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 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 Anaphase A: Melting Microtubules Move Chromosomes toward Spindle Poles

2017 ◽  
Author(s):  
Charles L. Asbury
Keyword(s):  
Fluorescence Microscopy ◽  
Mechanistic Models ◽  
Chromosome Movement ◽  
Load Bearing ◽  
Spindle Poles ◽  
Sister Chromatids ◽  
Spindle Elongation ◽  
Anaphase B ◽  
Chromosome Movements ◽  
Cellular Movement

The separation of sister chromatids during anaphase is the culmination of mitosis and one of the most strikingly beautiful examples of cellular movement. It consists of two distinct processes: Anaphase A, the movement of chromosomes toward spindle poles via shortening of the connecting fibers, and anaphase B, separation of the two poles from one another via spindle elongation. I focus here on anaphase A chromosome-to-pole movement. The chapter begins by summarizing classical observations of chromosome movements, which support the current understanding of anaphase mechanisms. Live cell fluorescence microscopy studies showed that poleward chromosome movement is associated with disassembly, or ‘melting’ of the kinetochore-attached microtubule fibers that link chromosomes to poles. Microtubule-marking techniques established that kinetochore-fiber disassembly often occurs through a ‘pac-man’ mechanism, where tubulin subunits are lost from kinetochore-attached plus ends and the kinetochore appears to consume its microtubule track as it moves poleward. In addition, kinetochore-fiber disassembly in many cells occurs partly through ‘flux’, where the microtubules flow continuously toward the poles and tubulin subunits are lost from minus ends. Molecular mechanistic models for how load-bearing attachments are maintained to disassembling microtubule ends, and how the forces are generated to drive pac-man and flux-based movements, are discussed.

Stability of nucleoside triphosphate levels in the red cells of the snake

1997 ◽  
Vol 200 (7) ◽  
pp. 1125-1131
Author(s):  
R Ingermann ◽  
D Bencic ◽  
J Herman
Keyword(s):  
Atpase Activity ◽  
Red Cells ◽  
Metabolic Inhibitors ◽  
Cell Swelling ◽  
Nucleoside Triphosphate ◽  
High Ph ◽  
High Concentrations ◽  
The Stability

Nucleated red cells in the nonpregnant garter snake (Thamnophis elegans) contain relatively high concentrations of nucleoside triphosphate (NTP), largely in the form of ATP, which is found at concentrations of approximately 10 mmol l-1 relative to cell volume and 15 mmol l-1 relative to cell water. During pregnancy, levels of NTP in adult red cells rise by approximately 50 % concomitant with an increase in blood progesterone level. The stability of the NTP pool within these red cells was assessed by maintaining cells in vitro at 20 °C, without exogenous nutrients, and in the presence and absence of the metabolic inhibitors iodoacetate and/or cyanide. After 96 h, NTP levels in adult red cells not exposed to the inhibitors had decreased by only approximately 10 %, while in the presence of both inhibitors NTP levels had fallen by less than 50 %. Red cell NTP levels were not affected by acute exposure to high concentrations of progesterone either in vivo or in vitro. NTP levels were much more labile when the cells were maintained in vitro at either low or high pH. Maintenance of red cells at pH 6.0 for 24 h resulted in a decrease in NTP levels of approximately 50 % and at pH 10.0 the levels fell by approximately 80 %, while buffers containing only ATP caused no detectable degradation. Incubation at low or high pH promoted some cell swelling; however, the magnitude of the decreases in intracellular NTP concentration prompted by these pH levels could not be mimicked by incubation of red cells in hypotonic buffer. Total nonspecific ATPase activity at pH 6.0 was approximately 55 % greater than that at pH 7.4, while at pH 10.0 it was approximately 6 % of that at pH 7.4. The pH-dependent decrease in intracellular NTP levels cannot, therefore, be due to stimulation of ATPase activity, at least not at high pH. Overall, the data are consistent with the hypothesis that an appreciable portion of the NTP within these cells is compartmentalized in a stable, but pH-sensitive, pool sequestered from intracellular ATP-hydrolyzing 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 Dissection of Swa2p/Auxilin Domain Requirements for Cochaperoning Hsp70 Clathrin-uncoating Activity In Vivo

2006 ◽  
Vol 17 (7) ◽  
pp. 3281-3290 ◽  
Author(s):  
Jing Xiao ◽  
Leslie S. Kim ◽  
Todd R. Graham
Keyword(s):  
Atpase Activity ◽  
Point Mutation ◽  
Mutational Analysis ◽  
Weak Interactions ◽  
Tetratricopeptide Repeat ◽  
Uba Domain ◽  
J Domain ◽  
Tpr Domain

The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p is a modular protein with three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal J-domain. In vitro, clathrin binding is mediated by multiple weak interactions, but a Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly.

Metabolic importance of Na+/K+-ATPase activity during sea urchin development.

1997 ◽  
Vol 200 (22) ◽  
pp. 2881-2892 ◽  
Author(s):  
P Leong ◽  
D Manahan
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Sea Urchin ◽  
Sodium Pump ◽  
Sea Water ◽  
Strongylocentrotus Purpuratus ◽  
Lytechinus Pictus ◽  
Stimulation Of

Early stages of animal development have high mass-specific rates of metabolism. The biochemical processes that establish metabolic rate and how these processes change during development are not understood. In this study, changes in Na+/K+-ATPase activity (the sodium pump) and rate of oxygen consumption were measured during embryonic and early larval development for two species of sea urchin, Strongylocentrotus purpuratus and Lytechinus pictus. Total (in vitro) Na+/K+-ATPase activity increased during development and could potentially account for up to 77 % of larval oxygen consumption in Strongylocentrotus purpuratus (pluteus stage) and 80 % in Lytechinus pictus (prism stage). The critical issue was addressed of what percentage of total enzyme activity is physiologically active in living embryos and larvae and thus what percentage of metabolism is established by the activity of the sodium pump during development. Early developmental stages of sea urchins are ideal for understanding the in vivo metabolic importance of Na+/K+-ATPase because of their small size and high permeability to radioactive tracers (86Rb+) added to sea water. A comparison of total and in vivo Na+/K+-ATPase activities revealed that approximately half of the total activity was utilized in vivo. The remainder represented a functionally active reserve that was subject to regulation, as verified by stimulation of in vivo Na+/K+-ATPase activity in the presence of the ionophore monensin. In the presence of monensin, in vivo Na+/K+-ATPase activities in embryos of S. purpuratus increased to 94 % of the maximum enzyme activity measured in vitro. Stimulation of in vivo Na+/K+-ATPase activity was also observed in the presence of dissolved alanine, presumably due to the requirement to remove the additional intracellular Na+ that was cotransported with alanine from sea water. The metabolic cost of maintaining the ionic balance was found to be high, with this process alone accounting for 40 % of the metabolic rate of sea urchin larvae (based on the measured fraction of total Na+/K+-ATPase that is physiologically active in larvae of S. purpuratus). Ontogenetic changes in pump activity and environmentally induced regulation of reserve Na+/K+-ATPase activity are important factors that determine a major proportion of the metabolic costs of sea urchin development.

Parthenogenesis and cytoskeletal organization in ageing mouse eggs

Development ◽  
1986 ◽  
Vol 95 (1) ◽  
pp. 131-145
Author(s):  
Michelle Webb ◽  
Sarah K. Howlett ◽  
Bernard Maro
Keyword(s):  
Critical Concentration ◽  
Metaphase Plate ◽  
Meiotic Spindle ◽  
Cytoskeletal Organization ◽  
Spindle Poles ◽  
Different Types ◽  
Mouse Egg ◽  
Parthenogenetic Embryos

The cytoskeletal organization of the mouse egg changes during ageing in vivo and in vitro. The earliest change observed is the disappearance of the microfilament-rich area overlying the meiotic spindle. This is followed by the migration of the spindle towards the centre of the egg. Finally the spindle breaks down and the chromosomes are no longer organized on a metaphase plate. This spindle disruption may result from changes in the microtubule nucleating material found at the spindle poles and from an increase in the critical concentration for tubulin polymerization. It is possible to correlate the changes in the cytoskeletal organization of the egg occurring during ageing with the different types of parthenogenetic embryos obtained after ethanol activation. These observations strengthen the hypothesis that the actin-rich cortical area that overlies the meiotic spindle forms a domain to which the meiotic cleavage furrow is restricted and provides some insights into the mechanisms by which different types of parthenogenetic embryos are generated.

scholarly journals A permeabilized cell model for studying cell division: a comparison of anaphase chromosome movement and cleavage furrow constriction in lysed PtK1 cells.

1981 ◽  
Vol 88 (3) ◽  
pp. 618-629 ◽  
Author(s):  
W Z Cande ◽  
K McDonald ◽  
R L Meeusen
Keyword(s):  
Cell Model ◽  
Chromosome Movement ◽  
Microtubule Depolymerization ◽  
Mitotic Apparatus ◽  
Permeabilized Cell ◽  
Culture Cells ◽  
Brij 58 ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Chromosome Movements

After lysis in a Brij 58-polyethylene glycol medium, PtK1 cells are permeable to small molecules, such as erythrosin B, and to proteins, such as rhodamine-labeled FAB, myosin subfragment-1, and tubulin. Holes are present in the plasma membrane, and the mitochondria are swollen and distorted, but other membrane-bounded organelles of the lysed cell model are not noticeably altered. After lysis, the mitotic apparatus is functional; chromosomes move poleward and the spindle elongates. Cells lysed while in cytokinesis will continue to divide for several minutes. Addition of crude tubulin extracts, MAP-free tubulin, or taxol to the lysis medium retards anaphase chromosome movements but does not affect cleavage. On the other hand, N-ethylmaleimide-modified myosin subfragment-1, phalloidin, and cytochalasin B inhibit cleavage but have no effect on anaphase chromosome movements under identical lysis conditions. These results suggest that actomyosin plays no functional role in anaphase chromosome movement in mammalian tissue culture cells and that microtubule depolymerization is a rate-limiting step for chromosome-to-pole movements.

Na-K-ATPase activity along the rabbit, rat, and mouse nephron

1979 ◽  
Vol 237 (2) ◽  
pp. F114-F120 ◽  
Author(s):  
A. I. Katz ◽  
A. Doucet ◽  
F. Morel
Keyword(s):  
Atpase Activity ◽  
Thick Ascending Limb ◽  
Sensitivity Limit ◽  
Henle's Loop ◽  
Collecting Tubule ◽  
Pars Recta ◽  
Hydrolysis Of ◽  
Total Enzyme

Na-K-ATPase activity along the rabbit, rat, and mouse nephron was determined with a micromethod that measures directly labeled phosphate released by the hydrolysis of [gamma-32P]ATP. Na-K-ATPase activity was highest in the rat, intermediate in the mouse, and lowest in the rabbit nephron. With the exception of rabbit cortical thick ascending limb, the enzyme profile was similar in the three species: Na-K-ATPase activity per millimeter tubule length was highest in the distal convoluted tubule and thick ascending limb of Henle's loop, intermediate in the proximal convoluted tubule, and lowest in the pars recta and collecting tubule. The enzyme was present in the thin limbs of Henle's loop, but its activity was very low and measurements were close to the sensitivity limit of the method. Both the absolute activity and the fraction of the total enzyme represented by Na-K-ATPase were severalfold higher than in kidney homogenates. Finally, the Na-K-ATPase activity measured in certain segments of the rat and rabbit nephron in this study seems sufficient to account in theory for the active component of the net sodium transport found in the corresponding region of the nephron with either in vivo or in vitro single tubule microperfusion techniques.

scholarly journals Ase1/Prc1-dependent spindle elongation corrects merotely during anaphase in fission yeast

2009 ◽  
Vol 187 (3) ◽  
pp. 399-412 ◽  
Author(s):  
Thibault Courtheoux ◽  
Guillaume Gay ◽  
Yannick Gachet ◽  
Sylvie Tournier
Keyword(s):  
Fission Yeast ◽  
Mechanical Model ◽  
Elongation Rate ◽  
Force Balance ◽  
Balance Model ◽  
Spindle Poles ◽  
Sister Chromatids ◽  
Spindle Elongation ◽  
Dependent Mechanism

Faithful segregation of sister chromatids requires the attachment of each kinetochore (Kt) to microtubules (MTs) that extend from opposite spindle poles. Merotelic Kt orientation is a Kt–MT misattachment in which a single Kt binds MTs from both spindle poles rather than just one. Genetic induction of merotelic Kt attachment during anaphase in fission yeast resulted in intra-Kt stretching followed by either correction or Kt disruption. Laser ablation of spindle MTs revealed that intra-Kt stretching and merotelic correction were dependent on MT forces. The presence of multiple merotelic chromosomes linearly antagonized the spindle elongation rate, and this phenomenon could be solved numerically using a simple force balance model. Based on the predictions of our mechanical model, we provide in vivo evidence that correction of merotelic attachment in anaphase is tension dependent and requires an Ase1/Prc1-dependent mechanism that prevents spindle collapse and thus asymmetric division and/or the appearance of the cut phenotype.

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