Genetic control of kinetochore-driven microtubule growth in Drosophila mitosis

Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but allows KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM) and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6 and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1 and Patronin positively regulate polymerization, bundling and stabilization of regrowing MTs until a bipolar spindle is reformed.

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Genes involved in centrosome-independent mitotic spindle assembly in Drosophila S2 cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1320013110 ◽

2013 ◽

Vol 110 (49) ◽

pp. 19808-19813 ◽

Cited By ~ 38

Author(s):

S. Moutinho-Pereira ◽

N. Stuurman ◽

O. Afonso ◽

M. Hornsveld ◽

P. Aguiar ◽

...

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly ◽

S2 Cells ◽

Drosophila S2 Cells ◽

Mitotic Spindle Assembly

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Thioredoxin peroxidases can foster cytoprotection or cell death in response to different stressors: over- and under-expression of thioredoxin peroxidase in Drosophila cells

Biochemical Journal ◽

10.1042/bj20021522 ◽

2003 ◽

Vol 371 (3) ◽

pp. 743-752 ◽

Cited By ~ 71

Author(s):

Svetlana N. RADYUK ◽

Rajindar S. SOHAL ◽

William C. ORR

Keyword(s):

Hydrogen Peroxide ◽

Heat Stress ◽

S2 Cells ◽

Free Radical Biol ◽

Drosophila Cell ◽

Functional Roles ◽

Thioredoxin Peroxidase ◽

Negative Effect ◽

Drosophila Cells

Recently, we identified a set of five genes constituting the peroxiredoxin gene family in Drosophila melanogaster [Radyuk, Klichko, Spinola, Sohal and Orr (2001) Free Radical Biol. Med. 31, 1090–1100]. This set includes two abundant thioredoxin peroxidase (TPx) species, namely Drosophila peroxiredoxin DPx-4783, a cytosolic TPx and DPx-5037, a mitochondrial TPx. Overexpression of either one of them in Drosophila S2 cells conferred increased resistance to toxicity induced by hydrogen peroxide, paraquat or cadmium. To understand further the functional roles of these enzymes in vivo, we report in the present study the effects of decreased expression, using RNA interference, on the response of S2 cells to different stressors. When either of the TPxs was blocked, cells became relatively more susceptible to oxidative stress caused by exposure to hydrogen peroxide or paraquat, but were unaffected when challenged with copper and heat stress. In contrast, TPx overexpressing cells were more susceptible to copper and heat stress when compared with control cells and exhibited DNA fragmentation. Furthermore, when cells were supplemented with N-acetyl-l-cysteine together with copper, there was a clear negative effect on cell survival, which was exacerbated by TPx overexpression. Manipulations in the levels of TPxs demonstrated that, under different stress conditions, these enzymes might have both beneficial and detrimental effects on Drosophila cell viability.

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Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis

The Journal of Cell Biology ◽

10.1083/jcb.200407090 ◽

2004 ◽

Vol 167 (5) ◽

pp. 831-840 ◽

Cited By ~ 199

Author(s):

Helder Maiato ◽

Conly L. Rieder ◽

Alexey Khodjakov

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly ◽

Spindle Pole ◽

S2 Cells ◽

Spindle Poles ◽

Drosophila S2 Cells ◽

Mitotic Spindle Assembly ◽

Normal Mitosis ◽

Proper Orientation ◽

Dependent Mechanism

It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

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Dynamics of Whole-Genome Contacts of Nucleoli in Drosophila Cells Suggests a Role for rDNA Genes in Global Epigenetic Regulation

Cells ◽

10.3390/cells9122587 ◽

2020 ◽

Vol 9 (12) ◽

pp. 2587

Author(s):

Nickolai A. Tchurikov ◽

Elena S. Klushevskaya ◽

Daria M. Fedoseeva ◽

Ildar R. Alembekov ◽

Galina I. Kravatskaya ◽

...

Keyword(s):

Gene Expression ◽

Regulation Of Gene Expression ◽

S2 Cells ◽

Heat Shock Treatment ◽

Rdna Genes ◽

Drosophila S2 Cells ◽

Mouse Cells ◽

Non Coding Rnas ◽

Large Groups ◽

Drosophila Cells

Chromosomes are organized into 3D structures that are important for the regulation of gene expression and differentiation. Important role in formation of inter-chromosome contacts play rDNA clusters that make up nucleoli. In the course of differentiation, heterochromatization of rDNA units in mouse cells is coupled with the repression or activation of different genes. Furthermore, the nucleoli of human cells shape the direct contacts with genes that are involved in differentiation and cancer. Here, we identified and categorized the genes located in the regions where rDNA clusters make frequent contacts. Using a 4C approach, we demonstrate that in Drosophila S2 cells, rDNA clusters form contacts with genes that are involved in chromosome organization and differentiation. Heat shock treatment induces changes in the contacts between nucleoli and hundreds of genes controlling morphogenesis. We show that nucleoli form contacts with regions that are enriched with active or repressive histone marks and where small non-coding RNAs are mapped. These data indicate that rDNA contacts are involved in the repression and activation of gene expression and that rDNA clusters orchestrate large groups of Drosophila genes involved in differentiation.

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