<i>Rossignol: An Edition and Translation</i>. J. L. Baird , John R. Kane

Margolis, Nadia

doi:10.2307/2847893

Cited by 4 publications

(11 citation statements)

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“…The kinetics of incorporation of [32P]P-MAP2 into microtubules suggests two different mechanisms : about 40 "/, of the total radioactivity added to the microtubule solution is found associated with these structures after approximately 30 min, while the rest of the radioactivity is incorporated in a linear manner at a lower rate. The incorporation rate of [32P]P-MAP2 in the second part of the curve is in good agreement with the theoretical rate which may be calculated by considering a mechanism for the incorporation of ["PI-P-MAP2 into microtubules similar to that described by Margolis and Wilson for tubulin [9], taking into account the relative proportion of MAP2 to tubulin in the microtubule protein.…”

Section: Phospho-protein [32p]p-map2 Was Prepared By Transferring [32supporting

confidence: 87%

“…The microtubule-assembly reaction was performed at 0.08 mM GTP, in the presence of acetate kinase and acetyl phosphate, as a GTP-regenerating system. The conditions used for microtubule assembly and maintenance of steady state over a long time course were those described by Margolis and Wilson [9]. Assembly was initiated by incubation of samples (0.5 ml) with approximately 1.5 mg protein/ml, at 30"C, and the incubation was extended for at least 90 min before the microtubule samples were used, to ensure that steady-state conditions were reached.…”

Section: Assembly Of Microtubules and Maintenance Of Steady-state Conmentioning

confidence: 99%

“…Since all incubations terminated at a single time, the addition of drugs, labelled molecules or compounds to chase the incorporation of label into microtubules, was performed in a staggered time sequence. At the end of the incubation the microtubules were pelleted and the microtubule pellets were washed extensively and resuspended in 0.5 ml of cold Pi/Glu buffer as described by Margolis and Wilson [9]. The radioactivity incorporated into microtubules was determined in each sample by liquid scintillation counting and the protein content was quantified by the method of Lowry et al [12].…”

Section: Treatment Of Microtuhule Samples and Analysis Of The Incorpomentioning

confidence: 99%

“…A dynamic equilibrium of microtubules with their tubulin subunits has been suggested to exist under conditions in vitro [20]. An elegant experiment by Margolis and Wilson [9] showed that such a dynamic exchange of subunits on microtubules involves polymerization and depolymerization or tubulin at the opposite ends of the microtubule, at equivalent rates. We have used this system to study the incorporation of phospho-protein [32P]P-MAP2 into microtubules at steady state.…”

Section: Phospho-protein [32p]p-map2 Was Prepared By Transferring [32mentioning

confidence: 99%

“…One method to obtain information about the possible role of protein MAP2 is to study how it is incorporated into microtubules. It has been shown recently that under conditions in vitro the microtubule reaches a steady state in which tubulin addition to and loss from microtubules occurs at opposite ends with equivalent rates [9]. Since protein MAP2 may be radioactively labelled by means of the endogeneous protein kinase reaction, we have taken advantage of this fact to study its incorporation into microtubules under steady-state conditions.…”

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confidence: 99%

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Incorporation of the High‐Molecular‐Weight Microtubule‐Associated Protein 2 (MAP2) into Microtubules at Steady State in vitro

Manso-Martínez

Villasanté

Ávila

1980

European Journal of Biochemistry

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Purified high‐molecular‐weight microtubule‐associated protein 2 (MAP2) labelled with [32P]‐phosphate has been obtained and used as a marker to study the incorporation of this protein into micfotubules at steady state in vitro. The incorporation kinetics of protein MAP2 show two different mechanisms of addition of this protein into microtubules. A fast incorporation, which proceeds essentially independently of microtubule assembly, indicates its addition into binding positions which may be available along the structure. Once the microtubule has been saturated with the protein, its incorporation proceeds from one end of the microtubule, in a polymerization‐dependent manner, at a slower rate. Loss of the protein from microtubules at steady state proceeds fundamentally at the opposite end.

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Section: Phospho-protein [32p]p-map2 Was Prepared By Transferring [32supporting

confidence: 87%

Section: Assembly Of Microtubules and Maintenance Of Steady-state Conmentioning

confidence: 99%

Section: Treatment Of Microtuhule Samples and Analysis Of The Incorpomentioning

confidence: 99%

Section: Phospho-protein [32p]p-map2 Was Prepared By Transferring [32mentioning

confidence: 99%

mentioning

confidence: 99%

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Incorporation of the High‐Molecular‐Weight Microtubule‐Associated Protein 2 (MAP2) into Microtubules at Steady State in vitro

Manso-Martínez

Villasanté

Ávila

1980

European Journal of Biochemistry

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Analysis of the treadmilling model during metaphase of mitosis using fluorescence redistribution after photobleaching.

Wadsworth¹,

Salmon²

1986

The Journal of Cell Biology

105

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Abstract. One recent hypothesis for the mechanism of chromosome movement during mitosis predicts that a continual, uniform, poleward flow or "treadmilling" of microtubules occurs within the half-spindle between the chromosomes and the poles during mitosis (Margolis, R. L., and L. Wilson, 1981, Nature (Lond.), 293:705-711). We have tested this treadmilling hypothesis using fluorescent analog cytochemistry and measurements of fluorescence redistribution after photobleaching to examine microtubule behavior during metaphase of mitosis. Mitotic BSC 1 mammalian tissue culture cells or newt lung epithelial cells were microinjected with brain tubulin labeled with 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF) to provide a fluorescent tracer of the endogenous tubulin pool. Using a laser microbeam, fluorescence in the half-spindle was photobleached in either a narrow 1.6 #m wide bar pattern across the half-spingle or in a circular area of 2.8 or 4.5 #m diameter. Fluorescence recovery in the spindle fibers, measured using video microscopy or photometric techniques, occurs as bleached DTAF-tubulin subunits within the microtubules are exchanged for unbleached DTAF-tubulin in the cytosol by steady-state microtubule assemblydisassembly pathways. Recovery of 75% of the bleached fluorescence follows first-order kinetics and has an average half-time of 37 sec, at 31-33°C. No translocation of the bleached bar region could be detected during fluorescence recovery, and the rate of recovery was independent of the size of the bleached spot. These results reveal that, for 75% of the halfspindle microtubules, FRAP does not occur by a synchronous treadmilling mechanism.T HE dynamic non-steady-state behavior of spindle microtubules in living cells has been clearly demonstrated by a variety of experimental approaches. For example, the majority of spindle fiber birefringence can be rapidly and reversibly abolished by cold, high-pressure, or anti-mitotic drugs (28). Spindle birefringence disappears uniformly throughout the half-spindle, primarily due to the depolymerization of non-kinetochore microtubules. Kinetochore microtubules, usually a small fraction of the total number of microtubules in the half-spindle, are differentially stable to such treatments (3,25,30).More recently, the steady-state behavior of spindle microtubules has been studied using the techniques of fluorescence analog cytochemistry (40) and fluorescence redistribution after photobleaching (FRAP) j (1,12,14,43). These studies have demonstrated that steady-state tubulin subunit exchange with the majority of spindle microtubules is remarkably rapid. In these experiments, mitotic tissue culture cells or sea urchin embryos were microinjected with fluorescein-labeled tubulin (13,16,42) to serve as a tracer of the endogenous tubulin A bbrevialions used in this paper: ARBs, areas of reduced birefringence; DTAF, 5-(4,6-dichlorotfiazin-2-yl) amino fluorescein; FRAP, fluorescence redistribution after photobleaching.pool. Fluorescent tubulin incorporated into spindle fibers ...

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Bistability and oscillations in cooperative microtubule and kinetochore dynamics in the mitotic spindle

Schwietert

Kierfeld

2020

New J. Phys.

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In the mitotic spindle microtubules attach to kinetochores via catch bonds during metaphase, and microtubule depolymerization forces give rise to stochastic chromosome oscillations. We investigate the cooperative stochastic microtubule dynamics in spindle models consisting of ensembles of parallel microtubules, which attach to a kinetochore via elastic linkers. We include the dynamic instability of microtubules and forces on microtubules and kinetochores from elastic linkers. A one-sided model, where an external force acts on the kinetochore is solved analytically employing a mean-field approach based on Fokker–Planck equations. The solution establishes a bistable force–velocity relation of the microtubule ensemble in agreement with stochastic simulations. We derive constraints on linker stiffness and microtubule number for bistability. The bistable force–velocity relation of the one-sided spindle model gives rise to oscillations in the two-sided model, which can explain stochastic chromosome oscillations in metaphase (directional instability). We derive constraints on linker stiffness and microtubule number for metaphase chromosome oscillations. Including poleward microtubule flux into the model we can provide an explanation for the experimentally observed suppression of chromosome oscillations in cells with high poleward flux velocities. Chromosome oscillations persist in the presence of polar ejection forces, however, with a reduced amplitude and a phase shift between sister kinetochores. Moreover, polar ejection forces are necessary to align the chromosomes at the spindle equator and stabilize an alternating oscillation pattern of the two kinetochores. Finally, we modify the model such that microtubules can only exert tensile forces on the kinetochore resulting in a tug-of-war between the two microtubule ensembles. Then, induced microtubule catastrophes after reaching the kinetochore are necessary to stimulate oscillations. The model can reproduce experimental results for kinetochore oscillations in PtK1 cells quantitatively.

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Rossignol: An Edition and Translation. J. L. Baird , John R. Kane

Cited by 4 publications

References 0 publications

Incorporation of the High‐Molecular‐Weight Microtubule‐Associated Protein 2 (MAP2) into Microtubules at Steady State in vitro

Incorporation of the High‐Molecular‐Weight Microtubule‐Associated Protein 2 (MAP2) into Microtubules at Steady State in vitro

Analysis of the treadmilling model during metaphase of mitosis using fluorescence redistribution after photobleaching.

Bistability and oscillations in cooperative microtubule and kinetochore dynamics in the mitotic spindle

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