scholarly journals Spatial Variation of Microtubule Depolymerization in Large Asters Suggests Regulation by MAP Depletion

2020 ◽  
Author(s):  
Keisuke Ishihara ◽  
Franziska Decker ◽  
Paulo Caldas ◽  
James F. Pelletier ◽  
Martin Loose ◽  
...  
Keyword(s):  
Steady State ◽  
Spatial Organization ◽  
Microtubule Assembly ◽  
Component Model ◽  
Microtubule Depolymerization ◽  
Physiological Regulation ◽  
Spatial Regulation ◽  
Important Target ◽  
Egg Extract ◽  
Difference Imaging

AbstractMicrotubule plus end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were almost two-fold higher in the aster interior compared to the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and MAPs in the interior cytosol compared to that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density.

scholarly journals Spatial Variation of Microtubule Depolymerization in Large Asters

2021 ◽  
pp. mbc.E20-11-0723
Author(s):  
Keisuke Ishihara ◽  
Franziska Decker ◽  
Paulo Caldas ◽  
James F. Pelletier ◽  
Martin Loose ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Microtubule Assembly ◽  
Microtubule Depolymerization ◽  
Physiological Regulation ◽  
Xenopus Egg Extract ◽  
Spatial Regulation ◽  
Important Target ◽  
Egg Extract ◽  
Xenopus Egg ◽  
Difference Imaging

Microtubule plus end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in  Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were higher in the aster interior compared to the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and MAPs in the interior cytosol compared to that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Reassembly of microtubules in Nitella tasmanica: assembly of cortical microtubules in branching clusters and its relevance to steady-state microtubule assembly

1989 ◽  
Vol 93 (4) ◽  
pp. 705-714
Author(s):  
GEOFFREY O. WASTENEYS ◽  
RICHARD E. WILLIAMSON
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Simple Model ◽  
Cortical Microtubule ◽  
Microtubule Assembly ◽  
Cortical Microtubules ◽  
Microtubule Depolymerization ◽  
Cortical Actin ◽  
Temperature Dependent ◽  
Standard Temperature

Giant internodal cells of Nitella tasmanica have cortical microtubules beneath the plasma membrane and endoplasmic microtubules associated with sub-cortical actin bundles and nuclei. We depolymerized the microtubules with oryzalin and followed their reassembly by immunofluorescence. At 18°C (the standard temperature of culture), microtubules were lost from young cells within 10 min and the first microtubules were detected in the cortex within 20 min of washing out the herbicide. Microtubules of older cells disassembled and re-formed more slowly. Continued cortical microtubule assembly was at acute angles to the first-formed microtubules, building branching clusters of microtubules. At 25°C, cortical microtubule assembly generated less extensively branched clusters and was completed more rapidly. Larger clusters but shorter MTs were generated in older cells. Reassembly of microtubules in the endoplasm only began 50 min after the removal of oryzalin. We therefore conclude that assembly proceeded independently in the cortex and endoplasm. Cortical assembly involves scattered assembly events initiating microtubules from which, as the latter elongate, further microtubules assemble as branches. We suggest that similar processes operate in steady-state cells and we explain with a simple model why branched clusters of microtubules are unusually large after microtubule depolymerization. By proposing that these processes show differential changes in activity with temperature and during cell ageing, we can account in qualitative terms for the age- and temperature-dependent differences in microtubule reassembly patterns.

Regulation of cell shape in Euglena gracilis. III. Involvement of stable microtubules

1985 ◽  
Vol 74 (1) ◽  
pp. 219-237
Author(s):  
C.L. Lachney ◽  
T.A. Lonergan
Keyword(s):  
Euglena Gracilis ◽  
Cell Shape ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Microtubule Depolymerization ◽  
Cytoplasmic Microtubule ◽  
Intact Cells ◽  
Calmodulin Antagonist ◽  
Cell Shapes ◽  
In Cells

The role of cytoplasmic microtubules in a recently reported biological clock-controlled rhythm in cell shape of the alga Euglena gracilis (strain Z) was examined using indirect immunofluorescence microscopy. The resulting fluorescent patterns indicated that, unlike many other cell systems, Euglena cells apparently change from round to long to round cell shape without associated cytoplasmic microtubule assembly and disassembly. Instead, the different cell shapes were correlated with microtubule patterns, which suggested that movement of stable microtubules to accomplish cell shape changes. In live intact cells, these microtubules were demonstrated by immunofluorescence to be stable to lowered temperature and elevated intracellular Ca2+ levels, treatments that are commonly used to depolymerize microtubules. In cells extracted in detergent at low temperature or in the presence of elevated Ca2+ levels, the fluorescent image of the microtubules was disrupted. Transmission electron microscopy confirmed the loss of one subset of pellicle microtubules. The difference in microtubule stability to these agents between live intact cells and cells extracted in detergent suggested the presence of a microtubule-stabilizing factor in live cells, which is released from the cell by extraction with detergent, thereby permitting microtubule depolymerization by Ca2+ or lowered temperature. The calmodulin antagonist trifluoperazine prevented the Ca2+-induced disruption of the fluorescent microtubule pattern in cells extracted in detergent. These results implied the involvement of calmodulin in the sensitivity to Ca2+ of the microtubules of cells extracted in detergent.

scholarly journals G2 phase chromatin lacks determinants of replication timing

2010 ◽  
Vol 189 (6) ◽  
pp. 967-980 ◽  
Author(s):  
Junjie Lu ◽  
Feng Li ◽  
Christopher S. Murphy ◽  
Michael W. Davidson ◽  
David M. Gilbert
Keyword(s):  
Spatial Organization ◽  
Replication Timing ◽  
S Phase ◽  
Decision Point ◽  
Genome Wide Analysis ◽  
Quiescent Cells ◽  
Egg Extract ◽  
Genome Wide ◽  
G2 Phase

DNA replication in all eukaryotes follows a defined replication timing program, the molecular mechanism of which remains elusive. Using a Xenopus laevis egg extract replication system, we previously demonstrated that replication timing is established during early G1 phase of the cell cycle (timing decision point [TDP]), which is coincident with the repositioning and anchorage of chromatin in the newly formed nucleus. In this study, we use this same system to show that G2 phase chromatin lacks determinants of replication timing but maintains the overall spatial organization of chromatin domains, and we confirm this finding by genome-wide analysis of rereplication in vivo. In contrast, chromatin from quiescent cells retains replication timing but exhibits disrupted spatial organization. These data support a model in which events at the TDP, facilitated by chromatin spatial organization, establish determinants of replication timing that persist independent of spatial organization until the process of chromatin replication during S phase erases those determinants.

Concerning the location of the GTP hydrolysis site on microtubules

1985 ◽  
Vol 63 (6) ◽  
pp. 422-429 ◽  
Author(s):  
Michael Caplow ◽  
John Shanks ◽  
Bruna Pegoraro Brylawski
Keyword(s):  
Steady State ◽  
Reaction Scheme ◽  
Product Inhibition ◽  
Microtubule Assembly ◽  
Assembly Process ◽  
Progressive Decrease ◽  
Reaction Conditions ◽  
Gtp Hydrolysis ◽  
Tubulin Subunit ◽  
A Site

The kinetics for GTP hydrolysis associated with microtubule assembly with microtubular protein has been analyzed under reaction conditions where tubulin–GDP does not readily assemble into microtubules. The GTPase rate is only slightly faster during the time when net microtubule assembly occurs, as compared with steady state. The slightly slower steady-state GTPase rate apparently results from GDP product inhibition, since the progressive decrease in the rate can be quantitatively accounted for using the previously determined GTP dissociation constant and the Ki value for GDP. Since the GTPase rate is not a function of the rate for net microtubule assembly, it is concluded that GTP hydrolysis is not required for tubulin subunit incorporation into microtubules. The constancy of the rate indicates that the GTPase reaction occurs at a site, the concentration of which does not change during the assembly process. This result is consistent with a reaction scheme in which GTP hydrolysis occurs primarily at microtubule ends. We propose that hydrolysis occurs at microtubule ends, at the interface between a long core of tubulin–GDP subunits and a short cap of tubulin–GTP subunits.

scholarly journals Calibration of a simple and a complex model of global marine biogeochemistry

2017 ◽  
Author(s):  
Iris Kriest
Keyword(s):  
Steady State ◽  
Model Calibration ◽  
Organic Phosphorus ◽  
Model Performance ◽  
Parametric Uncertainty ◽  
Complex Model ◽  
Component Model ◽  
Model Parameters ◽  
Model Assessment ◽  
High Level

Abstract. The assessment of the ocean biota's role in climate climate change is often carried out with global biogeochemical ocean models that contain many components, and involve a high level of parametric uncertainty. Examination the models' fit to climatologies of inorganic tracers, after the models have been spun up to steady state, is a common, but computationally expensive procedure to assess model performance and reliability. Using new tools that have become available for global model assessment and calibration in steady state, this paper examines two different model types – a complex seven-component model (MOPS), and a very simple two-component model (RetroMOPS) – for their fit to dissolved quantities. Before comparing the models, a subset of their biogeochemical parameters has been optimised against annual mean nutrients and oxygen. Both model types fit the observations almost equally well. The simple model, which contains only nutrients and dissolved organic phosphorus (DOP), is sensitive to the parameterisation of DOP production and decay. The spatio-temporal decoupling of nitrogen and oxygen, and processes involved in their uptake and release, renders oxygen and nitrate valuable tracers for model calibration. In addition, the non-conservative nature of these tracers (with respect to their upper boundary condition) introduces the global bias as a useful additional constraint on model parameters. Dissolved organic phosphorous at the surface behaves antagonistically to phosphate, and suggests that observations of this tracer – although difficult to measure – may be an important asset for model calibration.

scholarly journals Stability of microtubule attachment to metaphase kinetochores in PtK1 cells

1990 ◽  
Vol 96 (1) ◽  
pp. 9-15 ◽  
Author(s):  
L. Cassimeris ◽  
C.L. Rieder ◽  
G. Rupp ◽  
E.D. Salmon
Keyword(s):  
Attachment Site ◽  
Microtubule Assembly ◽  
Microtubule Depolymerization ◽  
Spindle Poles ◽  
Microtubule Attachment ◽  
Section Electron ◽  
The Mean ◽  
Serial Section Electron Microscopy ◽  
The Stability ◽  
Section Electron Microscopy

Kinetochore microtubules are known to be differentially stable to a variety of microtubule depolymerization agents compared to the non-kinetochore polar microtubules, but the dynamics of microtubule attachment to the kinetochore is currently controversial. We have examined the stability of kinetochore microtubules in metaphase PtK1 spindles at 23 degrees C when microtubule assembly is abruptly blocked with the drug nocodazole. Metaphase cells were incubated in medium containing 34 microM nocodazole for various times before fixation and processing either for immunofluorescence light microscopy or serial-section electron microscopy. Microtubules not associated with kinetochore fibers disappeared completely in less than 1 min. Kinetochore fibers persisted and shortened, as the spindle poles moved close to the chromosomes over a 10–20 min interval. During this shortening process, the number of kinetochore microtubules decreased slowly. The mean number of kinetochore microtubules was 24 +/− 5 in control cells and zero in cells incubated with nocodazole for 20 min. The half-time of microtubule attachment to the kinetochore was approximately 7.5 min. These results show that when microtubule assembly is blocked, kinetochore microtubules shorten more slowly and persist about 10 times longer than the labile polar microtubules. If kinetochore microtubules shorten by tubulin dissociation at their plus-ends like the non-kinetochore polar microtubules, then the microtubule surface lattice must be able to translocate through the kinetochore attachment site without frequent detachment occurring.

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