The Des-1 protein, required for central spindle assembly and cytokinesis, is associated with mitochondria along the meiotic spindle apparatus and with the contractile ring during male meiosis in Drosophila melanogaster

While Drosophila female meiosis is anastral, both meiotic divisions in Drosophila males exhibit prominent asters. We have identified a gene we call asterless (asl) that is required for aster formation during male meiosis. Ultrastructural analysis showed that asl mutants have morphologically normal centrioles. However, immunostaining with antibodies directed either to γ tubulin or centrosomin revealed that these proteins do not accumulate in the centrosomes, as occurs in wild-type. Thus, asl appears to specify a function required for the assembly of centrosomal material around the centrioles. Despite the absence of asters, meiotic cells of asl mutants manage to develop an anastral spindle. Microtubules grow from multiple sites around the chromosomes, and then focus into a peculiar bipolar spindle that mediates chromosome segregation, although in a highly irregular way. Surprisingly, asl spermatocytes eventually form a morphologically normal ana–telophase central spindle that has full ability to stimulate cytokinesis. These findings challenge the classical view on central spindle assembly, arguing for a self-organization of this structure from either preexisting or newly formed microtubules. In addition, these findings strongly suggest that the asters are not required for signaling cytokinesis.

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The Drosophila Kinesin-like Protein KLP67A Is Essential for Mitotic and Male Meiotic Spindle Assembly

Molecular Biology of the Cell ◽

10.1091/mbc.e03-05-0342 ◽

2004 ◽

Vol 15 (1) ◽

pp. 121-131 ◽

Cited By ~ 58

Author(s):

Rita Gandhi ◽

Silvia Bonaccorsi ◽

Diana Wentworth ◽

Stephen Doxsey ◽

Maurizio Gatti ◽

...

Keyword(s):

Cell Division ◽

Chromosome Segregation ◽

Mutational Analysis ◽

Spindle Assembly ◽

Male Meiosis ◽

Meiotic Spindle ◽

Drosophila Cell ◽

Centrosome Separation ◽

Mutant Cells

We have performed a mutational analysis together with RNA interference to determine the role of the kinesin-like protein KLP67A in Drosophila cell division. During both mitosis and male meiosis, Klp67A mutations cause an increase in MT length and disrupt discrete aspects of spindle assembly, as well as cytokinesis. Mutant cells exhibit greatly enlarged metaphase spindle as a result of excessive MT polymerization. The analysis of both living and fixed cells also shows perturbations in centrosome separation, chromosome segregation, and central spindle assembly. These data demonstrate that the MT plus end-directed motor KLP67A is essential for spindle assembly during mitosis and male meiosis and suggest that the regulation of MT plus-end polymerization is a key determinant of spindle architecture throughout cell division.

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Spindle assembly and cytokinesis in the absence of chromosomes during Drosophila male meiosis

The Journal of Cell Biology ◽

10.1083/jcb.200211029 ◽

2003 ◽

Vol 160 (7) ◽

pp. 993-999 ◽

Cited By ~ 48

Author(s):

Elisabetta Bucciarelli ◽

Maria Grazia Giansanti ◽

Silvia Bonaccorsi ◽

Maurizio Gatti

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly ◽

Male Meiosis ◽

Aurora B ◽

Aurora B Kinase ◽

Spindle Formation ◽

Central Spindle ◽

Morphological Transformations ◽

Bipolar Spindles ◽

Drosophila Mutants

Alarge body of work indicates that chromosomes play a key role in the assembly of both acentrosomal and centrosome-containing spindles. In animal systems, the absence of chromosomes either prevents spindle formation or allows the assembly of a metaphase-like spindle that fails to evolve into an ana-telophase spindle. Here, we show that Drosophila secondary spermatocytes can assemble morphologically normal spindles in the absence of chromosomes. The Drosophila mutants fusolo and solofuso are severely defective in chromosome segregation and produce secondary spermatocytes that are devoid of chromosomes. The centrosomes of these anucleated cells form robust asters that give rise to bipolar spindles that undergo the same ana-telophase morphological transformations that characterize normal spindles. The cells containing chromosome-free spindles are also able to assemble regular cytokinetic structures and cleave normally. In addition, chromosome-free spindles normally accumulate the Aurora B kinase at their midzones. This suggests that the association of Aurora B with chromosomes is not a prerequisite for its accumulation at the central spindle, or for its function during cytokinesis.

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Vesicles and actin are targeted to the cleavage furrow via furrow microtubules and the central spindle

The Journal of Cell Biology ◽

10.1083/jcb.200803096 ◽

2008 ◽

Vol 181 (5) ◽

pp. 777-790 ◽

Cited By ~ 40

Author(s):

Roger Albertson ◽

Jian Cao ◽

Tao-shih Hsieh ◽

William Sullivan

Keyword(s):

Drosophila Melanogaster ◽

Vesicle Transport ◽

Cleavage Furrow ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Distinct Population ◽

Contractile Ring ◽

Central Spindle ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor

During cytokinesis, cleavage furrow invagination requires an actomyosin-based contractile ring and addition of new membrane. Little is known about how this actin and membrane traffic to the cleavage furrow. We address this through live analysis of fluorescently tagged vesicles in postcellularized Drosophila melanogaster embryos. We find that during cytokinesis, F-actin and membrane are targeted as a unit to invaginating furrows through formation of F-actin–associated vesicles. F-actin puncta strongly colocalize with endosomal, but not Golgi-derived, vesicles. These vesicles are recruited to the cleavage furrow along the central spindle and a distinct population of microtubules (MTs) in contact with the leading furrow edge (furrow MTs). We find that Rho-specific guanine nucleotide exchange factor mutants, pebble (pbl), severely disrupt this F-actin–associated vesicle transport. These transport defects are a consequence of the pbl mutants' inability to properly form furrow MTs and the central spindle. Transport of F-actin–associated vesicles on furrow MTs and the central spindle is thus an important mechanism by which actin and membrane are delivered to the cleavage furrow.

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Novel roles for BicD in pronuclear fusion and meiosis II progression via localization of the CHC/TACC/Msps complex to MII spindles

10.1101/2020.12.22.423980 ◽

2020 ◽

Author(s):

Paula Vazquez-Pianzola ◽

Dirk Beuchle ◽

Gabriela Saro ◽

Greco Hernández ◽

Giovanna Maldonado ◽

...

Keyword(s):

Heavy Chain ◽

Spindle Assembly Checkpoint ◽

Coiled Coil ◽

Spindle Assembly ◽

Meiotic Spindle ◽

C Elegans ◽

Spindle Apparatus ◽

Spindle Microtubules ◽

Mitosis And Meiosis ◽

Overexpressed Gene

ABSTRACTVertebrate Clathrin heavy chain (Chc) plays a moonlighting function during mitosis. Chc forms a complex with TACC3 (Transforming Acidic Coiled Coil 3) and ch-TOG (colonic hepatic tumor overexpressed gene) at the spindle microtubules, forming inter microtubule bridges that stabilize the K-fibers. Since Drosophila Chc is a cargo of the dynein adaptor Bicaudal-D (BicD), we investigated whether BicD regulates Clathrin function at the spindle. We found that BicD localizes, like Chc, to centrosomes and spindles during mitosis and meiosis II, and that Chc interacts with Drosophila TACC (D-TACC). Using deGradFP to reduce the activity of BicD in mature eggs and very young embryos, we uncovered a novel function of BicD in meiosis II. The affected meiosis II products underwent abnormal rounds of additional replications and failed to carry out pronuclear fusion. Pointing to a mechanism, we found that the localization of Clathrin/D-TACC/Minispindles (Msps, homolog of ch-TOG) to the meiosis II spindles was impaired upon BicD knockdown. Furthermore, the meiotic products showed abnormal staining for PH3 and reduced recruitment of spindle assembly checkpoint (SAC) components. Altogether, our results support the notion that BicD performs a key activity in assembling the meiotic spindle apparatus. This function of BicD seems conserved in evolution because C. elegans embryos with reduced activities of these genes developed comparable phenotypes.

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C. elegans CLASP/CLS-2 negatively regulates membrane ingression throughout the oocyte cortex and is required for polar body extrusion

10.1101/2020.04.02.021675 ◽

2020 ◽

Author(s):

Aleesa J. Schlientz ◽

Bruce Bowerman

Keyword(s):

Polar Body ◽

Spindle Assembly ◽

Meiotic Spindle ◽

Contractile Apparatus ◽

Contractile Ring ◽

C Elegans ◽

Cell Divisions ◽

Polar Bodies ◽

Polar Body Extrusion ◽

The Relationship

AbstractThe requirements for oocyte meiotic cytokinesis during polar body extrusion are not well understood. In particular, the relationship between the oocyte meiotic spindle and polar body contractile ring dynamics remains largely unknown. We have used live cell imaging and spindle assembly defective mutants lacking the function of CLASP/CLS-2, kinesin-12/KLP-18, or katanin/MEI-1 to investigate the relationship between meiotic spindle structure and polar body extrusion in C. elegans oocytes. We show that spindle bipolarity and chromosome segregation are not required for polar body contractile ring formation and chromosome extrusion in klp-18 mutants, but oocytes with severe spindle assembly defects due to loss of CLS-2 or MEI-1 have penetrant and distinct polar body extrusion defects: CLS-2 is required early for contractile ring assembly or stability, while MEI-1 is required later for contractile ring constriction. We also show that CLS-2 negatively regulates membrane ingression throughout the oocyte cortex during meiosis I, and we explore the relationship between global cortical dynamics and oocyte meiotic cytokinesis.Author SummaryThe precursor cells that produce gametes—sperm and eggs in animals—have two copies of each chromosome, one from each parent. These precursors undergo specialized cell divisions that leave each gamete with only one copy of each chromosome; defects that produce incorrect chromosome number cause severe developmental abnormalities. In oocytes, these cell divisions are highly asymmetric, with extra chromosomes discarded into small membrane bound polar bodies, leaving one chromosome set within the much larger oocyte. How oocytes assemble the contractile apparatus that pinches off polar bodies remains poorly understood. To better understand this process, we have used the nematode Caenorhabditis elegans to investigate the relationship between the bipolar structure that separates oocyte chromosomes, called the spindle, and assembly of the contractile apparatus that pinches off polar bodies. We used a comparative approach, examining this relationship in three spindle assembly defective mutants. Bipolar spindle assembly and chromosome separation were not required for polar body extrusion, as it occurred normally in mutants lacking a protein called KLP-18. However, mutants lacking the protein CLS-2 failed to assemble the contractile apparatus, while mutants lacking the protein MEI-1 assembled a contractile apparatus that failed to fully constrict. We also found that CLS-2 down-regulates membrane ingression throughout the oocyte surface, and we explored the relationship between oocyte membrane dynamics and polar body extrusion.

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Endosome mediated delivery of ceramide phosphoethanolamine (CPE) with unique acyl chain anchors to the cleavage furrow is essential for male meiosis cytokinesis

10.1101/2021.04.27.441622 ◽

2021 ◽

Author(s):

Govind Kunduri ◽

Si-Hung Le ◽

Nagampalli Vijayakrishna ◽

Daniel Blankenberg ◽

Izumi Yoshihiro ◽

...

Keyword(s):

Plasma Membrane ◽

Acyl Chain ◽

Structural Analog ◽

Male Meiosis ◽

Cleavage Furrow ◽

Biophysical Properties ◽

Contractile Ring ◽

Central Spindle ◽

Daughter Cells ◽

Living Organisms

AbstractDivision of one cell into two daughter cells is fundamental in all living organisms. Cytokinesis, the final step of cell division, begins with the formation of an actomyosin contractile ring, positioned midway between the segregated chromosomes. Constriction of the ring with concomitant membrane deposition in a spatiotemporal precision generates a cleavage furrow that physically divides the cytoplasm. Unique lipids with specific biophysical properties have been shown to localize to midbodies however, their delivery mechanisms and biological roles were largely unknown. In this study, we show that Ceramide phosphoethanolamine (CPE), the structural analog of sphingomyelin, has unique acyl chain anchors in spermatocytes and is essential for meiosis cytokinesis. We found that disengagement of the central spindle from the contractile ring but not localization of phosphatidyl inositols (PIPs) at the plasma membrane was responsible for the male meiosis cytokinesis defect in CPE deficient animals. Further, we demonstrate that enrichment of CPE in Rab7 and Rab11 positive endosomes which in turn translocate to the cleavage furrows to promote cytokinesis. Our results implicate endosomal delivery of CPE to ingressing membranes is crucial for meiotic cytokinesis.

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The Drosophila kinesin-like protein KLP3A is a midbody component required for central spindle assembly and initiation of cytokinesis.

The Journal of Cell Biology ◽

10.1083/jcb.129.3.709 ◽

1995 ◽

Vol 129 (3) ◽

pp. 709-723 ◽

Cited By ~ 99

Author(s):

B C Williams ◽

M F Riedy ◽

E V Williams ◽

M Gatti ◽

M L Goldberg

Keyword(s):

Motor Protein ◽

Spindle Assembly ◽

Male Meiosis ◽

Cleavage Furrow ◽

Critical Component ◽

Central Spindle ◽

Late Anaphase ◽

Spindle Elongation ◽

Anaphase B ◽

Kinesin Superfamily

We describe here a new member of the kinesin superfamily in Drosophila, KLP3A (Kinesin-Like-Protein-at-3A). The KLP3A protein localizes to the equator of the central spindle during late anaphase and telophase of male meiosis. Mutations in the KLP3A gene disrupt the interdigitation of microtubules in spermatocyte central spindles. Despite this defect, anaphase B spindle elongation is not obviously aberrant. However, cytokinesis frequently fails after both meiotic divisions in mutant testes. Together, these findings strongly suggest that the KLP3A presumptive motor protein is a critical component in the establishment or stabilization of the central spindle. Furthermore, these results imply that the central spindle is the source of signals that initiate the cleavage furrow in higher cells.

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