Spatiotemporal control of functional specification and distribution of spindle microtubules with 13, 14 and 15 protofilaments during mitosis in the ciliate Nyctotherus

1985 ◽  
Vol 76 (1) ◽  
pp. 337-355
Author(s):  
U. Eichenlaub-Ritter
Keyword(s):  
Differential Sensitivity ◽  
Microtubule Depolymerization ◽  
Functional Specification ◽  
Microtubule Stability ◽  
Early Anaphase ◽  
Late Telophase ◽  
Spindle Microtubules ◽  
Elongation Phase ◽  
Spatiotemporal Control ◽  
Macronuclear Division

The formation of microtubules with more than 13 protofilaments in the ciliate Nyctotherus ovalis Leidy seems to be a highly ordered process. Such microtubules are restricted to the nucleoplasm and, moreover, to certain stages of nuclear division. They assemble during anaphase of micronuclear mitosis and during the elongation phase of macronuclear division. The number of microtubules with more than 13 protofilaments in the micronuclear nucleoplasm increases as anaphase progresses. Furthermore, assembly of microtubules with 14 and 15 protofilaments seems to proceed concomitantly with net disassembly of 13-protofilament microtubules, because the total amount of polymerized tubulin in the interpolar spindle region remains approximately constant between mid anaphase and late telophase. In addition, evidence for spatial control of the distribution of microtubules with different protofilament numbers in the micronuclear stembody has been found. The percentage of microtubules with 13 protofilaments per stembody cross-section is highest at the ends of the stembody, while the percentage of microtubules with either 14 or 15 protofilaments increases as the middle of the stembody is approached. Temporal control of polymerization of microtubules with high protofilament numbers seems to be exerted independently in the two types of nuclei. For example, when the macronucleus starts to elongate it contains microtubules with more than 13 protofilaments but the metaphase micronucleus still possesses only microtubules with 13 protofilaments at this stage. Control of fidelity of protofilament numbers is not lost in the early stages of micronuclear or macronuclear division when cells are exposed to 2H2O or media containing taxol. Even microtubules that reassemble during recovery of metaphase micronuclei from nocodazole-induced microtubule depolymerization, in either the absence or presence of 2H2O and taxol, possess 13 protofilaments. Similarly, if the introduction of microtubules with 14 and 15 protofilaments is inhibited during early micronuclear anaphase and delayed for 60 min by exposure to nocodazole, such microtubules still assemble during telophase when recovery is permitted. Microtubules that have been assembled under normal conditions show differential sensitivity to nocodazole. During metaphase, nocodazole induces disassembly of most microtubules. There is an increase in microtubule stability that coincides with the appearance of microtubules with high protofilament numbers during early anaphase. However, considerable numbers of 13-protofilament microtubules, as well as microtubules with 14 and 15 protofilaments, exhibit such stability during anaphase.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Microtubules regulate pancreatic β cell heterogeneity via spatiotemporal control of insulin secretion hot spots

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kathryn P Trogden ◽  
Justin S Lee ◽  
Kai M Bracey ◽  
Kung-Hsien Ho ◽  
Hudson McKinney ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Hot Spots ◽  
Cell Heterogeneity ◽  
Microtubule Depolymerization ◽  
Β Cell ◽  
Microtubule Stability ◽  
Mouse Islets ◽  
Regulation Of Secretion ◽  
Glucose Stimulated Insulin Secretion ◽  
Spatiotemporal Control

Heterogeneity of glucose-stimulated insulin secretion (GSIS) in pancreatic islets is physiologically important but poorly understood. Here, we utilize mouse islets to determine how microtubules affect secretion toward the vascular extracellular matrix at single cell and subcellular levels. Our data indicate that microtubule stability in the β-cell population is heterogenous, and that GSIS is suppressed in cells with highly stable microtubules. Consistently, microtubule hyper-stabilization prevents, and microtubule depolymerization promotes capacity of single β-cell for GSIS. Analysis of spatiotemporal patterns of secretion events shows that microtubule depolymerization activates otherwise dormant β-cells via initiation of secretion clusters (hot spots). Microtubule depolymerization also enhances secretion from individual cells, introducing both additional clusters and scattered events. Interestingly, without microtubules, the timing of clustered secretion is dysregulated, extending the first phase of GSIS and causing oversecretion. In contrast, glucose-induced Ca2+ influx was not affected by microtubule depolymerization yet required for secretion under these conditions, indicating that microtubule-dependent regulation of secretion hot spots acts in parallel with Ca2+ signaling. Our findings uncover a novel microtubule function in tuning insulin secretion hot spots, which leads to accurately measured and timed response to glucose stimuli and promotes functional β-cell heterogeneity.

scholarly journals Partially Processed pre-rRNA Is Preserved in Association with Processing Components in Nucleolus-derived Foci during Mitosis

1998 ◽  
Vol 9 (9) ◽  
pp. 2407-2422 ◽  
Author(s):  
Miroslav Dundr ◽  
Mark O.J. Olson
Keyword(s):  
Molecular Weight ◽  
Internal Transcribed Spacer ◽  
Rnase P ◽  
Rrna Processing ◽  
Rnase Mrp ◽  
External Transcribed Spacer ◽  
Early Anaphase ◽  
Late Telophase ◽  
Processing Site

Previous studies showed that components implicated in pre-rRNA processing, including U3 small nucleolar (sno)RNA, fibrillarin, nucleolin, and proteins B23 and p52, accumulate in perichromosomal regions and in numerous mitotic cytoplasmic particles, termed nucleolus-derived foci (NDF) between early anaphase and late telophase. The latter structures were analyzed for the presence of pre-rRNA by fluorescence in situ hybridization using probes for segments of pre-rRNA with known half-lives. The NDF did not contain the short-lived 5′-external transcribed spacer (ETS) leader segment upstream from the primary processing site in 47S pre-rRNA. However, the NDF contained sequences from the 5′-ETS core, 18S, internal transcribed spacer 1 (ITS1), and 28S segments and also had detectable, but significantly reduced, levels of the 3′-ETS sequence. Northern analyses showed that in mitotic cells, the latter sequences were present predominantly in 45S-46S pre-rRNAs, indicating that high-molecular weight processing intermediates are preserved during mitosis. Two additional essential processing components were also found in the NDF: U8 snoRNA and hPop1 (a protein component of RNase MRP and RNase P). Thus, the NDF appear to be large complexes containing partially processed pre-rRNA associated with processing components in which processing has been significantly suppressed. The NDF may facilitate coordinated assembly of postmitotic nucleoli.

scholarly journals Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression

2019 ◽  
Vol 12 (594) ◽  
pp. eaaw2939 ◽  
Author(s):  
Rozita Adib ◽  
Jessica M. Montgomery ◽  
Joseph Atherton ◽  
Laura O’Regan ◽  
Mark W. Richards ◽  
...  
Keyword(s):  
Double Mutant ◽  
Constitutive Activation ◽  
Mitotic Kinases ◽  
Terminal Domain ◽  
Microtubule Stability ◽  
Cryo Electron Microscopy ◽  
Chromosome Congression ◽  
Spindle Microtubules ◽  
Mitotic Phosphorylation

EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo–electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser144 and Ser146 in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. In addition, constitutive activation of NEK6 or NEK7 reduced the association of EML4 with interphase microtubules. Together, these data support a model in which NEK6- and NEK7-dependent phosphorylation promotes the dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression.

scholarly journals Microtubule aging probed by microfluidics-assisted tubulin washout

2016 ◽  
Vol 27 (22) ◽  
pp. 3563-3573 ◽  
Author(s):  
Christian Duellberg ◽  
Nicholas Ian Cade ◽  
Thomas Surrey
Keyword(s):  
Molecular Mechanism ◽  
Growth Phase ◽  
Morphological Changes ◽  
Threshold Model ◽  
Theoretical Models ◽  
Age Dependence ◽  
Microtubule Depolymerization ◽  
Microtubule Stability ◽  
Age Dependent

Microtubules switch stochastically between phases of growth and shrinkage. The molecular mechanism responsible for the end of a growth phase, an event called catastrophe, is still not understood. The probability for a catastrophe to occur increases with microtubule age, putting constraints on the possible molecular mechanism of catastrophe induction. Here we used microfluidics-assisted fast tubulin washout experiments to induce microtubule depolymerization in a controlled manner at different times after the start of growth. We found that aging can also be observed in this assay, providing valuable new constraints against which theoretical models of catastrophe induction can be tested. We found that the data can be quantitatively well explained by a simple kinetic threshold model that assumes an age-dependent broadening of the protective cap at the microtubule end as a result of an evolving tapered end structure; this leads to a decrease of the cap density and its stability. This analysis suggests an intuitive picture of the role of morphological changes of the protective cap for the age dependence of microtubule stability.

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.

scholarly journals Ultrastructural immunocytochemical localization of calmodulin in cultured cells.

1983 ◽  
Vol 31 (4) ◽  
pp. 445-461 ◽  
Author(s):  
M C Willingham ◽  
J Wehland ◽  
C B Klee ◽  
N D Richert ◽  
A V Rutherford ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Performic Acid ◽  
Microtubule Organizing Center ◽  
Interphase Cells ◽  
Organizing Center ◽  
Late Telophase ◽  
Cytoplasmic Microtubules ◽  
Spindle Microtubules ◽  
And Function ◽  
Fibroblastic Cells

Using an antibody prepared against performic acid-treated calmodulin, we have localized calmodulin in cultured fibroblastic cells by immunofluorescence and immunoelectron microscopy. In interphase cells, calmodulin was found to be diffusely distributed throughout the cytosol. An increased amount of calmodulin was found in the pericentriolar region of interphase cells. No significant aggregation of calmodulin was found in association with microfilaments, peripheral cytoplasmic microtubules or clathrin-coated structures. Calmodulin was present in moderate amounts in microvilli, ruffles, and zeiotic blebs of the cell surface. In motitic cells, calmodulin was found concentrated in the pericentriolar region, and appeared to concentrate along radiating spindle microtubules proximal to the centrioles. Redistribution of calmodulin was seen between early and late telophase, in which the pericentriolar pattern of calmodulin in early telophase shifted to an aggregation on the intercellular bridge, with a large part of the midbody portion of the bridge being devoid of calmodulin. These results show that calmodulin is distributed throughout the cytosol, but is markedly concentrated in the region of the microtubule organizing center in interphase cells, as well as in elements of the mitotic spindle apparatus. This distribution suggests that calmodulin has a regulatory role in the organization and function of microtubules during interphase, as well as during mitosis.

Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis

1992 ◽  
Vol 102 (3) ◽  
pp. 401-416 ◽  
Author(s):  
M.A. Jordan ◽  
D. Thrower ◽  
L. Wilson
Keyword(s):  
Hela Cells ◽  
Mitotic Arrest ◽  
Mitotic Spindles ◽  
Microtubule Depolymerization ◽  
Metaphase Chromosomes ◽  
Low Concentrations ◽  
Subtle Effect ◽  
Full Complement ◽  
Spindle Microtubules

Inhibition of mitosis by many drugs that bind to tubulin has been attributed to depolymerization of microtubules. However, we found previously that low concentrations of vinblastine and vincristine blocked mitosis in HeLa cells with little or no depolymerization of spindle microtubules, and spindles appeared morphologically normal or nearly normal. In the present study, we characterized the effects of vinblastine, podophyllotoxin and nocodazole over broad concentration ranges on mitotic spindle organization in HeLa cells. These three drugs are known to affect the dynamics of microtubule polymerization in vitro and to depolymerize microtubules in cells. We wanted to probe further whether mitotic inhibition by these drugs is brought about by a more subtle effect on the microtubules than net microtubule depolymerization. We compared the effects of vinblastine, podophyllotoxin and nocodazole on the organization of spindle microtubules, chromosomes and centrosomes, and on the total mass of microtubules. Spindle organization was examined by immunofluorescence microscopy, and microtubule polymer mass was assayed on isolated cytoskeletons by a quantitative enzyme-linked immunoadsorbence assay for tubulin. As the drug concentration was increased, the organization of mitotic spindles changed in the same way with all three drugs. The changes were associated with mitotic arrest, but were not necessarily accompanied by net microtubule depolymerization. With podophyllotoxin, mitotic arrest was accompanied by microtubule depolymerization. In contrast, with vinblastine and nocodazole, mitotic arrest occurred in the presence of a full complement of spindle microtubules. All three drugs induced a nearly identical rearrangement of spindle microtubules, an increasingly aberrant organization of metaphase chromosomes, and fragmentation of centrosomes. The data suggest that these anti-mitotic drugs block mitosis primarily by inhibiting the dynamics of spindle microtubules rather than by simply depolymerizing the microtubules.

scholarly journals THE DISTRIBUTION OF SPINDLE MICROTUBULES DURING MITOSIS IN CULTURED HUMAN CELLS

1971 ◽  
Vol 49 (2) ◽  
pp. 468-497 ◽  
Author(s):  
J. Richard McIntosh ◽  
Story C. Landis
Keyword(s):  
Electron Microscopy ◽  
Hela Cells ◽  
Metaphase Plate ◽  
Human Cells ◽  
Late Anaphase ◽  
Monolayer Cultures ◽  
Spindle Axis ◽  
Early Anaphase ◽  
Cultured Human Cells ◽  
Spindle Microtubules

WI-38 and HeLa cells in mitosis have been selected from fixed monolayer cultures and serially sectioned for electron microscopy. Sections perpendicular to the spindle axis permit counting of the number of microtubules at each position on the spindle axis and hence the preparation of tubule distribution profiles. Errors intrinsic to this method are discussed. The changes in the tubule distributions from one mitotic stage to another provide evidence concerning the behavior of the spindle tubules during mitosis. The ratio of the number of tubules passing the chromosomes on the metaphase plate to the maximum number in each half spindle is about 1/2. This ratio changes little in early anaphase, and then decreases in late anaphase at about the same time that a zone of increased tubule number develops at the middle of the interzone. The region where the stem bodies form contains about 3/2 the number of tubules seen elsewhere in the interzone. This ratio is almost constant as the mid-body forms in telophase and then increases to 2/1 in early interphase before the final stages of cytokinesis occur.

scholarly journals Kinesin-8 from Fission Yeast: A Heterodimeric, Plus-End–directed Motor that Can Couple Microtubule Depolymerization to Cargo Movement

2009 ◽  
Vol 20 (3) ◽  
pp. 963-972 ◽  
Author(s):  
Paula M. Grissom ◽  
Thomas Fiedler ◽  
Ekaterina L. Grishchuk ◽  
Daniela Nicastro ◽  
Robert R. West ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Metaphase Plate ◽  
Sf9 Cells ◽  
Microtubule Depolymerization ◽  
Chromosome Congression ◽  
Motor Activities ◽  
Spindle Microtubules ◽  
Anaphase B

Fission yeast expresses two kinesin-8s, previously identified and characterized as products of the klp5+ and klp6+ genes. These polypeptides colocalize throughout the vegetative cell cycle as they bind cytoplasmic microtubules during interphase, spindle microtubules, and/or kinetochores during early mitosis, and the interpolar spindle as it elongates in anaphase B. Here, we describe in vitro properties of these motor proteins and some truncated versions expressed in either bacteria or Sf9 cells. The motor-plus-neck domain of Klp6p formed soluble dimers that cross-linked microtubules and showed both microtubule-activated ATPase and plus-end–directed motor activities. Full-length Klp5p and Klp6p, coexpressed in Sf9 cells, formed soluble heterodimers with the same activities. The latter recombinant protein could also couple microbeads to the ends of shortening microtubules and use energy from tubulin depolymerization to pull a load in the minus end direction. These results, together with the spindle localizations of these proteins in vivo and their requirement for cell viability in the absence of the Dam1/DASH kinetochore complex, support the hypothesis that fission yeast kinesin-8 contributes both to chromosome congression to the metaphase plate and to the coupling of spindle microtubules to kinetochores during anaphase A.

scholarly journals Opto-Katanin: An Optogenetic Tool for Localized Microtubule Disassembly

2021 ◽  
Author(s):  
Joyce C.M. Meiring ◽  
Ilya Grigoriev ◽  
Wilco Nijenhuis ◽  
Lukas C. Kapitein ◽  
Anna Akhmanova
Keyword(s):  
Intracellular Transport ◽  
Experimental Manipulation ◽  
Microtubule Depolymerization ◽  
Microtubule Associated Proteins ◽  
Microtubule Severing ◽  
Associated Proteins ◽  
Dividing Cells ◽  
Chromosome Separation ◽  
Organelle Morphology ◽  
Spatiotemporal Control

Microtubules are major cytoskeletal filaments that drive chromosome separation during cell division, serve as rails for intracellular transport and as a scaffold for organelle positioning. Experimental manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubule integrity are currently lacking. Here, we exploit the dependence of the mammalian microtubule-severing protein katanin on microtubule-targeting co-factors to generate a light-activated system for localized microtubule disassembly that we named opto-katanin. Targeted illumination with blue light induces rapid and localized opto-katanin recruitment and local microtubule depolymerization, which is quickly reversible after stopping light-induced activation. Opto-katanin can be employed to locally perturb microtubule-based transport and organelle morphology in dividing cells and differentiated neurons with high spatiotemporal precision. We show that different microtubule-associated proteins can be used to recruit opto-katanin to microtubules and induce severing, paving the way for spatiotemporally precise manipulation of specific microtubule subpopulations.

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