson ◽  
W C Earnshaw
Keyword(s):  
Dna Binding ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
In Vitro Assays ◽  
Centromeric Heterochromatin ◽  
Structural Protein ◽  
Chromosomal Site

The kinetochore in eukaryotes serves as the chromosomal site of attachment for microtubules of the mitotic spindle and directs the movements necessary for proper chromosome segregation. In mammalian cells, the kinetochore is a highly differentiated trilaminar structure situated at the surface of the centromeric heterochromatin. CENP-C is a basic, DNA-binding protein that localizes to the inner kinetochore plate, the region that abuts the heterochromatin. Microinjection experiments using antibodies specific for CENP-C have demonstrated that this protein is required for the assembly and/or stability of the kinetochore as well as for a timely transition through mitosis. From these observations, it has been suggested that CENP-C is a structural protein that is involved in the organization or the kinetochore. In this report, we wished to identify and map the functional domains of CENP-C. Analysis of CENP-C truncation mutants expressed in vivo demonstrated that CENP-C possesses an autonomous centromere-targeting domain situated at the central region of the CENP-C polypeptide. Similarly, in vitro assays revealed that a region of CENP-C with the ability to bind DNA is also located at the center of the CENP-C molecule, where it overlaps the centromere-targeting domain.

scholarly journals The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time

2002 ◽  
Vol 157 (7) ◽  
pp. 1139-1149 ◽  
Author(s):  
Jordan W. Raff ◽  
Kim Jeffers ◽  
Jun-yong Huang
Keyword(s):  
Cell Cycle ◽  
Triple Point ◽  
Mitotic Spindle ◽  
Cyclin B ◽  
Second Phase ◽  
Point Mutant ◽  
Two Phases ◽  
Drosophila Cells

In Drosophila cells cyclin B is normally degraded in two phases: (a) destruction of the spindle-associated cyclin B initiates at centrosomes and spreads to the spindle equator; and (b) any remaining cytoplasmic cyclin B is degraded slightly later in mitosis. We show that the APC/C regulators Fizzy (Fzy)/Cdc20 and Fzy-related (Fzr)/Cdh1 bind to microtubules in vitro and associate with spindles in vivo. Fzy/Cdc20 is concentrated at kinetochores and centrosomes early in mitosis, whereas Fzr/Cdh1 is concentrated at centrosomes throughout the cell cycle. In syncytial embryos, only Fzy/Cdc20 is present, and only the spindle-associated cyclin B is degraded at the end of mitosis. A destruction box–mutated form of cyclin B (cyclin B triple-point mutant [CBTPM]–GFP) that cannot be targeted for destruction by Fzy/Cdc20, is no longer degraded on spindles in syncytial embryos. However, CBTPM–GFP can be targeted for destruction by Fzr/Cdh1. In cellularized embryos, which normally express Fzr/Cdh1, CBTPM–GFP is degraded throughout the cell but with slowed kinetics. These findings suggest that Fzy/Cdc20 is responsible for catalyzing the first phase of cyclin B destruction that occurs on the mitotic spindle, whereas Fzr/Cdh1 is responsible for catalyzing the second phase of cyclin B destruction that occurs throughout the cell. These observations have important implications for the mechanisms of the spindle checkpoint.

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