How motor proteins influence microtubule polymerization dynamics

2000 ◽  
Vol 113 (24) ◽  
pp. 4379-4389 ◽  
Author(s):  
A.W. Hunter ◽  
L. Wordeman
Keyword(s):  
Intracellular Transport ◽  
Motor Proteins ◽  
Force Production ◽  
Nucleotide Hydrolysis ◽  
Microtubule Polymerization ◽  
Microtubule Stability ◽  
Microtubule Growth ◽  
Chromosome Movements ◽  
Shed Light

The interplay between microtubules and microtubule-based motors is fundamental to basic aspects of cellular function, such as the intracellular transport of organelles and alterations in cellular morphology during cell locomotion and division. Motor proteins are unique in that they couple nucleotide hydrolysis to force production that can do work. The force transduction by proteins belonging to the kinesin and dynein superfamilies has been thought only to power movement of these motors along the surface of microtubules; however, a growing body of evidence, both genetic and biochemical, suggests that motors can also directly influence the polymerization dynamics of microtubules. For example, at the vertebrate kinetochore, motors interact directly with microtubule ends and modulate polymerization dynamics to orchestrate chromosome movements during mitosis. Although a role for motors in regulating microtubule length has been established, the mechanisms used by motors to promote microtubule growth or shrinkage are unclear, as is an understanding of why cells might choose motors to control dynamics rather than a variety of non-motor proteins known to affect microtubule stability. Elucidation of the exact mechanisms by which motors alter the exchange of tubulin subunits at microtubule ends in vitro may shed light on how microtubule stability is regulated to produce the array of dynamic behavior seen in cells.

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.

Melanosomes on the move: a model to understand organelle dynamics

2011 ◽  
Vol 39 (5) ◽  
pp. 1191-1196 ◽  
Author(s):  
Alistair N. Hume ◽  
Miguel C. Seabra
Keyword(s):  
Intracellular Transport ◽  
Motor Proteins ◽  
Molecular Mechanisms ◽  
Pigment Cells ◽  
Future Prospects ◽  
Dynamic Nature ◽  
Live Cell Microscopy ◽  
Intracellular Organelles ◽  
Cell Microscopy ◽  
Shed Light

Advances in live-cell microscopy have revealed the extraordinarily dynamic nature of intracellular organelles. Moreover, movement appears to be critical in establishing and maintaining intracellular organization and organellar and cellular function. Motility is regulated by the activity of organelle-associated motor proteins, kinesins, dyneins and myosins, which move cargo along polar MT (microtubule) and actin tracks. However, in most instances, the motors that move specific organelles remain mysterious. Over recent years, pigment granules, or melanosomes, within pigment cells have provided an excellent model for understanding the molecular mechanisms by which motor proteins associate with and move intracellular organelles. In the present paper, we discuss recent discoveries that shed light on the mechanisms of melanosome transport and highlight future prospects for the use of pigment cells in unravelling general molecular mechanisms of intracellular transport.

scholarly journals Purification of functional Plasmodium falciparum tubulin allows for the identification of parasite-specific microtubule inhibitors

2021 ◽  
Author(s):  
William Graham Hirst ◽  
Dominik Fachet ◽  
Benno Kuropka ◽  
Christoph Weise ◽  
Kevin J Saliba ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Drug Targets ◽  
Cytoskeletal Proteins ◽  
Microtubule Inhibitors ◽  
Microtubule Polymerization ◽  
Molecular Building Block ◽  
Structural Divergence ◽  
Microtubule Growth ◽  
Exciting Possibility

Cytoskeletal proteins are essential for parasite proliferation, growth, and transmission, and therefore represent promising drug targets. While αβ-tubulin, the molecular building block of microtubules, is an established drug target in a variety of cancers, we still lack substantial knowledge of the biochemistry of parasite tubulins, which would allow us to exploit the structural divergence between parasite and human tubulins. Indeed, mechanistic insights have been limited by the lack of purified, functional parasite tubulin. In this study, we isolated Plasmodium falciparum tubulin that is assembly-competent and shows specific microtubule dynamics in vitro. We further present mechanistic evidence that two compounds selectively interact with parasite over host microtubules and inhibit Plasmodium microtubule polymerization at substoichiometric compound concentrations. The ability of compounds to selectively disrupt protozoan microtubule growth without affecting human microtubules provides the exciting possibility for the targeted development of novel antimalarials.

scholarly journals Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells

2014 ◽  
Vol 204 (7) ◽  
pp. 1111-1121 ◽  
Author(s):  
Emmanuel Gallaud ◽  
Renaud Caous ◽  
Aude Pascal ◽  
Franck Bazile ◽  
Jean-Philippe Gagné ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Microtubule Associated Proteins ◽  
Microtubule Polymerization ◽  
Sister Chromatids ◽  
Centrosome Separation ◽  
Associated Proteins ◽  
Microtubule Growth ◽  
Mitotic Spindle Assembly

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.

scholarly journals Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate.

1991 ◽  
Vol 114 (1) ◽  
pp. 73-81 ◽  
Author(s):  
R A Walker ◽  
N K Pryer ◽  
E D Salmon
Keyword(s):  
Dynamic Instability ◽  
Elongation Rate ◽  
Gtp Hydrolysis ◽  
Microtubule Stability ◽  
Assembly Mechanism ◽  
High Elongation ◽  
Microtubule Growth ◽  
Hydrolysis Of ◽  
Brain Tubulin

Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 microM) was assembled at 37 degrees C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution). Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length. Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated lifetime of the stable cap after infinite dilution was less than 3-4 s for plus and minus ends, much shorter than the approximately 200 s observed at steady state (Walker, R. A., E. T. O'Brien, N. K. Pryer, M. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. 1988. J. Cell Biol. 107:1437-1448.). We conclude that during elongation, both plus and minus ends are stabilized by a short region (approximately 200 dimers or less) and that the size of the stable cap is independent of 10-fold variation in elongation rate. These results eliminate models of dynamic instability which predict extensive "build-up" stabilizing caps and support models which constrain the cap to the elongating tip. We propose that the cell may take advantage of such an assembly mechanism by using "catastrophe factors" that can promote frequent catastrophe even at high elongation rates by transiently binding to microtubule ends and briefly inhibiting GTP-tubulin association.

scholarly journals XMAP215 promotes microtubule catastrophe by disrupting the growing microtubule end

2020 ◽  
Author(s):  
Veronica Farmer ◽  
Göker Arpağ ◽  
Sarah Hall ◽  
Marija Zanic
Keyword(s):  
Growth Rate ◽  
Growth Rates ◽  
Fast Growth ◽  
Fast Growing ◽  
Microtubule Stability ◽  
Large Fluctuations ◽  
Microtubule Growth ◽  
Enhanced Stability

ABSTRACTThe GTP-tubulin cap is widely accepted to protect microtubules against catastrophe. The GTP-cap size is thought to increase with the microtubule growth rate, presumably endowing fast-growing microtubules with enhanced stability. It is unknown what GTP-cap properties permit frequent microtubule catastrophe despite fast growth. Here, we investigate microtubules grown in vitro in the presence and absence of the microtubule polymerase XMAP215. Using EB1 as a GTP-cap marker, we find that GTP-cap size increases regardless of whether growth acceleration is achieved by increasing tubulin concentration or by XMAP215. In spite of the increased mean GTP-cap size, microtubules grown with XMAP215 display increased catastrophe frequency, in contrast to microtubules grown with more tubulin, for which catastrophe is abolished. However, microtubules polymerized with XMAP215 have large fluctuations in growth rate and EB1 intensity; display tapered and curled ends; and undergo catastrophe at faster growth rates and with higher EB1 end-localization. Our results underscore the role of growth irregularities in overall microtubule stability.

scholarly journals EB3-informed dynamics of the microtubule stabilizing cap during stalled growth

2021 ◽  
Author(s):  
Maurits Kok ◽  
Florian Huber ◽  
Svenja-Marei Kalisch ◽  
Marileen Dogterom
Keyword(s):  
Growth Velocity ◽  
Hydrolysis Rate ◽  
Exact Relation ◽  
Gtp Hydrolysis ◽  
Lifetime Distributions ◽  
Microtubule Stability ◽  
In Vitro Reconstitution ◽  
1D Model ◽  
Microtubule Growth

Microtubule stability is known to be governed by a stabilizing GTP/GDP-Pi cap, but the exact relation between growth velocity, GTP hydrolysis and catastrophes remains unclear. We investigate the dynamics of the stabilizing cap through in vitro reconstitution of microtubule dynamics in contact with micro-fabricated barriers, using the plus-end binding protein GFP-EB3 as a marker for the nucleotide state of the tip. The interaction of growing microtubules with steric objects is known to slow down microtubule growth and accelerate catastrophes. We show that the lifetime distributions of stalled microtubules, as well as the corresponding lifetime distributions of freely growing microtubules, can be fully described with a simple phenomenological 1D model based on noisy microtubule growth and a single EB3-dependent hydrolysis rate. This same model is furthermore capable of explaining both the previously reported mild catastrophe dependence on microtubule growth rates and the catastrophe statistics during tubulin washout experiments.

scholarly journals Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis

2005 ◽  
Vol 170 (7) ◽  
pp. 1047-1055 ◽  
Author(s):  
Kazuhisa Kinoshita ◽  
Tim L. Noetzel ◽  
Laurence Pelletier ◽  
Karl Mechtler ◽  
David N. Drechsel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Xenopus Laevis ◽  
Microtubule Assembly ◽  
Aurora A ◽  
Specific Mechanism ◽  
Microtubule Polymerization ◽  
Egg Extracts ◽  
Microtubule Growth

Centrosomes act as sites of microtubule growth, but little is known about how the number and stability of microtubules emanating from a centrosome are controlled during the cell cycle. We studied the role of the TACC3–XMAP215 complex in this process by using purified proteins and Xenopus laevis egg extracts. We show that TACC3 forms a one-to-one complex with and enhances the microtubule-stabilizing activity of XMAP215 in vitro. TACC3 enhances the number of microtubules emanating from mitotic centrosomes, and its targeting to centrosomes is regulated by Aurora A–dependent phosphorylation. We propose that Aurora A regulation of TACC3 activity defines a centrosome-specific mechanism for regulation of microtubule polymerization in mitosis.

scholarly journals Chromosome movement in lysed mitotic cells is inhibited by vanadate.

1978 ◽  
Vol 79 (2) ◽  
pp. 573-580 ◽  
Author(s):  
W Z Cande ◽  
S M Wolniak
Keyword(s):  
Atpase Activity ◽  
Metabolic Inhibitors ◽  
Chromosome Movement ◽  
Microtubule Polymerization ◽  
Spindle Poles ◽  
Brij 58 ◽  
Spindle Elongation ◽  
Chromosome Movements

Mitotic PtK1 cells, lysed at anaphase into a carbowax 20 M Brij 58 solution, continue to move chromosomes toward the spindle poles and to move the spindle poles apart at 50% in vivo rates for 10 min. Chromosome movements can be blocked by adding metabolic inhibitors to the lysis medium and inhibition of movement can be reversed by adding ATP to the medium. Vanadate at micromolar levels reversibly inhibits dynein ATPase activity and movement of demembranated flagella and cilia. It does not affect glycerinated myofibril contraction or myosin ATPase activty at less than millimolar concentrations. Vanadate at 10--100 micron reversibly inhibits anaphase movement of chromosomes and spindle elongation. After lysis in vanadate, spindles lose their fusiform appearance and become more barrel shaped. In vitro microtubule polymerization is insensitive to vanadate.

scholarly journals Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity

2016 ◽  
Vol 113 (17) ◽  
pp. 4788-4793 ◽  
Author(s):  
Monica Markovski ◽  
Jessica L. Bohrhunter ◽  
Tania J. Lupoli ◽  
Tsuyoshi Uehara ◽  
Suzanne Walker ◽  
...  
Keyword(s):  
Polymerase Activity ◽  
Activation Mechanism ◽  
Phenotypic Analysis ◽  
Outer Membranes ◽  
Division Site ◽  
Physiological Relevance ◽  
Membrane Lipoproteins ◽  
Shed Light

To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a ofEscherichia colirequire the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b–LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed.

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