9 results on '"Singer, Robert H."'
Search Results
2. Fluorescence in situ hybridization: past, present and future.
- Author
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Levsky, Jeffrey M. and Singer, Robert H.
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FLUORESCENCE in situ hybridization , *IN situ hybridization , *FLUORESCENCE microscopy - Abstract
Discusses the development of fluorescence in situ hybridization (FISH), the assay of choice for localization of specific nucleic acids sequences in native context. Methodologies and modifications that have introduced to optimize the detection of DNA and RNA; Pervasiveness of the technique; Basic principles of FISH; Computer image processing; Low-noise hybridization probes.
- Published
- 2003
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3. Dynamic visualization of transcription and RNA subcellular localization in zebrafish.
- Author
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Campbell, Philip D., Chao, Jeffrey A., Singer, Robert H., and Marlow, Florence L.
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GENETIC transcription , *RNA , *ZEBRA danio , *BACTERIOPHAGES , *COAT proteins (Viruses) - Abstract
Live imaging of transcription and RNA dynamics has been successful in cultured cells and tissues of vertebrates but is challenging to accomplish in vivo. The zebrafish offers important advantages to study these processes - optical transparency during embryogenesis, genetic tractability and rapid development. Therefore, to study transcription and RNA dynamics in an intact vertebrate organism, we have adapted the MS2 RNA-labeling system to zebra fish. By using this binary system to co-express a fluorescent MS2 bacteriophage coat protein (MCP) and an RNA of interest tagged with multiple copies of the RNA hairpin MS2-binding site (MBS), live-cell imaging of RNA dynamics at single RNA molecule resolution has been achieved in other organisms. Here, using a Gateway compatible MS2 labeling system, we generated stable transgenic Zebra fish lines expressing MCP, validated the MBS-MCP interaction and applied the system to investigate zygotic genome activation (ZGA) and RNA localization in primordial germ cells (PGCs) in Zebra fish. Although cleavage stage cells are initially transcriptionally silent, we detect transcription of MS2-tagged transcripts driven by the βactin promoter at ~3-3.5 h post-fertilization, consistent with the previously reported ZGA. Furthermore, we show that MS2-tagged nanos3 3'UTR transcripts localize to PGCs, where they are diffusely cytoplasmic and within larger cytoplasmic accumulations reminiscent of those displayed by endogenous nanos3. These tools provide a new avenue for live-cell imaging of RNA molecules in an intact vertebrate. Together with new techniques for targeted genome editing, this system will be a valuable tool to tag and study the dynamics of endogenous RNAs during zebra fish developmental processes. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
4. Specific interaction of KIF11 with ZBP1 regulates the transport of b-actin mRNA and cell motility.
- Author
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Song, Tingting, Zheng, Yi, Wang, Yarong, Katz, Zachary, Liu, Xin, Chen, Shaoying, Singer, Robert H., and Gu, Wei
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KINESIN , *Z-DNA , *ACTIN , *MESSENGER RNA , *CELL motility , *DNA-binding proteins - Abstract
ZBP1-modulated localization of β-actin mRNA enables a cell to establish polarity and structural asymmetry. Although the mechanism of β-actin mRNA localization has been well established, the underlying mechanism of how a specific molecular motor contributes to the transport of the ZBP1 (also known as IGF2BP1) complex in non-neuronal cells remains elusive. In this study, we report the isolation and identification of KIF11, a microtubule motor, which physically interacts with ZBP1 and is a component of β-actin messenger ribonucleoprotein particles (mRNPs). We show that KIF11 colocalizes with the β-actin mRNA, and the ability of KIF11 to transport β-actin mRNA is dependent on ZBP1. We characterize the corresponding regions of ZBP1 and KIF11 that mediate the interaction of the two proteins in vitro and in vivo. Disruption of the in vivo interaction of KIF11 with ZBP1 delocalizes β-actin mRNA and affects cell migration. Our study reveals a molecular mechanism by which a particular microtubule motor mediates the transport of an mRNP through direct interaction with an mRNA-binding protein. [ABSTRACT FROM AUTHOR]
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- 2015
- Full Text
- View/download PDF
5. Regulation of local expression of cell adhesion and motility-related mRNAs in breast cancer cells by IMP1/ ZBP1.
- Author
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Wei Gu, Katz, Zachary, Bin Wu, Hye Yoon Park, Deling Li, Stanley Lin, Wells, Amber L., and Singer, Robert H.
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CELL adhesion , *CELLULAR mechanics , *JUNCTIONAL complexes (Epithelium) , *MEMBRANE fusion , *CELL communication - Abstract
Metastasis involves tumor cell detachment from the primary tumor, and acquisition of migratory and invasive capabilities. These capabilities are mediated by multiple events, including loss of cell-cell contact, an increase in focal adhesion turnover and failure to maintain a normal cell polarity. We have previously reported that silencing of the expression of the zipcode-binding protein IMP1/ZBP1 in breast tumor patients is associated with metastasis. IMP1/ZBP1 selectively binds to a group of mRNAs that encode important mediators for cell adhesion and motility. Here, we show that in both T47D and MDA231 human breast carcinoma cells IMP1/ZBP1 functions to suppress cell invasion. Binding of ZBP1 to the mRNAs encoding E-cadherin, b-actin, a-actinin and the Arp2/3 complex facilitates localization of the mRNAs, which stabilizes cell-cell connections and focal adhesions. Our studies suggest a novel mechanism through which IMP1/ZBP1 simultaneously regulates the local expression of many cell-motility-related mRNAs to maintain cell adherence and polarity, decrease focal adhesion turnover and maintain a persistent and directional motility. [ABSTRACT FROM AUTHOR]
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- 2012
- Full Text
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6. Blocking β-catenin binding to the ZBP1 promoter represses ZBP1 expression, leading to increased proliferation and migration of metastatic breast-cancer cells.
- Author
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Wei Gu, Feng Pan, and Singer, Robert H.
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BREAST cancer , *CANCER cells , *CELL tumors , *CELL proliferation , *EMBRYOLOGY , *CELL lines , *MESSENGER RNA - Abstract
ZBP1 (zipcode-binding protein 1, also known as IMP-1) is an mRNA regulator, functioning in mRNA localization, stability and translational control. ZBP1 is actively expressed during embryogenesis and tumorigenesis, but its expression is repressed in metastatic breast-cancer cell lines and tumors. In this article, we show that downregulation of ZBP1 expression results from its promoter methylation, an epigenetic process that remodels the chromatin structure and frequently represses gene activity. Demethylation of the ZBP1 promoter in metastatic cells reactivated ZBP1 expression, owing to restoration of the interaction of the ZBP1 promoter with β-catenin. Loss of ZBP1 function not only increased growth ability of metastatic cells, but also promoted cell migration. We identified a number of mRNAs that were selectively associated with ZBP1 in breast-cancer cells. Many of these are involved in cell motility and in cell-cycle regulation, and displayed altered expression patterns in the absence of ZBP1. These data suggest that repression of ZBP1 deregulates its associated mRNAs, leading to the phenotypic changes of breast cancers. [ABSTRACT FROM AUTHOR]
- Published
- 2009
7. Localization of all seven messenger RNAs for the actin-polymerization nucleator Arp2/3 complex in the protrusions of fibroblasts.
- Author
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Mingle, Lisa A., Okuhama, Nataly N., Jian Shi, Singer, Robert H., Condeelis, John, and Gang Liu
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MESSENGER RNA , *PROTEINS , *ACTIN , *POLYMERIZATION , *FIBROBLASTS , *CELL migration - Abstract
The actin-related protein 2⁄3 (Arp2⁄3) complex is a crucial actin polymerization nucleator and is localized to the leading protrusions of migrating cells. However, how the multiprotein complex is targeted to the protrusions remains unknown. Here, we demonstrate that mRNAs for the seven subunits of the Arp2⁄3 complex are localized to the protrusions in fibroblasts, supporting a hypothesis that the Arp2⁄3 complex is targeted to its site of function by mRNA localization. Depletion of serum from culture medium inhibits Arp2⁄3-complex mRNA localization to the protrusion, whereas serum stimulation leads to significant mRNA localization within 30 minutes. The effect of serum suggests that Arp2⁄3-complex mRNA localization is a cellular response to extracellular stimuli. The localization of the Arp2⁄3 complex mRNAs is dependent on both actin filaments and microtubules, because disruption of either cytoskeletal system (with cytochalasin D and colchicine, respectively) inhibited the localization of all seven subunit mRNAs. In addition, myosin inhibitors significantly inhibit Arp2 mRNA localization in chicken embryo fibroblasts, suggesting a myosin motor dependent mechanism for Arp2⁄3-complex mRNA localization. [ABSTRACT FROM AUTHOR]
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- 2005
- Full Text
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8. The life of an mRNA in space and time.
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Ben-Ari, Ya'ara, Brody, Yehuda, Kinor, Noa, Mor, Amir, Tsukamoto, Toshiro, Spector, David L., Singer, Robert H., and Yaron Shav-Tal
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CYTOPLASM , *ACTIN , *CYTOSKELETON , *EUKARYOTIC cells , *RNA polymerases , *MESSENGER RNA - Abstract
Nuclear transcribed genes produce mRNA transcripts destined to travel from the site of transcription to the cytoplasm for protein translation. Certain transcripts can be further localized to specific cytoplasmic regions. We examined the life cycle of a transcribed β-actin mRNA throughout gene expression and localization, in a cell system that allows the in vivo detection of the gene locus, the transcribed mRNAs and the cytoplasmic β-actin protein that integrates into the actin cytoskeleton. Quantification showed that RNA polymerase II elongation progressed at a rate of 3.3 kb/minute and that transactivator binding to the promoter was transient (40 seconds), and demonstrated the unique spatial structure of the coding and non-coding regions of the integrated gene within the transcription site. The rates of gene induction were measured during interphase and after mitosis, demonstrating that daughter cells were not synchronized in respect to transcription initiation of the studied gene. Comparison of the spatial and temporal kinetics of nucleoplasmic and cytoplasmic mRNA transport showed that the ?-actin-localization response initiates from the existing cytoplasmic mRNA pool and not from the newly synthesized transcripts arising after gene induction. It was also demonstrated that mechanisms of random movement were predominant in mediating the efficient translocation of mRNA in the eukaryotic cell. [ABSTRACT FROM AUTHOR]
- Published
- 2010
9. ZBP1 enhances cell polarity and reduces chemotaxis.
- Author
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Lapidus, Kyle, Wyckoff, Jeffrey, mouneimne, Ghassan, Lorenz, Mike, Soon, Lillian, Condeelis, John S., and Singer, Robert H.
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CARRIER proteins , *CELL polarity , *CHEMOTAXIS , *IMAGING systems , *CELL motility - Abstract
The interaction of β-actin mRNA with zipcode-binding protein 1 (ZBP1) is necessary for its localization to the lamellipod of fibroblasts and plays a crucial role in cell polarity and motility. Recently, we have shown that low ZBP1 levels correlate with tumor-cell invasion and metastasis. In order to establish a cause and effect relationship, we expressed ZBP1 in a metastatic rat mammary adenocarcinoma cell line (MTLn3) that has low endogenous ZBP1 levels and delocalized β-actin mRNA. This leads to localization of β-actin mRNA, and eventually reduces the chemotactic potential of the cells as well as their ability to move and orient towards vessels in tumors. To determine how ZBP1 leads to these two apparently contradictory aspects of cell behavior -- increased cell motility but decreased chemotaxis -- we examined cell motility in detail, both in cell culture and in vivo in tumors. We found that ZBP1 expression resulted in tumor cells with a stable polarized phenotype, and reduced their ability to move in response to a gradient in culture. To connect these results on cultured cells to the reduced metastatic ability of these cells, we used multiphoton imaging in vivo to examine tumor cell behavior in primary tumors. We found that ZBP1 expression actually reduced tumor cell motility and chemotaxis, presumably mediating their decreased metastatic potential by reducing their ability to respond to signals necessary for invasion. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
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