scholarly journals γ-Tubulin ring complexes regulate microtubule plus end dynamics

2009 ◽  
Vol 187 (3) ◽  
pp. 327-334 ◽  
Author(s):  
Anaïs Bouissou ◽  
Christel Vérollet ◽  
Aureliana Sousa ◽  
Paula Sampaio ◽  
Michel Wright ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Interference ◽  
Live Imaging ◽  
S2 Cells ◽  
Imaging Analysis ◽  
Mitotic Progression ◽  
Small Complex ◽  
Drosophila S2 Cells ◽  
Ring Complex ◽  
Ring Complexes

γ-Tubulin is critical for the initiation and regulation of microtubule (MT) assembly. In Drosophila melanogaster, it acts within two main complexes: the γ-tubulin small complex (γ-TuSC) and the γ-tubulin ring complex (γ-TuRC). Proteins specific of the γ-TuRC, although nonessential for viability, are required for efficient mitotic progression. Until now, their role during interphase remained poorly understood. Using RNA interference in Drosophila S2 cells, we show that the γ-TuRC is not critical for overall MT organization. However, depletion of any component of this complex results in an increase of MT dynamics. Combined immunofluorescence and live imaging analysis allows us to reveal that the γ-TuRC localizes along interphase MTs and that distal γ-tubulin spots match with sites of pause or rescue events. We propose that, in addition to its role in nucleation, the γ-TuRC associated to MTs may regulate their dynamics by limiting catastrophes.

scholarly journals Mechanisms for focusing mitotic spindle poles by minus end–directed motor proteins

2005 ◽  
Vol 171 (2) ◽  
pp. 229-240 ◽  
Author(s):  
Gohta Goshima ◽  
François Nédélec ◽  
Ronald D. Vale
Keyword(s):  
Rna Interference ◽  
High Resolution ◽  
Computer Modeling ◽  
Mitotic Spindle ◽  
Motor Proteins ◽  
Spindle Pole ◽  
S2 Cells ◽  
Spindle Poles ◽  
Drosophila S2 Cells ◽  
High Resolution Microscopy

During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end–directed motor proteins. Here, we have characterized the roles of two minus end–directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end–tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end–directed motors cooperate to ensure spindle pole coalescence during mitosis.

scholarly journals Characterization of a Drosophila Centrosome Protein CP309 That Shares Homology with Kendrin and CG-NAP

2004 ◽  
Vol 15 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Shin-ichi Kawaguchi ◽  
Yixian Zheng
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Coiled Coil ◽  
Animal Cells ◽  
Microtubule Nucleation ◽  
Small Complex ◽  
Biochemical Studies ◽  
Ring Complex ◽  
Centrosome Protein

The centrosome in animal cells provides a major microtubule-nucleating site that regulates the microtubule cytoskeleton temporally and spatially throughout the cell cycle. We report the identification in Drosophila melanogaster of a large coiled-coil centrosome protein that can bind to calmodulin. Biochemical studies reveal that this novel Drosophila centrosome protein, centrosome protein of 309 kDa (CP309), cofractionates with the γ-tubulin ring complex and the centrosome-complementing activity. We show that CP309 is required for microtubule nucleation mediated by centrosomes and that it interacts with the γ-tubulin small complex. These findings suggest that the microtubule-nucleating activity of the centrosome requires the function of CP309.

scholarly journals Recruitment of Cdc20 to the Kinetochore Requires BubR1 but Not Mad2 in Drosophila melanogaster

2010 ◽  
Vol 30 (13) ◽  
pp. 3384-3395 ◽  
Author(s):  
Deyu Li ◽  
Gary Morley ◽  
Michael Whitaker ◽  
Jun-Yong Huang
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Interference ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Cyclin B ◽  
Stable Complex ◽  
S2 Cells ◽  
Anaphase Promoting Complex ◽  
Surveillance Mechanism ◽  
Initial Binding

ABSTRACT To prevent aneuploidy, cells require a mitotic surveillance mechanism, the spindle assembly checkpoint (SAC). The SAC prevents metaphase/anaphase transition by blocking the ubiquitylation and destruction of cyclin B and securin via the Cdc20-activated anaphase-promoting complex or cyclosome (APC/C)-mediated proteolysis pathway. This checkpoint involves the kinetochore proteins Mad2, BubR1, and Cdc20. Mad2 and BubR1 are inhibitors of the APC/C, but Cdc20 is an activator. Exactly how the SAC regulates Cdc20 via unattached kinetochores remains unclear; in vertebrates, most current models suggest that kinetochore-bound Mad2 is required for initial binding to Cdc20 to form a stable complex that includes BubR1. Here, we show that the Mad2 kinetochore dimerization recruitment mechanism is conserved and that the recruitment of Cdc20 to kinetochores in Drosophila requires BubR1 but not Mad2. BubR1 and Mad2 can bind to Cdc20 independently, and the interactions are enhanced after cells are arrested at mitosis by the depletion of Cdc27 using RNA interference (RNAi) in S2 cells or by MG132 treatment in syncytial embryos. These findings offer an explanation of why BubR1 is more important than Mad2 for SAC function in flies. These findings could lead to a better understanding of vertebrate SAC mechanisms.

scholarly journals Use of RNA interference in Drosophila S2 cells to identify host pathways controlling compartmentalization of an intracellular pathogen

2005 ◽  
Vol 102 (38) ◽  
pp. 13646-13651 ◽  
Author(s):  
L. W. Cheng ◽  
J. P. M. Viala ◽  
N. Stuurman ◽  
U. Wiedemann ◽  
R. D. Vale ◽  
...  
Keyword(s):  
Rna Interference ◽  
Intracellular Pathogen ◽  
S2 Cells ◽  
Drosophila S2 Cells ◽  
Host Pathways

scholarly journals CDK5RAP2 stimulates microtubule nucleation by the γ-tubulin ring complex

2010 ◽  
Vol 191 (6) ◽  
pp. 1089-1095 ◽  
Author(s):  
Yuk-Kwan Choi ◽  
Pengfei Liu ◽  
Siu Kwan Sze ◽  
Chao Dai ◽  
Robert Z. Qi
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Interference ◽  
Schizosaccharomyces Pombe ◽  
Binding Domain ◽  
Deficient Mutant ◽  
Microtubule Nucleation ◽  
Ring Complex ◽  
Regulatory Functions

CDK5RAP2 is a human microcephaly protein that contains a γ-tubulin complex (γ-TuC)–binding domain conserved in Drosophila melanogaster centrosomin and Schizosaccharomyces pombe Mto1p and Pcp1p, which are γ-TuC–tethering proteins. In this study, we show that this domain within CDK5RAP2 associates with the γ-tubulin ring complex (γ-TuRC) to stimulate its microtubule-nucleating activity and is therefore referred to as the γ-TuRC–mediated nucleation activator (γ-TuNA). γ-TuNA but not its γ-TuC–binding-deficient mutant stimulates microtubule nucleation by purified γ-TuRC in vitro and induces extensive, γ-TuRC-dependent nucleation of microtubules in a microtubule regrowth assay. γ-TuRC bound to γ-TuNA contains NME7, FAM128A/B, and actin in addition to γ-tubulin and GCP2–6. RNA interference–mediated depletion of CDK5RAP2 impairs both centrosomal and acentrosomal microtubule nucleation, although γ-TuRC assembly is unaffected. Collectively, these results suggest that the γ-TuNA found in CDK5RAP2 has regulatory functions in γ-TuRC–mediated microtubule nucleation.

scholarly journals The augmin complex plays a critical role in spindle microtubule generation for mitotic progression and cytokinesis in human cells

2009 ◽  
Vol 106 (17) ◽  
pp. 6998-7003 ◽  
Author(s):  
Ryota Uehara ◽  
Ryu-suke Nozawa ◽  
Akiko Tomioka ◽  
Sabine Petry ◽  
Ronald D. Vale ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Critical Role ◽  
Critical Factor ◽  
Human Cells ◽  
S2 Cells ◽  
Mitotic Progression ◽  
Spindle Microtubule ◽  
Ring Complex

The mitotic spindle is constructed from microtubules (MTs) nucleated from centrosomes, chromosome proximal regions, and preexisting spindle MTs. Augmin, a recently identified protein complex, is a critical factor in spindle MT-based MT generation in Drosophila S2 cells. Previously, we identified one subunit of human augmin. Here, by using mass spectrometry, we identified the full human augmin complex of 8 subunits and show that it interacts with the γ-tubulin ring complex (γ-TuRC). Unlike augmin-depleted S2 cells, in which the defect in spindle-mediated MT generation is mostly compensated by centrosomal MTs, augmin knockdown alone in HeLa cells triggers the spindle checkpoint, reduces tension on sister kinetochores, and severely impairs metaphase progression. Human augmin knockdown also reduces the number of central spindle MTs during anaphase and causes late-stage cytokinesis failure. A link between augmin and γ-TuRC is likely critical for these functions, because a γ-TuRC mutant that attenuates interaction with augmin does not restore function in vivo. These results demonstrate that MT generation mediated by augmin and γ-TuRC is critical for chromosome segregation and cytokinesis in human cells.

scholarly journals An RNA Interference Screen Identifies a Novel Regulator of Target of Rapamycin That Mediates Hypoxia Suppression of Translation in Drosophila S2 Cells

2008 ◽  
Vol 19 (10) ◽  
pp. 4051-4061 ◽  
Author(s):  
Soo-Jung Lee ◽  
Renny Feldman ◽  
Patrick H. O'Farrell
Keyword(s):  
Protein Synthesis ◽  
Rna Interference ◽  
Fluorescent Protein ◽  
Tyrosine Phosphatase ◽  
Protein Translation ◽  
Target Of Rapamycin ◽  
Dependent Manner ◽  
S2 Cells ◽  
A Genome ◽  
Drosophila S2 Cells

In addition to its central role in energy production, oxygen has pervasive regulatory actions. Hypoxia (oxygen limitation) triggers the shutdown of major cellular processes, including gene expression. We carried out a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells for functions required to down-regulate translation during hypoxia. RNAi knockdown of specific genes allowed induction of a green fluorescent protein (GFP) reporter gene and continued protein synthesis during hypoxia. Among the identified genes, Tsc1 and Tsc2, which together form the tuberose sclerosis complex that negatively regulates target of rapamycin (TOR) kinase, gave an especially strong effect. This finding is consistent with the involvement of TOR in promoting translation. Another gene required for efficient inhibition of protein translation during hypoxia, the protein tyrosine phosphatase 61F (Ptp61F), down-regulates TOR activity under hypoxia. Lack of Ptp61F or Tsc2 improves cell survival under prolonged hypoxia in a TOR-dependent manner. Our results identify Ptp61F as a novel modulator of TOR activity and suggest that its function during hypoxia contributes to the down-regulation of protein synthesis.

scholarly journals A novel role for dp115 in the organization of tER sites in Drosophila

2003 ◽  
Vol 162 (2) ◽  
pp. 185-198 ◽  
Author(s):  
Vangelis Kondylis ◽  
Catherine Rabouille
Keyword(s):  
Rna Interference ◽  
Protein Transport ◽  
Immunofluorescence Microscopy ◽  
Morphological Changes ◽  
S2 Cells ◽  
Golgi Stack ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Drosophila S2 Cells ◽  
Exocytic Pathway

Here, we describe that depletion of the Drosophila homologue of p115 (dp115) by RNA interference in Drosophila S2 cells led to important morphological changes in the Golgi stack morphology and the transitional ER (tER) organization. Using conventional and immunoelectron microscopy and confocal immunofluorescence microscopy, we show that Golgi stacks were converted into clusters of vesicles and tubules, and that the tERs (marked by Sec23p) lost their focused organization and were now dispersed throughout the cytoplasm. However, we found that this morphologically altered exocytic pathway was nevertheless largely competent in anterograde protein transport using two different assays. The effects were specific for dp115. Depletion of the Drosophila homologues of GM130 and syntaxin 5 (dSed5p) did not lead to an effect on the tER organization, though the Golgi stacks were greatly vesiculated in the cells depleted of dSed5p. Taken together, these studies suggest that dp115 could be implicated in the architecture of both the Golgi stacks and the tER sites.

scholarly journals Regulation of a Drosophila melanogaster cGMP-specific phosphodiesterase by prenylation and interaction with a prenyl-binding protein

2008 ◽  
Vol 414 (3) ◽  
pp. 363-374 ◽  
Author(s):  
Jonathan P. Day ◽  
Vaughn Cleghon ◽  
Miles D. Houslay ◽  
Shireen-A. Davies
Keyword(s):  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Apical Membrane ◽  
Binding Protein ◽  
Membrane Targeting ◽  
Type I ◽  
S2 Cells ◽  
Post Translational Modification ◽  
Drosophila S2 Cells

Post-translational modification by isoprenylation is a pivotal process for the correct functioning of many signalling proteins. The Drosophila melanogaster cGMP-PDE (cGMP-specific phosphodiesterase) DmPDE5/6 possesses a CaaX-box prenylation signal motif, as do several novel cGMP-PDEs from insect and echinoid species (in CaaX, C is cysteine, a is an aliphatic amino acid and X is ‘any’ amino acid). DmPDE5/6 is prenylated in vivo at Cys1128 and is localized to the plasma membrane when expressed in Drosophila S2 cells. Site-directed mutagenesis of the prenylated cysteine residue (C1128S-DmPDE5/6), pharmacological inhibition of prenylation or co-expression of DmPrBP (Drosophila prenyl-binding protein)/δ each alters the subcellular localization of DmPDE5/6. Thus prenylation constitutes a critical post-translational modification of DmPDE5/6 for membrane targeting. Co-immunoprecipitation and subcellular-fractionation experiments have shown that DmPDE5/6 interacts with DmPrBP/δ in Drosophila S2 cells. Transgenic lines allow targeted expression of tagged prenylation-deficient C1128S-DmPDE5/6 in Type I (principal) cells in Drosophila Malpighian tubules, an in vivo model for DmPDE5/6 function. In contrast with wild-type DmPDE5/6, which was exclusively associated with the apical membrane, the C1128S-DmPDE5/6 mutant form was located primarily in the cytosol, although some residual association occurred at the apical membrane. Despite the profound change in intracellular localization of C1128S-DmPDE5/6, active transport of cGMP is affected in the same way as it is by DmPDE5/6. This suggests that, in addition to prenylation and interaction with DmPrBP/δ, further functional membrane-targeting signals exist within DmPDE5/6.

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