scholarly journals Kinetochore microtubule dynamics and the metaphase-anaphase transition.

1995 ◽  
Vol 131 (3) ◽  
pp. 721-734 ◽  
Author(s):  
Y Zhai ◽  
P J Kronebusch ◽  
G G Borisy
Keyword(s):  
Dynamic Behavior ◽  
Mammalian Cells ◽  
Chromosome Movement ◽  
Relative Contribution ◽  
Anaphase Onset ◽  
Early Anaphase ◽  
Order Of Magnitude ◽  
Spindle Elongation ◽  
Dramatic Shift ◽  
The Stability

We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation. Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state. A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process. The movement of the activated zone was also measured along with chromosome movement and spindle elongation. To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase. We determined values for the turnover of kMTs (t1/2 = 7.1 +/- 2.4 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs. In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 37.5 +/- 8.5 min at 30 degrees C). Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value. In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16%. These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition. A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore. The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.

Characterization of mitotic motors by their relative sensitivity to AMP-PNP

1989 ◽  
Vol 94 (3) ◽  
pp. 425-441
Author(s):  
G.M. Lee
Keyword(s):  
Fluorescence Intensity ◽  
Molecular Motors ◽  
Lucifer Yellow ◽  
Relative Sensitivity ◽  
Chromosome Movement ◽  
Anaphase Onset ◽  
Mitotic Motors ◽  
Spindle Elongation ◽  
Tubulin Immunofluorescence

The relative sensitivities of the motors for mitotic chromosome movements and saltatory motion were compared using a nonhydrolyzable analog of ATP, AMP-PNP. K+AMP-PNP was microinjected into PtKl cells at the time of nuclear envelope disassembly or at anaphase onset. To produce a dose-response curve for the effect of AMP-PNP on the rate of movement, the intracellular concentration of AMP-PNP in individual cells was measured. The volume injected into each cell was determined by adding dextrans labeled with Lucifer Yellow to the injection buffer, measuring the injected cell's fluorescence intensity, and then comparing the value with the fluorescence intensity of known volumes of Lucifer Yellow dextran solution. AMP-PNP produced a 50% inhibition of spindle elongation at 0.2 mM, of saltatory motion at 0.8 mM, and of chromosome movement at 8.6 mM. Prometaphase chromosome movement and anaphase chromosome-to-pole movement were similarly inhibited by AMP-PNP. Equivalent volumes of injection buffer containing 1% Lucifer Yellow dextran had no effect on chromosome movement, spindle elongation or saltatory motion. Although AMP-PNP occasionally produced shorter anaphase spindles, tubulin immunofluorescence revealed the presence of abundant spindle microtubules. Metaphase cells treated with very high cell concentrations of AMP-PNP had spindles with unusually long astral microtubules; thus microtubules are stabilized rather than broken down by AMP-PNP. In conclusion, spindle elongation is four times more sensitive than saltatory motion to AMP-PNP and 40 times more sensitive than chromosome movement. When these sensitivities to AMP-PNP are considered with the results from other studies, it can be concluded that the molecular motors for spindle elongation, chromosome movement and saltatory motion are different.

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 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 Ase1 domains dynamically slow anaphase spindle elongation and recruit Bim1 to the midzone

2020 ◽  
Author(s):  
Ezekiel C. Thomas ◽  
Amber Ismael ◽  
Jeffrey K. Moore
Keyword(s):  
Elongation Rate ◽  
Modular System ◽  
Binding Domains ◽  
Anaphase Onset ◽  
Carboxy Terminal Domain ◽  
Late Anaphase ◽  
Early Anaphase ◽  
Spindle Elongation ◽  
Carboxy Terminal ◽  
And Control

ABSTRACTHow cells regulate microtubule crosslinking activity to control the rate and duration of spindle elongation during anaphase is poorly understood. In this study, we test the hypothesis that PRC1/Ase1 proteins use distinct microtubule-binding domains to control spindle elongation rate. Using budding-yeast Ase1, we identify unique contributions for the spectrin and carboxy-terminal domains during different phases of spindle elongation. We show that the spectrin domain uses conserved, basic residues to promote the recruitment of Ase1 to the midzone before anaphase onset and slow spindle elongation during early anaphase. In contrast, a partial Ase1 carboxy-terminal truncation fails to form a stable midzone in late anaphase, produces faster elongation rates after early anaphase, and exhibits frequent spindle collapses. We find that the carboxy-terminal domain interacts with the plus-end tracking protein EB1/Bim1 and recruits Bim1 to the midzone to maintain midzone length. Overall, our results suggest that the Ase1 domains provide cells with a modular system to tune midzone activity and control elongation rates.

scholarly journals Functional implications of cold-stable microtubules in kinetochore fibers of insect spermatocytes during anaphase.

1980 ◽  
Vol 85 (3) ◽  
pp. 853-865 ◽  
Author(s):  
E D Salmon ◽  
D A Begg
Keyword(s):  
Dynamic Equilibrium ◽  
Chromosome Movement ◽  
Temperature Dependent ◽  
Abrupt Changes ◽  
Functional Implications ◽  
Early Anaphase ◽  
Physiological Pathways ◽  
Cold Stability ◽  
Rate Limiting ◽  
Steady State Balance

In normal anaphase of crane fly spermatocytes, the autosomes traverse most of the distance to the poles at a constant, temperature-dependent velocity. Concurrently, the birefringent kinetochore fibers shorten while retaining a constant birefringent retardation (BR) and width over most of the fiber length as the autosomes approach the centrosome region. To test the dynamic equilibrium model of chromosome poleward movement, we abruptly cooled or heated primary spermatocytes of the crane fly Nephrotoma ferruginea (and the grasshopper Trimerotropis maritima) during early anaphase. According to this model, abrupt cooling should induce transient depolymerization of the kinetochore fiber microtubules, thus producing a transient acceleration in the poleward movement of the autosomal chromosomes, provided the poles remain separated. Abrupt changes in temperature from 22 degrees C to as low as 4 degrees C or as high as 31 degrees C in fact produced immediate changes in chromosome velocity to new constant velocities. No transient changes in velocity were observed. At 4 degrees C (10 degrees C for grasshopper cells), chromosome movement ceased. Although no nonkinetochore fiber BR remained at these low temperatures, kinetochore fiber BR had changed very little. The cold stability of the kinetochore fiber microtubules, the constant velocity character of chromosome movement, and the observed Arrhenius relationship between temperature and chromosome velocity indicate that a rate-limiting catalyzed process is involved in the normal anaphase depolymerization of the spindle fiber microtubules. On the basis of our birefringence observations, the kinetochore fiber microtubules appear to exist in a steady-state balance between comparatively irreversible, and probably different, physiological pathways of polymerization and depolymerization.

Retrospective Genomics Suggests the Disappearance of a Tiger Shark (Galeocerdo Cuvier) Population Off South-Eastern Australia

2021 ◽  
Author(s):  
Alice Manuzzi ◽  
Belen Jiménez-Mena ◽  
Romina Henriques ◽  
Bonnie J. Holmes ◽  
Julian Pepperell ◽  
...  
Keyword(s):  
Anthropogenic Disturbance ◽  
East Coast ◽  
Tiger Shark ◽  
Galeocerdo Cuvier ◽  
Relative Contribution ◽  
Eastern Australia ◽  
Genomic Analyses ◽  
Dramatic Shift ◽  
Well Differentiated ◽  
Two Populations

Abstract Over the last century, many populations of sharks have been reduced in numbers by overexploitation or attempts to mitigate human-shark interactions. Still, there is a general perception that populations of large ocean predators cover wide areas and therefore their diversity is less susceptible to local anthropogenic disturbance. Here we report retrospective genomic analyses of DNA using archived and contemporary samples of tiger shark (Galeocerdo cuvier) from eastern Australia. Using SNP loci, we documented a significant overall change in genetic composition of tiger sharks born over the last century. The change was most likely due to a shift over time in the relative contribution of two well differentiated, but hitherto cryptic populations. Our data strongly indicate a dramatic shift in relative contribution of the two populations to the overall tiger shark abundance of the east coast of Australia, possibly associated with differences in direct or indirect exploitation rates.

Electrolysis soy protein isolate-based oleogels prepared with an emulsion-templated approach

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Dianyu Yu ◽  
Yan Chen ◽  
Xing Chen ◽  
Yunyan Huang ◽  
Liqi Wang ◽  
...  
Keyword(s):  
Soy Protein ◽  
Xanthan Gum ◽  
Binding Capacity ◽  
Soy Protein Isolate ◽  
Single Layer ◽  
Protein Isolate ◽  
Regulatory Limits ◽  
Order Of Magnitude ◽  
The Stability ◽  
Oil Binding

Abstract This research focuses on the use of protein-polyphenol complex and protein-polyphenol: polysaccharide complexes to prepare oleogels through an emulsion-templated approach. Electrolysis soy protein isolate (ESPI) could be effectively adsorbed on the surface of a single-layer emulsion to increase the particle size. The order of the negative charges of the emulsion after adding polysaccharides was xanthan gum (XG)> pectin> carboxymethyl cellulose (CMC). Rheological behavior showed that the stability of the double-layer emulsions increased, and the viscoelasticity increased around one order of magnitude with the addition of polysaccharides. The oil binding capacity (OBC) of the oleogel prepared by adding polysaccharides increased to more than 97%. The peroxide value (PV) and anisidine value (AV) of XG oleogel were the minimum values in all samples. The AV and POV were within the regulatory limits of China after storage for 21 days. This provides a reference to design of ESPI-based oleogel for different applications.

scholarly journals Probability in transcriptional regulation and its implications for leukocyte differentiation and inducible gene expression

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2323-2328 ◽  
Author(s):  
David A. Hume
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Mammalian Cells ◽  
Cell Lineage ◽  
Monoallelic Expression ◽  
Stem Cell Lineage ◽  
The Stability ◽  
Stochastic Phenomena ◽  
Inducible Genes ◽  
Probabilistic Nature

The phenotype of individual hematopoietic cells, like all other differentiated mammalian cells, is determined by selective transcription of a subset of the genes encoded within the genome. This overview summarizes the recent evidence that transcriptional regulation at the level of individual cells is best described in terms of the regulation of the probability of transcription rather than the rate. In this model, heterogeneous gene expression among populations of cells arises by chance, and the degree of heterogeneity is a function of the stability of the mRNA and protein products of individual genes. The probabilistic nature of transcriptional regulation provides one explanation for stochastic phenomena, such as stem cell lineage commitment, and monoallelic expression of inducible genes, such as lymphokines and cytokines.

Delayed cyclin A and B1 degradation in non-transformed mammalian cells

1995 ◽  
Vol 108 (7) ◽  
pp. 2599-2608 ◽  
Author(s):  
F. Girard ◽  
A. Fernandez ◽  
N. Lamb
Keyword(s):  
Mammalian Cells ◽  
Marine Invertebrates ◽  
Cyclin A ◽  
Cdc2 Kinase ◽  
Marked Contrast ◽  
Anaphase Onset ◽  
Significant Difference ◽  
Primary Fibroblasts ◽  
Transformed Cell Lines ◽  
Derivatives Of

Cyclins A and B are known to exhibit significant differences in their function, cellular distribution and timing of degradation at mitosis. On the basis of observations in marine invertebrates and Xenopus, it was proposed that cyclin destruction triggers cdc2 kinase inactivation and anaphase onset. However, this model has recently been questioned, both in Xenopus and in budding yeast. In this report, we present evidence for delayed degradation of both cyclins A and B1 in non-transformed mammalian cells. Indeed, by means of indirect immunofluorescence and confocal microscopy, we show that cyclins A and B1 are present up to anaphase in REF52, Hs68, human primary fibroblasts and NRK epithelial cells. In marked contrast, cyclin A is shown to be degraded within metaphase and cyclin B just at the transition to anaphase in HeLa and two transformed cell lines, derivatives of normal NRK and REF52. These results further support the notion that cyclin destruction might be not correlated with anaphase onset in normal cells and highlight a significant difference in the fate of mitotic cyclins between transformed and non-transformed cells.

scholarly journals Dual functions of Rack1 in regulating Hedgehog pathway

2020 ◽  
Vol 27 (11) ◽  
pp. 3082-3096 ◽  
Author(s):  
Yan Li ◽  
Xiaohan Sun ◽  
Dongqing Gao ◽  
Yan Ding ◽  
Jinxiao Liu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cellular Localization ◽  
Multiple Roles ◽  
Dual Roles ◽  
Cubitus Interruptus ◽  
Trimeric Complex ◽  
Physiological Processes ◽  
Underlying Mechanisms ◽  
The Stability ◽  
Fine Tune

Abstract Hedgehog (Hh) pathway plays multiple roles in many physiological processes and its dysregulation leads to congenital disorders and cancers. Hh regulates the cellular localization of Smoothened (Smo) and the stability of Cubitus interruptus (Ci) to fine-tune the signal outputs. However, the underlying mechanisms are still unclear. Here, we show that the scaffold protein Rack1 plays dual roles in Hh signaling. In the absence of Hh, Rack1 promotes Ci and Cos2 to form a Ci–Rack1–Cos2 complex, culminating in Slimb-mediated Ci proteolysis. In the presence of Hh, Rack1 dissociates from Ci–Rack1–Cos2 complex and forms a trimeric complex with Smo and Usp8, leading to Smo deubiquitination and cell surface accumulation. Furthermore, we find the regulation of Rack1 on Hh pathway is conserved from Drosophila to mammalian cells. Our findings demonstrate that Rack1 plays dual roles during Hh signal transduction and provide Rack1 as a potential drug target for Hh-related diseases.

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