Actin in spindles of Haemanthus katherinae endosperm. II. Distribution of actin in chromosomal spindle fibres, determined by analysis of serial sections

1979 ◽  
Vol 37 (1) ◽  
pp. 349-371
Author(s):  
A. Forer ◽  
W.T. Jackson ◽  
A. Engberg
Keyword(s):  
Functional Role ◽  
Spindle Pole ◽  
Serial Sections ◽  
Heavy Meromyosin ◽  
A Cell ◽  
Spindle Microtubules ◽  
Spindle Structure ◽  
Spindle Fibres

We have studied the arrangements of actin-containing filaments in 13 bundles of kinetochore microtubules in glycerinated, heavy meromyosin-treated Haemanthus endosperm cells: 7 bundles were in a cell at anaphase, and 6 were in a cell at metaphase. Actin-containing filaments were present in each of the 13 bundles of kinetochore microtubules: they were in amongst the microtubules in the bundle and seemed to be associated with the microtubules. Actin-containing filaments in each bundle seemed to terminate at the kinetochores. Actin-containing filaments associated with the kinetochore microtubules were of consistent polarity (the arrowheads pointed towards the kinetochores) whereas those associated with other microtubles and those not associated with microtubules did not have consistent polarity (some pointed towards the spindle pole, others pointed away from it). Roughly, there were as many individual stretches of actin-containing filaments identified per bundle of kinetochore microtubules as there were microtubules which terminated at the kinetochore. These data suggest that actin-containing filaments in spindles have a functional role. We used 2 glycerination procedures in our studies (one for each cell), and neither seemed to disrupt the basic microtubule arrangements: the arrangements of spindle microtubules seen after glycerination of Haemanthus endosperm were identical to those described previously by others in non-glycerinated glutaraldehyde-fixed Haemanthus endosperm. Thus we argue that spindle structure is not disrupted by the procedures, and therefore that the arrangements of actin-containing filaments are not artifacts of the glycerination procedures. The only difference between microtubules in glycerinated cells and microtubules in untreated cells is that there seem to be fewer in the glycerinated cells. The possible role of actin-containing filaments in the spindle is discussed.

The role of gamma-tubulin in mitotic spindle formation and cell cycle progression in Aspergillus nidulans

1997 ◽  
Vol 110 (5) ◽  
pp. 623-633 ◽  
Author(s):  
M.A. Martin ◽  
S.A. Osmani ◽  
B.R. Oakley
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Cycle Progression ◽  
Spindle Microtubules ◽  
Gamma Tubulin

gamma-Tubulin has been hypothesized to be essential for the nucleation of the assembly of mitotic spindle microtubules, but some recent results suggest that this may not be the case. To clarify the role of gamma-tubulin in microtubule assembly and cell-cycle progression, we have developed a novel variation of the gene disruption/heterokaryon rescue technique of Aspergillus nidulans. We have used temperature-sensitive cell-cycle mutations to synchronize germlings carrying a gamma-tubulin disruption and observe the phenotypes caused by the disruption in the first cell cycle after germination. Our results indicate that gamma-tubulin is absolutely required for the assembly of mitotic spindle microtubules, a finding that supports the hypothesis that gamma-tubulin is involved in spindle microtubule nucleation. In the absence of functional gamma-tubulin, nuclei are blocked with condensed chromosomes for about the length of one cell cycle before chromatin decondenses without nuclear division. Our results indicate that gamma-tubulin is not essential for progression from G1 to G2, for entry into mitosis nor for spindle pole body replication. It is also not required for reactivity of spindle pole bodies with the MPM-2 antibody which recognizes a phosphoepitope important to mitotic spindle formation. Finally, it does not appear to be absolutely required for cytoplasmic microtubule assembly but may play a role in the formation of normal cytoplasmic microtubule arrays.

scholarly journals Analysis of the distribution of spindle microtubules in the diatom Fragilaria.

1978 ◽  
Vol 79 (3) ◽  
pp. 737-763 ◽  
Author(s):  
D H Tippit ◽  
D Schulz ◽  
J D Pickett-Heaps
Keyword(s):  
Spatial Arrangement ◽  
Spindle Pole ◽  
Serial Sections ◽  
Late Prophase ◽  
Central Spindle ◽  
Sources Of Error ◽  
Late Anaphase ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Intercellular Variability

The spindle of the colonial diatom Fragilaria contains two distinct sets of spindle microtubules (MTs): (a) MTs comprising the central spindle, which is composed of two half-spindles interdigitated to form a region of "overlap"; (b) MTs which radiate laterally from the poles. The central spindles from 28 cells are reconstructed by tracking each MT of the central spindle through consecutive serial sections. Because the colonies of Fragilaria are flat ribbons of contiguous cells (clones), it is possible, by using single ribbons of cells, to compare reconstructed spindles at different mitotic stages with minimal intercellular variability. From these reconstructions we have determined: (a) the changes in distribution of MTs along the spindle during mitosis; (b) the change in the total number of MTs during mitosis; (c) the length of each MT (measured by the number of sections each traverses) at different mitotic stages; (d) the frequency of different classes of MTs (i.e., free, continuous, etc.); (e) the spatial arrangement of MTs from opposite poles in the overlap; (f) the approximate number of MTs, separate from the central spindle, which radiate from each spindle pole. From longitudinal sections of the central spindle, the lengths of the whole spindle, half-spindle, and overlap were measured from 80 cells at different mitotic stages. Numerous sources of error may create inaccuracies in these measurements; these problems are discussed. The central spindle at prophase consists predominantly of continuous MTs (pole to pole). Between late prophase and prometaphase, spindle length increases, and the spindle is transformed into two half-spindles (mainly polar MTs) interdigitated to form the overlap. At late anaphase-telophase, the overlap decreases concurrent with spindle elongation. Our interpretation is that the MTs of the central spindle slide past one another at both late prophase and late anaphase. These changes in MT distribution have the effect of elongating the spindle and are not involved in the poleward movement of the chromosomes. Some aspects of tracking spindle MTs, the interaction of MTs in the overlap, formation of the prophase spindle, and our interpretation of rearrangements of MTs, are discussed.

Unorthodox Mitosis in Trichonympha Agilis: Kinetochore Differentiation and Chromosome Movement

1973 ◽  
Vol 13 (2) ◽  
pp. 511-552
Author(s):  
DONNA F. KUBAI
Keyword(s):  
Nuclear Envelope ◽  
Direct Contact ◽  
Direct Interaction ◽  
Stage Iv ◽  
Spindle Pole ◽  
Stage Ii ◽  
Nuclear Surface ◽  
Chromosome Movement ◽  
Serial Sections ◽  
Spindle Microtubules

Changes in rostral structures and the nuclear events which occur in dividing cells of Trichonympha agilis (obtained from experimentally refaunated termites) were studied by means of electron microscopy of serial sections. It is possible to characterize 5 stages of division: Stage I. During this earliest recognizable division stage, the bilaterally symmetrical hemirostra have begun to separate and spindle microtubules appear in the intervening space. As in interphase, the kinetochore regions of chromosomes are distinguishable as fibrillar masses underlying the intact nuclear envelope; and, in individual sections, they are often seen to occur in pairs. These pairs are taken to be sister kinetochores. Stage II. The extranuclear spindle has become established between the posterior ends of well separated hemirostral tubes. Elaboration of daughter rostral structures begins and will continue through the subsequent stages of division. Kinetochores differentiate, becoming bipartite structures consisting of a fibrillar element underlain by a dense disk. The fibrillar kinetochore element is associated with the still-intact nuclear envelope which lies between kinetochores and cytoplasmic spindle microtubules. Reconstruction from serial sections shows all kinetochores to be disposed in pairs which are distributed randomly over the nuclear surface. Stage III. The fibrillar elements of kinetochores are enclosed in evaginations of the nuclear envelope, while the disk elements have come to lie in the plane of the nuclear surface. Kinetochores remain separated from the extranuclear spindle microtubules by the intact nuclear envelope. The distribution of kinetochores has changed relative to that seen in stage II: kinetochores no longer appear to be paired, and they are confined to that hemisphere of the nuclear surface closest to the spindle. Stage IV. The nuclear envelope opens at the sites of kinetochores, leaving the dense disk kinetochore element inserted in pore-like discontinuities of the nuclear envelope and the fibrillar element in the cytoplasm. Direct interaction between fibrillar kinetochore element and extranuclear spindle microtubules is, however, not yet established. Stage V. The cytoplasmically situated fibrillar elements of ‘inserted’ kinetochores are now in direct contact with spindle microtubules. As seen in reconstructions of the nucleus from serial sections, kinetochores have become segregated in 2 groups on the nuclear surface, one near each spindle pole. It is during this stage that final elaboration of rostral structures takes place. On the basis of the observed changes in kinetochore distribution which occur between stages II and III while the intact nuclear envelope prevents any direct interaction between intra-nuclear kinetochores and extranuclear spindle microtubules, it is suggested that kinetochore-membrane interaction is involved in early chromosome movement in Trichonympha agilis. Only during stage V, when direct contact between kinetochores and spindle microtubules is established, may the microtubules assume their usual role in chromosome movement.

Actin in spindles of Haemanthus katherinae endosperm. I. General results using various glycerination methods

1979 ◽  
Vol 37 (1) ◽  
pp. 323-347
Author(s):  
A. Forer ◽  
W.T. Jackson
Keyword(s):  
Phase Contrast ◽  
Close Association ◽  
Rapid Expansion ◽  
Heavy Meromyosin ◽  
Electron Microscopic ◽  
Contrast Microscopy ◽  
One Step ◽  
Triton X ◽  
Spindle Structure ◽  
Spindle Fibres

We have studied actin-containing filaments in spindles in Haemanthus endosperm cells glycerinated by various methods; the actin-containing filaments were identified by their reaction with rabbit skeletal muscle heavy meromyosin (HMM) to form ‘decorated’ filaments. Actin-containing filaments in the spindle were seen in amongst microtubules in bundles (both non-kinetochore microtubule bundles and kinetochore microtuble bundles) and were also seen not associated with microtubules. There were very few extra-spindle actin-containing filaments in these cells. Actin-containing filaments seemed to interact with microtubules, because the filaments remained close to and parallel to microtubules even when the microtubules were sharply curved. Because of the close association between microtubules and actin-containing filaments we could not identify all the actin-containing filaments present in microtubule bundles: microtubules obscured actin-containing filaments. We studied Haemanthus endosperm cells as they were glycerinated. For some of these observations we used phase-contrast microscopy. Glycerination caused the cells to shrink, initially, and this was followed by rapid expansion, but the cells did not expand to as large a volume as before glycerination. Spindle structure was maintained despite these changes in cell size. Evidences for this are that relative chromosome positions were maintained during glycerination, that spindle birefringence was maintained during glycerination, and that individual chromosomal spindle fibres remained birefringent during glycerination. Electron-microscopic observations supported this in that kinetochore microtubule bundles and non-kinetochore microtubule bundle were maintained during glycerination, as was the helical arrangement of spindle ribosomes into polyribosomes. One-step glycerination procedures were used (cells were treated with mixtures containing 25% glycerol, Triton-X-100 and HMM), and such procedures might be of general use. Living cells were embedded in fibrin clots in making light-microscopic observations; this procedure, too, might be of general use.

O-171 Altered meiotic spindle morphology and composition in in vitro matured oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
P Karamtzioti ◽  
G Tiscornia ◽  
D Garcia ◽  
A Rodriguez ◽  
I Vernos ◽  
...  
Keyword(s):  
Metaphase Plate ◽  
Spindle Pole ◽  
Meiotic Spindle ◽  
Developmental Potential ◽  
Software Analysis ◽  
Spindle Microtubules ◽  
Non Parametric

Abstract Study question How does the meiotic spindle tubulin PTMs of MII oocytes matured in vitro compare to that of MII oocytes matured in vivo? Summary answer MII cultured in vitro present detyrosinated tubulin in the spindle microtubules, while MII oocytes matured in vivo do not. What is known already A functional spindle is required for chromosomal segregation during meiosis, but the role of tubulin post-translational modifications (PTMs) in spindle meiotic dynamics remains poorly characterized. In contrast with GVs matured in vitro within the cumulus oophorous, in vitro maturation of denuded GVs to the MII stage (GV-MII) is associated with spindle abnormalities, chromosome misalignment and compromised developmental potential. Although aneuploidy rates in GV-MII are not higher than in vivo matured MII, disorganized chromosomes may contribute to compromised developmental potential. However, to date, spindle PTMs morphology of GV-MII has not been compared to that of in vivo cultured MII oocytes. Study design, size, duration GV (n = 125), and MII oocytes (n = 24) were retrieved from hormonally stimulated women, aged 20 to 35 years old. GVs were matured to the MII stage in vitro in G-2 PLUS medium for 30h; the maturation rate was 68,2%; the 46 GV-MII oocytes obtained were vitrified, stored, and warmed before fixing and subjecting to immunofluorescent analysis. In vivo matured MII oocytes donated to research were used as controls. Participants/materials, setting, methods Women were stimulated using a GnRH antagonist protocol, with GnRH agonist trigger. Trigger criterion was ≥2 follicles ≥18mm; oocytes were harvested 36h later. Spindle microtubules were incubated with antibodies against alpha tubulin and tubulin PTMs (acetylation, tyrosination, polyglutamylation, Δ2-tubulin, and detyrosination); chromosomes were stained with Hoechst 33342 and samples subjected to confocal immunofluorescence microscopy (ZEISS LSM780), with ImageJ software analysis. Differences in spindle morphometric parameters were assessed by non-parametric Kruskal–Wallis and Fisher’s exact tests. Main results and the role of chance Qualitatively, Δ2-tubulin, tyrosination and polyglutamylation were similar for both groups. Acetylation was also present in both groups, albeit in different patterns: while in vivo matured MII oocytes showed acetylation at the poles, GV-MII showed a symmetrical distribution of signal intensity, but discontinuous signal on individual microtubule tracts, suggesting apparent islands of acetylation. In contrast, detyrosination was detected in in vivo matured MII oocytes but was absent from GV-MII. Regarding spindle pole morphology, of the four possible phenotypes described in the literature (double flattened and double focused; flattened-focused, focused-flattened, with the first word characterizing the cortex side of the spindle), we observed double flat shaped spindle poles in 86% of GV-MII oocytes (25/29) as opposed to 40.5% (15/37) for the in vivo matured MII oocytes (p = 0.0004, Fisher’s exact test). Further morphometric analysis of the spindle size (maximum projection, major and minor axis length) and the metaphase plate position (proximal to distal ratio, angle) revealed decreased spindle size in GV-MII oocytes (p = 0.019, non parametric Kruskal- Wallis test). Limitations, reasons for caution Oocytes retrieved from hyperstimulation cycles could be intrinsically impaired since they failed to mature in vivo. Our conclusions should not be extrapolated to IVM in non-stimulated cycles, as in this model, the cumulus oophorus is a major factor in oocyte maturation and correlation with spindle dynamics has been inferred. Wider implications of the findings The metaphase II spindle stability compared to the mitotic or metaphase I meiotic one justifies the presence of PTMs such as acetylation and glutamylation, which are found in stable, long-lived microtubules. The significance of the absence of detyrosinated microtubules in the MII-GV group remains to be determined Trial registration number not applicable

scholarly journals Novel insights into mammalian embryonic neural stem cell division: focus on microtubules

2015 ◽  
Vol 26 (24) ◽  
pp. 4302-4306 ◽  
Author(s):  
Felipe Mora-Bermúdez ◽  
Wieland B. Huttner
Keyword(s):  
Stem Cell ◽  
Cell Division ◽  
Neural Stem Cell ◽  
Spindle Orientation ◽  
Stem Cell Divisions ◽  
A Cell ◽  
Spindle Microtubules ◽  
Stem Cell Division ◽  
Embryonic Neural Stem Cell

During stem cell divisions, mitotic microtubules do more than just segregate the chromosomes. They also determine whether a cell divides virtually symmetrically or asymmetrically by establishing spindle orientation and the plane of cell division. This can be decisive for the fate of the stem cell progeny. Spindle defects have been linked to neurodevelopmental disorders, yet the role of spindle orientation for mammalian neurogenesis has remained controversial. Here we explore recent advances in understanding how the microtubule cytoskeleton influences mammalian neural stem cell division. Our focus is primarily on the role of spindle microtubules in the development of the cerebral cortex. We also highlight unique characteristics in the architecture and dynamics of cortical stem cells that are tightly linked to their mode of division. These features contribute to setting these cells apart as mitotic “rule breakers,” control how asymmetric a division is, and, we argue, are sufficient to determine the fate of the neural stem cell progeny in mammals.

Embigin Regulates HSPC Homing and Quiescence and Acts As a Cell Surface Marker for a Niche Factor-Enriched Subset of Osteolineage Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 663-663 ◽  
Author(s):  
Lev Silberstein ◽  
Peter V. Kharchenko ◽  
Youmna Sami Kfoury ◽  
Francois Mercier ◽  
Raphael Turcotte ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Functional Role ◽  
Hematopoietic Cells ◽  
Surface Marker ◽  
Bone Marrow Niche ◽  
A Cell ◽  
Niche Factor ◽  
Osteolineage Cells

Abstract Background. Discovery of niche-derived HSPC regulators is critical for further development of novel therapeutic approaches to promote HSPC regeneration. We have previously reported a proximity-based approach to the study of the bone marrow niche, which is based on the transcriptional comparison of osteolineage cells (OLCs) located close (proximal OLCs) or further away from transplanted HSPC (Blood 2014;124: 773), which lead to identification of IL18 as a quiescence regulator of early progenitors. We now report the results of functional validation for another molecule - Embigin - identified through this strategy as a hematopoietic regulator. Results. Embigin is a cell adhesion molecule with poorly characterized function. Our analysis showed that Embigin expression in proximal OLCs was significantly higher. Since the genetic tools for this molecule do not exist, we used a neutralizing antibody against Embigin to investigate the effect of Embigin blockade on primitive hematopoietic cells. We found that injection of anti-Embigin resulted in mobilization of myeloid progenitors and colony-forming cells (CFC) into the blood. In contrast, Embigin blockade was associated with a homing defect when LKS cells [known to express Embigin] were either pre-incubated with anti-Embigin antibody or injected into anti-Embigin pretreated animals, overall suggesting that Embigin regulates retention and localization of primitive hematopoietic cells in the bone marrow. Moreover, animals treated with anti-Embigin antibody had a higher frequency and proliferative activity of primitive hematopoietic cells, as demonstrated by cell cycle and BrdU incorporation studies and an increased CFC, consistent with Embigin-mediated localization also affecting HSPC cell cycling. Bone marrow from anti-Embigin treated mice reconstituted poorly when competitively transplanted with untreated animal marrow into irradiated recipients, likely due to the impaired homing and increased cell cycling. Finally, pre-treating irradiated recipients with anti-Embigin resulted in increased proliferation of transplanted WT LKS cells. Collectively, these data are consistent with Embigin serving as a regulator of HSPC localization and quiescence. Given a functional role of Embigin in the bone marrow niche and an overlapping pattern of expression with VCAM1, a known niche-derived HSPC regulator, in proximal OLCs we explored the use of Embigin in conjunction with VCAM1 as cell surface markers for prospective isolation of niche factor-enriched OLC subset by flow cytometry. Using this strategy, we were able to purify a rare OLC subset of CD45- Ter119-V CAM-1+E mbigin+ cells (termed VE cells), which were enriched for niche factor expression as compared to their non-VE counterparts. VE cells were transcriptionally distinct from other, previously defined niche subsets such as nestin-GFPdim mesenchymal stem cells, nestin-GFPbright pericytes and N-cadherin-positive osteoblastic cells. In particular, VE cells expressed higher levels of most niche factors than nestin-GFPbright cells - a cell population recently characterized as regulating HSPC quiescence. Interestingly, the expression profile of VE cells from animals transplanted with LT-HSCs and those which were irradiated but injected with saline alone demonstrated upregulation of cell adhesion molecules in the LT-HSC-injected group, suggesting that VE cells are involved in bidirectional communication within the niche. To investigate a functional role of VE cells, we performed co-culture experiments in which we compared the effect of VE and non-VE cells on HSPC proliferation and engraftment. We found that in the presence of VE cells, HSPCs proliferated at slower rate and generated a lower hematopoietic colony number, consistent with quiescence-inducing effect. Upon transplantation, VE-cultured HSPCs generated a higher level of chimerism compared to those cultured on non-VE layer, indicating a superior ability of VE cells to support engraftment and reconstitution properties of HSPC during the in vitro culture. Conclusion. Our work defines Embgin as a previously unrecognized hematopoietic regulator and a cell surface marker for a niche factor-enriched subset of the osteolineage cells which regulates HSPC quiescence. Pharmacological blockade of Embigin signaling may serve as a potential therapeutic tool to enhance hematopoietic regeneration. Disclosures No relevant conflicts of interest to declare.

scholarly journals Interpolar spindle microtubules in PTK cells.

1993 ◽  
Vol 123 (6) ◽  
pp. 1475-1489 ◽  
Author(s):  
D N Mastronarde ◽  
K L McDonald ◽  
R Ding ◽  
J R McIntosh
Keyword(s):  
Electron Microscopy ◽  
Cross Section ◽  
Specific Interaction ◽  
Spindle Pole ◽  
Serial Sections ◽  
Spindle Poles ◽  
Late Anaphase ◽  
Computer Image Processing ◽  
Spindle Microtubules ◽  
Anaphase B

Spindle microtubules (MTs) in PtK1 cells, fixed at stages from metaphase to telophase, have been reconstructed using serial sections, electron microscopy, and computer image processing. We have studied the class of MTs that form an interdigitating system connecting the two spindle poles (interpolar MTs or ipMTs) and their relationship to the spindle MTs that attach to kinetochores (kMTs). Viewed in cross section, the ipMTs cluster with antiparallel near neighbors throughout mitosis; this bundling becomes much more pronounced as anaphase proceeds. While the minus ends of most kMTs are near the poles, those of the ipMTs are spread over half of the spindle length, with at least 50% lying > 1.5 microns from the poles. Longitudinal views of the ipMT bundles demonstrate a major rearrangement of their plus ends between mid- and late anaphase B. However, the minus ends of these MTs do not move appreciably farther from the spindle midplane, suggesting that sliding of these MTs contributes little to anaphase B. The minus ends of ipMTs are markedly clustered in the bundles of kMTs throughout anaphase A. These ends lie close to kMTs much more frequently than would be expected by chance, suggesting a specific interaction. As sister kinetochores separate and kMTs shorten, the minus ends of the kMTs remain associated with the spindle poles, but the minus ends of many ipMTs are released from the kMT bundles, allowing the spindle pole and the kMTs to move away from the ipMTs as the spindle elongates.

Spindle scaling mechanisms

2020 ◽  
Vol 64 (2) ◽  
pp. 383-396
Author(s):  
Lara K. Krüger ◽  
Phong T. Tran
Keyword(s):  
Cell Size ◽  
Spindle Assembly ◽  
Dependent Manner ◽  
Post Translational Modification ◽  
Size Dependent ◽  
A Cell ◽  
Chromosome Separation ◽  
Spindle Elongation ◽  
Protein Gradients ◽  
Spindle Structure

Abstract The mitotic spindle robustly scales with cell size in a plethora of different organisms. During development and throughout evolution, the spindle adjusts to cell size in metazoans and yeast in order to ensure faithful chromosome separation. Spindle adjustment to cell size occurs by the scaling of spindle length, spindle shape and the velocity of spindle assembly and elongation. Different mechanisms, depending on spindle structure and organism, account for these scaling relationships. The limited availability of critical spindle components, protein gradients, sequestration of spindle components, or post-translational modification and differential expression levels have been implicated in the regulation of spindle length and the spindle assembly/elongation velocity in a cell size-dependent manner. In this review, we will discuss the phenomenon and mechanisms of spindle length, spindle shape and spindle elongation velocity scaling with cell size.

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