Knock down analysis reveals critical phases for specific oskar noncoding RNA functions during Drosophila oogenesis

Abstract The oskar transcript, acting as a noncoding RNA, contributes to a diverse set of pathways in the Drosophila ovary, including karyosome formation, positioning of the microtubule organizing center, integrity of certain ribonucleoprotein particles, control of nurse cell divisions, restriction of several proteins to the germline, and progression through oogenesis. How oskar mRNA acts to perform these functions remains unclear. Here we use a knock down approach to identify the critical phases when oskar is required for three of these functions. The existing transgenic shRNA for removal of oskar mRNA in the germline targets a sequence overlapping a regulatory site bound by Bruno1 protein to confer translational repression, and was ineffective during oogenesis. Novel transgenic shRNAs targeting other sites were effective at strongly reducing oskar mRNA levels and reproducing phenotypes associated with the absence of the mRNA. Using GAL4 drivers active at different developmental stages of oogenesis, we found that early loss of oskar mRNA reproduced defects in karyosome formation and positioning of the microtubule organizing center, but not arrest of oogenesis. Loss of oskar mRNA at later stages was required to prevent progression through oogenesis. The noncoding function of oskar mRNA is thus required for more than a single event.

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Three-Dimensional reconstruction of isolated centrosomes by automated electron tomography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100169286 ◽

1994 ◽

Vol 52 ◽

pp. 310-311

Author(s):

M.B. Braunfeld ◽

M. Moritz ◽

B.M. Alberts ◽

J.W. Sedat ◽

D.A. Agard

Keyword(s):

Electron Tomography ◽

Three Dimensional ◽

Vital Role ◽

Complex Nature ◽

Animal Cells ◽

Microtubule Organizing Center ◽

Nucleation Sites ◽

Dimensional Reconstruction ◽

Organizing Center ◽

Resolution Model

In animal cells, the centrosome functions as the primary microtubule organizing center (MTOC). As such the centrosome plays a vital role in determining a cell's shape, migration, and perhaps most importantly, its division. Despite the obvious importance of this organelle little is known about centrosomal regulation, duplication, or how it nucleates microtubules. Furthermore, no high resolution model for centrosomal structure exists.We have used automated electron tomography, and reconstruction techniques in an attempt to better understand the complex nature of the centrosome. Additionally we hope to identify nucleation sites for microtubule growth.Centrosomes were isolated from early Drosophila embryos. Briefly, after large organelles and debris from homogenized embryos were pelleted, the resulting supernatant was separated on a sucrose velocity gradient. Fractions were collected and assayed for centrosome-mediated microtubule -nucleating activity by incubating with fluorescently-labeled tubulin subunits. The resulting microtubule asters were then spun onto coverslips and viewed by fluorescence microscopy.

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Faculty Opinions recommendation of Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1160734.621851 ◽

2009 ◽

Author(s):

Tsutomu Takeuchi ◽

Hideto Kameda

Keyword(s):

T Cells ◽

Microtubule Organizing Center ◽

Organizing Center

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Faculty Opinions recommendation of SPD-2/CEP192 and CDK Are Limiting for Microtubule-Organizing Center Function at the Centrosome.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725598359.793535884 ◽

2017 ◽

Author(s):

Andrew Fry

Keyword(s):

Microtubule Organizing Center ◽

Organizing Center

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Genetic interactions between CDC31 and KAR1, two genes required for duplication of the microtubule organizing center in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/137.2.407 ◽

1994 ◽

Vol 137 (2) ◽

pp. 407-422 ◽

Cited By ~ 8

Author(s):

E A Vallen ◽

W Ho ◽

M Winey ◽

M D Rose

Keyword(s):

Copy Number ◽

Gene Dosage ◽

Spindle Pole Body ◽

Direct Interaction ◽

Spindle Pole ◽

High Copy Number ◽

Temperature Sensitive ◽

Microtubule Organizing Center ◽

Recessive Mutations ◽

Organizing Center

Abstract KAR1 encodes an essential component of the yeast spindle pole body (SPB) that is required for karyogamy and SPB duplication. A temperature-sensitive mutation, kar1-delta 17, mapped to a region required for SPB duplication and for localization to the SPB. To identify interacting SPB proteins, we isolated 13 dominant mutations and 3 high copy number plasmids that suppressed the temperature sensitivity of kar1-delta 17. Eleven extragenic suppressor mutations mapped to two linkage groups, DSK1 and DSK2. The extragenic suppressors were specific for SPB duplication and did not suppress karyogamy-defective alleles. The major class, DSK1, consisted of mutations in CDC31. CDC31 is required for SPB duplication and encodes a calmodulin-like protein that is most closely related to caltractin/centrin, a protein associated with the Chlamydomonas basal body. The high copy number suppressor plasmids contained the wild-type CDC31 gene. One CDC31 suppressor allele conferred a temperature-sensitive defect in SPB duplication, which was counter-suppressed by recessive mutations in KAR1. In spite of the evidence for a direct interaction, the strongest CDC31 alleles, as well as both DSK2 alleles, suppressed a complete deletion of KAR1. However, the CDC31 alleles also made the cell supersensitive to KAR1 gene dosage, arguing against a simple bypass mechanism of suppression. We propose a model in which Kar1p helps localize Cdc31p to the SPB and that Cdc31p then initiates SPB duplication via interaction with a downstream effector.

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Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements

The Journal of Cell Biology ◽

10.1083/jcb.83.3.623 ◽

1979 ◽

Vol 83 (3) ◽

pp. 623-632 ◽

Cited By ~ 28

Author(s):

M Schliwa ◽

U Euteneuer ◽

W Herzog ◽

K Weber

Keyword(s):

Complete Removal ◽

Cell System ◽

The State ◽

Microtubule Assembly ◽

Microtubule Organizing Center ◽

Functional Changes ◽

Organizing Center ◽

Pigment Distribution ◽

And Function ◽

In Cells

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.

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Murine Cytomegalovirus Capsid Assembly Is Dependent on US22 Family Gene M140 in Infected Macrophages

Journal of Virology ◽

10.1128/jvi.00325-09 ◽

2009 ◽

Vol 83 (15) ◽

pp. 7449-7456 ◽

Cited By ~ 9

Author(s):

Laura K. Hanson ◽

Jacquelyn S. Slater ◽

Victoria J. Cavanaugh ◽

William W. Newcomb ◽

Lisa L. Bolin ◽

...

Keyword(s):

Dna Cleavage ◽

Early Gene ◽

Murine Cytomegalovirus ◽

Genome Replication ◽

Microtubule Organizing Center ◽

Viral Dna ◽

Dnase Treatment ◽

Organizing Center ◽

Significant Impairment ◽

The Stability

ABSTRACT Macrophages are an important target cell for infection with cytomegalovirus (CMV). A number of viral genes that either are expressed specifically in this cell type or function to optimize CMV replication in this host cell have now been identified. Among these is the murine CMV (MCMV) US22 gene family member M140, a nonessential early gene whose deletion (RVΔ140) leads to significant impairment in virus replication in differentiated macrophages. We have now determined that the defect in replication is at the stage of viral DNA encapsidation. Although the rate of RVΔ140 genome replication and extent of DNA cleavage were comparable to those for revertant virus, deletion of M140 resulted in a significant reduction in the number of viral capsids in the nucleus, and the viral DNA remained sensitive to DNase treatment. These data are indicative of incomplete virion assembly. Steady-state levels of both the major capsid protein (M86) and tegument protein M25 were reduced in the absence of the M140 protein (pM140). This effect may be related to the localization of pM140 to an aggresome-like, microtubule organizing center-associated structure that is known to target misfolded and overexpressed proteins for degradation. It appears, therefore, that pM140 indirectly influences MCMV capsid formation in differentiated macrophages by regulating the stability of viral structural proteins.

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Peroxynitrite Deteriorates Oocyte Quality through Disassembly of Microtubule Organizing Center

Free Radical Biology and Medicine ◽

10.1016/j.freeradbiomed.2015.10.300 ◽

2015 ◽

Vol 87 ◽

pp. S114

Author(s):

Sana Khan ◽

Faten Shaeib ◽

Mili Thakur ◽

Roohi Jeelani ◽

Husam M Abu-Soud

Keyword(s):

Oocyte Quality ◽

Microtubule Organizing Center ◽

Organizing Center

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Protease-Dependent Uncoating of a Complex Retrovirus

Journal of Virology ◽

10.1128/jvi.79.14.9244-9253.2005 ◽

2005 ◽

Vol 79 (14) ◽

pp. 9244-9253 ◽

Cited By ~ 26

Author(s):

Jacqueline Lehmann-Che ◽

Marie-Lou Giron ◽

Olivier Delelis ◽

Martin Löchelt ◽

Patricia Bittoun ◽

...

Keyword(s):

Nuclear Import ◽

Viral Genome ◽

Productive Infection ◽

Wild Type Virus ◽

Target Cells ◽

Wild Type ◽

Microtubule Organizing Center ◽

Viral Protease ◽

Virus Life Cycle ◽

Organizing Center

ABSTRACT Although retrovirus egress and budding have been partly unraveled, little is known about early stages of the replication cycle. In particular, retroviral uncoating, a process during which incoming retroviral cores are altered to allow the integration of the viral genome into host chromosomes, is poorly understood. To get insights into these early events of the retroviral cycle, we have used foamy complex retroviruses as a model. In this report, we show that a protease-defective foamy retrovirus is noninfectious, although it is still able to bud and enter target cells efficiently. Similarly, a retrovirus mutated in an essential viral protease-dependent cleavage site in the central part of Gag is noninfectious. Following entry, wild-type and mutant retroviruses are able to traffic along microtubules towards the microtubule-organizing center (MTOC). However, whereas nuclear import of Gag and of the viral genome was observed for the wild-type virus as early as 8 hours postinfection, incoming capsids and genome from mutant viruses remained at the MTOC. Interestingly, a specific viral protease-dependent Gag cleavage product was detected only for the wild-type retrovirus early after infection, demonstrating that cleavage of Gag by the viral protease at this stage of the virus life cycle is absolutely required for productive infection, an unprecedented observation among retroviruses.

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CEP110 and ninein are located in a specific domain of the centrosome associated with centrosome maturation

Journal of Cell Science ◽

10.1242/jcs.115.9.1825 ◽

2002 ◽

Vol 115 (9) ◽

pp. 1825-1835 ◽

Cited By ~ 1

Author(s):

Young Y. Ou ◽

Gary J. Mack ◽

Meifeng Zhang ◽

Jerome B. Rattner

Keyword(s):

Cell Cycle ◽

Protein Interactions ◽

Protein Function ◽

Similar Distribution ◽

Microtubule Organizing Center ◽

Specific Domain ◽

The Reformation ◽

Mother And Daughter ◽

Organizing Center ◽

Pericentriolar Material

The mammalian centrosome consists of a pair of centrioles surrounded by pericentriolar material (PCM). The architecture and composition of the centrosome, especially the PCM, changes during the cell cycle. Recently, a subset of PCM proteins have been shown to be arranged in a tubular conformation with an open and a closed end within the centrosome. The presence of such a specific configuration can be used as a landmark for mapping proteins in both a spatial and a temporal fashion. Such mapping studies can provide information about centrosome organization, protein dynamics,protein-protein interactions as well as protein function. In this study, the centrosomal proteins CEP110 and ninein were mapped in relationship to the tubular configuration. Both proteins were found to exhibit a similar distribution pattern. In the mother centrosome, they were found at both ends of the centrosome tube, including the site of centrosome duplication. However,in the daughter centrosome they were present only at the closed end. At the closed end of the mother and daughter centrosome tube, both CEP110 and ninein co-localized with the centriolar protein CEP250/c-Nap1, which confirms ninein's centriole association and places CEP110 in association with this structure. Importantly, the appearance of CEP110 and ninein at the open end of the daughter centrosome occurred during the telophase-G1 transition of the next cell cycle, concomitant with the maturation of the daughter centrosome into a mother centrosome. Microinjection of antibodies against either CEP110 or ninein into metaphase HeLa cells disrupted the reformation of the tubular conformation of proteins within the centrosome following cell division and consequently led to dispersal of centrosomal material throughout the cytosol. Further, microinjection of antibodies to either CEP110 or ninein into metaphase PtK2 cells not only disrupted the tubular configuration within the centrosome but also affected the centrosome's ability to function as a microtubule organizing center (MTOC). This MTOC function was also disrupted when the antibodies were injected into postmitotic cells. Taken together, our results indicate that: (1) a population of CEP110 and ninein is located in a specific domain within the centrosome, which corresponds to the open end of the centrosome tube and is the site of protein addition associated with maturation of a daughter centrosome into a mother centrosome; and (2) the addition of CEP110 and ninein are essential for the reformation of specific aspects of the interphase centrosome architecture following mitosis as well as being required for the centrosome to function as a MTOC.

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