scholarly journals Variable pathways for developmental changes of mitosis and cytokinesis in Physarum polycephalum.

1991 ◽  
Vol 113 (3) ◽  
pp. 591-604 ◽  
Author(s):  
L Solnica-Krezel ◽  
T G Burland ◽  
W F Dove
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Physarum Polycephalum ◽  
Developmental Changes ◽  
Binucleate Cell ◽  
Mitotic Spindles ◽  
Tubulin Isotypes ◽  
Binucleate Cells ◽  
Microscopic Studies ◽  
Mitotic Figures

The development of a uninucleate ameba into a multinucleate, syncytial plasmodium in myxomycetes involves a change from the open, astral mitosis of the ameba to the intranuclear, anastral mitosis of the plasmodium, and the omission of cytokinesis from the cell cycle. We describe immunofluorescence microscopic studies of the amebal-plasmodial transition (APT) in Physarum polycephalum. We demonstrate that the reorganization of mitotic spindles commences in uninucleate cells after commitment to plasmodium formation, is completed by the binucleate stage, and occurs via different routes in individual developing cells. Most uninucleate developing cells formed mitotic spindles characteristic either of amebae or of plasmodia. However, chimeric mitotic figures exhibiting features of both amebal and plasmodial mitoses, and a novel star microtubular array were also observed. The loss of the ameba-specific alpha 3-tubulin and the accumulation of the plasmodium-specific beta 2-tubulin isotypes during development were not sufficient to explain the changes in the organization of mitotic spindles. The majority of uninucleate developing cells undergoing astral mitoses (amebal and chimeric) exhibited cytokinetic furrows, whereas cells with the anastral plasmodial mitosis exhibited no furrows. Thus, the transition from astral to anastral mitosis during the APT could be sufficient for the omission of cytokinesis from the cell cycle. However, astral mitosis may not ensure cytokinesis: some cells undergoing amebal or chimeric mitosis contained unilateral cytokinetic furrows or no furrow at all. These cells would, most probably, fail to divide. We suggest that a uninucleate committed cell undergoing amebal or chimeric mitosis can either divide or else form a binucleate cell. In contrast, a uninucleate cell with a mitotic spindle of the plasmodial type gives rise only to a binucleate cells. Further, the decision to enter mitosis after commitment to the APT is independent of the developmental changes in the organization of the mitotic spindle and cytokinesis.

The localization of the divergent beta 2-tubulin isotype in the microtubular arrays of Physarum polycephalum

1989 ◽  
Vol 94 (2) ◽  
pp. 217-226
Author(s):  
M. Diggins-Gilicinski ◽  
L. Solnica-Krezel ◽  
T.G. Burland ◽  
E.C. Paul ◽  
W.F. Dove
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Polyclonal Antibody ◽  
Mitotic Spindle ◽  
Subcellular Distribution ◽  
Physarum Polycephalum ◽  
Mitotic Spindles ◽  
Tubulin Isotypes ◽  
Tubulin Isotype ◽  
Beta 2

The beta 2-tubulin isotype of Physarum polycephalum is only 83% identical in amino acid sequence with the constitutively expressed beta 1B-tubulin and the myxamoeba-specific beta 1A-tubulin isotypes. A polyclonal antibody specific for beta 2-tubulin was used to monitor the subcellular distribution of the beta 2-tubulin antigen in the mitotic spindle of the mature plasmodium - the sole microtubular array in that stage of Physarum. By immunofluorescence, the beta 2-tubulin antigen was detected throughout this anastral mitotic spindle, at all stages of mitosis. Physarum myxamoebae contain astral mitotic spindles and cytoskeletal microtubules. No beta 2-tubulin antigen was detected in the myxamoebal stage. However, as cultures of myxamoebae developed into plasmodia, the beta 2-tubulin antigen was found in the astral mitotic spindles and cytoskeletons in developing cells. Thus, the presence of the plasmodial beta 2-tubulin isotype in a mitotic spindle does not determine a closed, anastral mitosis.

scholarly journals The distribution of cytoplasmic microtubules throughout the cell cycle of the centric diatom Stephanopyxis turris: their role in nuclear migration and positioning the mitotic spindle during cytokinesis.

1986 ◽  
Vol 102 (5) ◽  
pp. 1688-1698 ◽  
Author(s):  
L Wordeman ◽  
K L McDonald ◽  
W Z Cande
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Nuclear Migration ◽  
Spindle Pole ◽  
Centric Diatom ◽  
Mitotic Spindles ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Cytoplasmic Microtubule ◽  
Cytoplasmic Microtubules

The cell cycle of the marine centric diatom Stephanopyxis turris consists of a series of spatially and temporally well-ordered events. We have used immunofluorescence microscopy to examine the role of cytoplasmic microtubules in these events. At interphase, microtubules radiate out from the microtubule-organizing center, forming a network around the nucleus and extending much of the length and breadth of the cell. As the cell enters mitosis, this network breaks down and a highly ordered mitotic spindle is formed. Peripheral microtubule bundles radiate out from each spindle pole and swing out and away from the central spindle during anaphase. Treatment of synchronized cells with 2.5 X 10(-8) M Nocodazole reversibly inhibited nuclear migration concurrent with the disappearance of the extensive cytoplasmic microtubule arrays associated with migrating nuclei. Microtubule arrays and mitotic spindles that reformed after the drug was washed out appeared normal. In contrast, cells treated with 5.0 X 10(-8) M Nocodazole were not able to complete nuclear migration after the drug was washed out and the mitotic spindles that formed were multipolar. Normal and multipolar spindles that were displaced toward one end of the cell by the drug treatment had no effect on the plane of division during cytokinesis. The cleavage furrow always bisected the cell regardless of the position of the mitotic spindle, resulting in binucleate/anucleate daughter cells. This suggests that in S. turris, unlike animal cells, the location of the plane of division is cortically determined before mitosis.

scholarly journals The Catastrophe-promoting Activity of Ectopic Op18/Stathmin Is Required for Disruption of Mitotic Spindles But Not Interphase Microtubules

2001 ◽  
Vol 12 (1) ◽  
pp. 73-83 ◽  
Author(s):  
Per Holmfeldt ◽  
Niklas Larsson ◽  
Bo Segerman ◽  
Bonnie Howell ◽  
Justin Morabito ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Polymerization Rate ◽  
Terminal Region ◽  
Phosphorylation Sites ◽  
Mitotic Spindles ◽  
Intact Cells ◽  
Spindle Formation ◽  
Tubulin Binding

Oncoprotein18/stathmin (Op18) is a microtubule (MT) destabilizing protein that is inactivated during mitosis by phosphorylation at four Ser-residues. Op18 has at least two functions; the N-terminal region is required for catastrophe-promotion (i.e., transition from elongation to shortening), while the C-terminal region is required to inhibit MT-polymerization rate in vitro. We show here that a “pseudophosphorylation” derivative of Op18 (i.e., four Ser- to Glu-substitutions at phosphorylation sites) exhibits a selective loss of catastrophe-promoting activity. This is contrasted to authentic phosphorylation, which efficiently attenuates all activities except tubulin binding. In intact cells, overexpression of pseudophosphorylated Op18, which is not phosphorylated by endogenous kinases, is shown to destabilize interphase MTs but to leave spindle formation untouched. To test if the mitotic spindle is sensitive only to the catastrophe-promoting activity of Op18 and resistant to C-terminally associated activities, N- and C-terminal truncations with defined activity-profiles were employed. The cell-cycle phenotypes of nonphosphorylatable mutants (i.e., four Ser- to Ala-substitutions) of these truncation derivatives demonstrated that catastrophe promotion is required for interference with the mitotic spindle, while the C-terminally associated activities are sufficient to destabilize interphase MTs. These results demonstrate that specific Op18 derivatives with defined activity-profiles can be used as probes to distinguish interphase and mitotic MTs.

3-D reconstruction and analysis of mammalian mitotic spindle structure

1992 ◽  
Vol 50 (1) ◽  
pp. 578-579
Author(s):  
Kent McDonald ◽  
David Mastronarde ◽  
Rubai Ding ◽  
Eileen O'Toole ◽  
J. Richard McIntosh
Keyword(s):  
Cross Sections ◽  
Mitotic Spindle ◽  
Experimental Studies ◽  
Video Camera ◽  
Three Dimensions ◽  
Mitotic Spindles ◽  
Black And White ◽  
Reconstruction Methods ◽  
Spindle Structure ◽  
Tissue Culture Line

Mammalian spindles are generally large and may contain over a thousand microtubules (MTs). For this reason they are difficult to reconstruct in three dimensions and many researchers have chosen to study the smaller and simpler spindles of lower eukaryotes. Nevertheless, the mammalian spindle is used for many experimental studies and it would be useful to know its detailed structure.We have been using serial cross sections and computer reconstruction methods to analyze MT distributions in mitotic spindles of PtK cells, a mammalian tissue culture line. Images from EM negatives are digtized on a light box by a Dage MTI video camera containing a black and white Saticon tube. The signal is digitized by a Parallax 1280 graphics device in a MicroVax III computer. Microtubules are digitized at a magnification such that each is 10-12 pixels in diameter.

Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

1990 ◽  
Vol 48 (3) ◽  
pp. 166-167
Author(s):  
J. Holy ◽  
G. Schatten
Keyword(s):  
Confocal Microscopy ◽  
Mitotic Spindle ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Two Dimensions ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Cleavage Division ◽  
Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

scholarly journals Cell cycle dependent change in the endogenous phosphorylation of nucleolar proteins of Physarum polycephalum

FEBS Letters ◽  
1981 ◽  
Vol 124 (1) ◽  
pp. 53-56 ◽  
Author(s):  
Toshiko Shibayama ◽  
Shouzou Sawai ◽  
Kazuyasu Nakaya ◽  
Yasuharu Nakamura
Keyword(s):  
Cell Cycle ◽  
Physarum Polycephalum ◽  
Nucleolar Proteins ◽  
Dependent Change ◽  
Cycle Dependent

Biochemical and morphological characterization of the nuclear matrix during the synchronous cell cycle of Physarum polycephalum

1993 ◽  
Vol 105 (4) ◽  
pp. 1121-1130 ◽  
Author(s):  
S. Lang ◽  
T. Decristoforo ◽  
W. Waitz ◽  
P. Loidl
Keyword(s):  
Cell Cycle ◽  
Nuclear Matrix ◽  
Physarum Polycephalum ◽  
Nuclear Periphery ◽  
S Phase ◽  
Cycle Phase ◽  
Electron Microscopic ◽  
Nuclear Lamins ◽  
Synchronous Cell ◽  
Matrix Bound

We have investigated biochemical and ultrastructural aspects of the nuclear matrix during the naturally synchronous cell cycle of Physarum polycephalum. The morphology of the in situ nuclear matrix exhibited significant cell cycle changes as revealed by electron microscopic examination, especially during the progression of nuclei through mitosis and S-phase. In mitosis the interchromatin matrix was found to be retracted to the nuclear periphery; during S-phase this interchromatin matrix gradually resembled, concomitant with the reconstruction of a nucleolar remnant structure. During the G2-period no significant changes in matrix morphology were observed. The pattern of nuclear matrix proteins was invariant during the cell cycle; no cycle phase-specific proteins could be detected. In vivo labelling of plasmodia with [35S]methionine/cysteine showed that only a few proteins are synthesized and assembled into nuclear matrix structures in a cell cycle-dependent way; the majority of proteins were synthesized almost continuously. This was also shown for nuclear lamins homologues. In contrast to bulk nuclear histones, those histones that remain tightly bound to the nuclear matrix were synthesized and assembled into nuclear structures in the very first hour of S-phase; assembly was terminated in mid-S-phase, indicating that nuclear matrix-bound chromatin is replicated early in S-phase. Comparison of the acetylation pattern of matrix-bound histone H4 with bulk nuclear H4 revealed a largely elevated acetate content of matrix H4. The percentage of acetylated subspecies was entirely different from that in bulk nuclear H4, indicating that matrix-associated histones represent a subpopulation of nuclear histones with distinct properties, reflecting specific structural requirements of matrix-attached chromatin.

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly

1998 ◽  
Vol 111 (5) ◽  
pp. 557-572 ◽  
Author(s):  
C. Roghi ◽  
R. Giet ◽  
R. Uzbekov ◽  
N. Morin ◽  
I. Chartrain ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Pericentriolar Material ◽  
Spindle Microtubules ◽  
Cycle Dependent

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 ‘invades’ the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

scholarly journals Human Enhancer of Invasion-Cluster, a Coiled-Coil Protein Required for Passage through Mitosis

2004 ◽  
Vol 24 (9) ◽  
pp. 3957-3971 ◽  
Author(s):  
Margret B. Einarson ◽  
Edna Cukierman ◽  
Duane A. Compton ◽  
Erica A. Golemis
Keyword(s):  
Cell Cycle ◽  
Coiled Coil ◽  
Cell Cycle Checkpoints ◽  
Mitotic Spindles ◽  
Human Genes ◽  
Hydroxyurea Treatment ◽  
Evolutionarily Conserved ◽  
Defects Analysis ◽  
Spindle Function

ABSTRACT In a cross-species overexpression approach, we used the pseudohyphal transition of Saccharomyces cerevisiae as a model screening system to identify human genes that regulate cell morphology and the cell cycle. Human enhancer of invasion-cluster (HEI-C), encoding a novel evolutionarily conserved coiled-coil protein, was isolated in a screen for human genes that induce agar invasion in S. cerevisiae. In human cells, HEI-C is primarily localized to the spindle during mitosis. Depletion of HEI-C in vivo with short interfering RNAs results in severe mitotic defects. Analysis by immunofluorescence, flow cytometry analysis, and videomicroscopy indicates that HEI-C-depleted cells form metaphase plates with normal timing after G2/M transition, although in many cases cells have disorganized mitotic spindles. Subsequently, severe defects occur at the metaphase-anaphase transition, characterized by a significant delay at this stage or, more commonly, cellular disintegration accompanied by the display of classic biochemical markers of apoptosis. These mitotic defects occur in spite of the fact that HEI-C-depleted cells retain functional cell cycle checkpoints, as these cells arrest normally following nocodazole or hydroxyurea treatment. These results place HEI-C as a novel regulator of spindle function and integrity during the metaphase-anaphase transition.

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