scholarly journals Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila

Open Biology ◽  
2014 ◽  
Vol 4 (5) ◽  
pp. 140047 ◽  
Author(s):  
Matthew S. Savoian ◽  
David M. Glover
Keyword(s):  
Mitotic Spindle ◽  
Cell Types ◽  
Male Meiosis ◽  
Negative Regulation ◽  
Mitotic Spindles ◽  
Animal Cells ◽  
Spindle Formation ◽  
Formation Pathway ◽  
Fixed Cell ◽  
Mitotic Cells

Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin's spindle association, and this in turn dictates γ-tubulin distribution and spindle density. Polo's negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.

scholarly journals The Nek6 and Nek7 Protein Kinases Are Required for Robust Mitotic Spindle Formation and Cytokinesis

2009 ◽  
Vol 29 (14) ◽  
pp. 3975-3990 ◽  
Author(s):  
Laura O'Regan ◽  
Andrew M. Fry
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Serine Threonine Kinase ◽  
Spindle Formation ◽  
Spindle Poles ◽  
And Function ◽  
Interfering Rna ◽  
Reduced Activity

ABSTRACT Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.

scholarly journals A Spindle Checkpoint Functions during Mitosis in the Early Caenorhabditis elegans Embryo

2005 ◽  
Vol 16 (3) ◽  
pp. 1056-1070 ◽  
Author(s):  
Sandra E. Encalada ◽  
John Willis ◽  
Rebecca Lyczak ◽  
Bruce Bowerman
Keyword(s):  
Caenorhabditis Elegans ◽  
Chromosome Segregation ◽  
Spindle Checkpoint ◽  
Metaphase Plate ◽  
Time Lapse ◽  
Cell Types ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Animal Cells ◽  
Dividing Cells

During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.

Clathrin in mitotic spindles

2000 ◽  
Vol 279 (2) ◽  
pp. C369-C374 ◽  
Author(s):  
Curtis T. Okamoto ◽  
Jeana McKinney ◽  
Young Y. Jeng
Keyword(s):  
Mitotic Spindle ◽  
Regulatory Mechanism ◽  
Staining Pattern ◽  
Brefeldin A ◽  
Polyclonal Antiserum ◽  
Mitotic Spindles ◽  
Madin Darby Canine Kidney ◽  
Western Blots ◽  
Darby Canine Kidney ◽  
Mitotic Cells

Subconfluent cultures of Madin-Darby canine kidney (MDCK) and CV-1 cells were immunostained with two monoclonal antibodies (MAbs), MAb X-22 and MAb 23, against clathrin heavy chain and with polyclonal antiserum against a conserved region of all mammalian clathrin light chains. In interphase MDCK and CV-1 cells, staining by all three antibodies resulted in the characteristic intracellular punctate vesicular and perinuclear staining pattern. In mitotic cells, all three anti-clathrin antibodies strongly stained the mitotic spindle. Staining of clathrin in the mitotic spindle was colocalized with anti-tubulin staining of microtubular arrays in the spindle. Staining of the mitotic spindle was evident in mitotic cells from prometaphase to telophase and in spindles in mitotic cells released from a thymidine-nocodazole block. In CV-1 cells, staining of clathrin in the mitotic spindle was not affected by brefeldin A. On Western blots, clathrin was detected, but not enriched, in isolated spindles. The immunodetection of clathrin in the mitotic spindle may suggest a novel role for clathrin in mitosis. Alternatively, the recruitment of clathrin to the spindle may suggest a novel regulatory mechanism for localization of clathrin in mitotic cells.

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.

scholarly journals Selective autophagy maintains centrosome integrity and accurate mitosis by turnover of centriolar satellites

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Søs Grønbæk Holdgaard ◽  
Valentina Cianfanelli ◽  
Emanuela Pupo ◽  
Matteo Lambrughi ◽  
Michal Lubas ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Chromosomal Instability ◽  
Vital Role ◽  
Animal Cells ◽  
Spindle Formation ◽  
Selective Autophagy ◽  
Lir Motif

Abstract The centrosome is the master orchestrator of mitotic spindle formation and chromosome segregation in animal cells. Centrosome abnormalities are frequently observed in cancer, but little is known of their origin and about pathways affecting centrosome homeostasis. Here we show that autophagy preserves centrosome organization and stability through selective turnover of centriolar satellite components, a process we termed doryphagy. Autophagy targets the satellite organizer PCM1 by interacting with GABARAPs via a C-terminal LIR motif. Accordingly, autophagy deficiency results in accumulation of large abnormal centriolar satellites and a resultant dysregulation of centrosome composition. These alterations have critical impact on centrosome stability and lead to mitotic centrosome fragmentation and unbalanced chromosome segregation. Our findings identify doryphagy as an important centrosome-regulating pathway and bring mechanistic insights to the link between autophagy dysfunction and chromosomal instability. In addition, we highlight the vital role of centriolar satellites in maintaining centrosome integrity.

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.

EML4 and NUDC in mitotic spindle formation

Reactome ◽  
2020 ◽  
Author(s):  
Susanne Bechstedt ◽  
Andrew M. Fry ◽  
Kellie J Lucken ◽  
Laura O'Regan
Keyword(s):  
Mitotic Spindle ◽  
Spindle Formation

Fine structure of the kinetochores in six species of the Coleoptera

Genome ◽  
1997 ◽  
Vol 40 (3) ◽  
pp. 379-385
Author(s):  
Klaus Werner Wolf
Keyword(s):  
Direct Contact ◽  
Karyotype Evolution ◽  
Coccinella Septempunctata ◽  
Cell Types ◽  
Male Meiosis ◽  
Serial Sections ◽  
Cell Type ◽  
Present Survey ◽  
Transmission Electron ◽  
Size Variability

Kinetochore structure was examined in a total of 6 species from 5 different families of the Coleoptera using transmission electron microscopy of ultrathin serial sections. Metaphase spermatogonia and primary and secondary spermatocytes were studied in Tenebrio molitor (Tenebrionidae) to determine whether kinetochore structure varies depending on the cell type. In all three cell types, the kinetochore microtubules (MTs) were in direct contact with the chromosomal surface, and kinetochore plates were not detectable. In the other species, only metaphase I spermatocytes were examined. As in T. molitor, distinct kinetochore plates were also absent in Adelocera murina (Elateridae), Agapanthia villosoviridescens (Cerambycidae), and Coccinella septempunctata (Coccinellidae). However, bivalents in male meiosis of two representatives of the Chrysomelidae, Agelastica alni and Chrysolina graminis, showed roughly spherical kinetochores at their poleward surfaces. Microtubules were in contact with this material. Thus, although the present survey covers only a small number of species, it is clear that at least two kinetochore types occur in the Coleoptera. The cytological findings are discussed in the context of chromosome number and genome size variability in the Coleopteran families studied. It is suggested that properties of the kinetochores could play a role in karyotype evolution in the Coleoptera.Key words: bivalent, microtubule, meiosis, metaphase, spermatocyte.

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