Robert Singer

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..We conclude that aligning cell behavior in vivo with patterns of gene expression can lead to new insights into the microenvironment of carcinoma cells in the primary tumor and the molecular mechanisms behind cell behavior...

..This observation resolves a controversy, showing that the energetic requirements of nuclear mRNP trafficking are consistent with a diffusional model...

..This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured...

..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...

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..ZBP1 then associates with the RNA throughout the nuclear export and cytoplasmic localization process...

..These results reveal a novel function for an mRNA localization element and its binding protein in the regulation of dendritic morphology and synaptic growth via dendritic filopodia...

..The activity-dependent trafficking of ZBP1 and its dynamic movements within dendritic spines provide new evidence to implicate RNA binding proteins as regulators of mRNA transport to activated synapses in response to synaptic activity...

..We discuss how these new insights may lead to a better understanding of the molecular basis of the invasive behavior of tumor cells in vivo, which may result in new strategies for the diagnosis and treatment of metastasis...

..This is the first time that the activity of full-length N-WASP has been visualized in vivo, and this has lead to new insights for N-WASP function...

..Our results indicate that CT-RT allows microarray profiling of severely degraded RNA that could not be analyzed by previous methods...

..Using the nucleus as the substrate for parallel gene analysis, we provide a platform for the fusion of genomics and cell biology: "cellular genomics."..

..Additionally, localization of beta-actin mRNA and translation activity was enhanced at cell contacts and facilitated the formation of intercellular junctions...

..ZBP1 reexpression also inhibited metastasis from tumors. These experiments support the involvement in metastasis of the pathways identified in invasive cells, which are regulated by ZBP1...

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..These sequential events provide both temporal and spatial control over beta-actin mRNA translation, which is important for cell migration and neurite outgrowth...

..These data suggest that ZBP2, although predominantly a nuclear protein, has a role in the cytoplasmic localization of beta-actin mRNA...

..These data suggest that repression of ZBP1 deregulates its associated mRNAs, leading to the phenotypic changes of breast cancers...

..Individual mRNA molecules switched frequently among these movements, suggesting that mRNAs undergo continuous cycles of anchoring, diffusion, and active transport...

..The ZBP1 protein contains four KH RNA binding domains and a classic RBD RNA binding domain. It also contains a putative nuclear import and export sequence, suggesting a nuclear phase in this protein's function...

..beta-Catenin mediates the transcription of the ZBP1 gene, while ZBP1 promotes the stability of beta-catenin mRNA...

..Such observations provide motivation to investigate possible transport defects or inefficiency of SMN associated RNPs in motor neuron axons in SMA...

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..The approach has sufficient spatial resolution to be applied to the detection of chromosomal translocations or the identification of cancer cells...

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..Our systems approach, quantifying both polymerase and mRNA kinetics on a defined DNA template in vivo with high temporal resolution, opens new avenues for studying regulation of transcriptional processes in vivo...

..These findings highlight a mechanism for stimulus-induced dendritic mRNA transport and link its impairment in a mouse model of FXS to altered developmental morphologic plasticity...

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..These findings provide new insight into the molecular composition of distinct SMN multiprotein complexes in neurons and motivation to investigate deficiencies of localized RNPs in SMA...

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..Microarray analysis of the set1 mutants reveals genomic clustering of mis-expressed genes, suggesting a requirement for Set1 in the regulation of chromatin-mediated events at gene clusters...

..The RNA looping induced by ZBP1 binding provides a mechanism for specific recognition and may facilitate the assembly of post-transcriptional regulatory complexes by remodeling the bound transcript...

..This demonstrates yet another case where the mRNA's cytoplasmic fate is determined by the RNAPII complex...

..Taken together, these results implicate EF1alpha in the anchoring of beta-actin mRNA to the protrusion in crawling cells...

..Hence the physiological consequences of beta-actin mRNA delocalization affect the stability of the cell phenotype...

..We propose that Puf6p is a protein that functions in the translational control of ASH1 mRNA, and this translational inhibition is necessary before localization can proceed...

..We review advances in these methods with the goal of identifying the particular strengths and weaknesses of the various approaches in their ability to follow the movements of mRNAs from transcription sites to translation sites...

..These findings provide further evidence in support of a neuronal function for SMN and motivation to investigate for impaired assembly and/or localization of mRNP complexes as an underlying cause of SMA...

..This allows the visualization and quantification of gene expression and intracellular trafficking events of the studied molecules and the associated enzymatic processes in individual cells, in real time...

..This approach provides a novel ability to identify and characterize unique minor cell populations in the tumor that may exhibit relative resistance to chemotherapy...

..In support of this, Ash1p asymmetry could be rescued by slowing its translation...

..RNA localization during development is required for proper sorting of developmental determinants. Direct visualization of endogenous transcribed RNA is now possible and should provide new insights into how this process occurs...

..In the future, this technique is likely to have significant further impact on live-cell imaging and on medical diagnostics...

..These data directly demonstrate the existence of multiple expression modes used to modulate the transcriptome...

..These proteins might link nuclear events, such as RNA splicing, with the subsequent cytoplasmic localization of specific transcripts...

..For this reason, understanding of spatial relationships between mRNA and mRNA turnover factors should enrich our understanding of this process...

..This chapter presents an overview of general approaches for single particle tracking and some methodologies that are used for single mRNA detection...

..By contrast, an understanding of the dynamics of an individual gene's expression in real time is just beginning to emerge following recent technological developments...

..They provide a molecular explanation for the phosphorylation-dependent nuclear import of nuclear localization signal-containing cargo proteins...

..Thus, the close coupling between imaging and mathematical theory has established single-cell analysis as an essential branch of systems biology...

..We have described a novel approach for evaluating the effects of chemopreventive agents on the expression of a gene critical in colonic tumorigenesis...

..Different mechanisms have been identified that enable localized mRNAs to target different subcellular compartments, and recent advances in understanding these pathways is reviewed here...

..Identifying regulatory sites on transport factors as well as novel physiological functions for well-known translation factors has provided significant advances in how spatially controlled translation impacts cell function...

..This study represents the first direct visualization of transcriptional pulsing in eukaryotes. Discontinuity of transcription may allow greater flexibility in the gene-expression decisions of a cell...

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..These studies suggest that PRP1 associated with the secretory vesicle scaffold serves an important role in Ca2+-regulated exocytosis and cell invasion...

..Finally, a fluorescence microscopy approach on a single living cell is described to visualize the in vivo localization of mRNAs involved in mitochondria biogenesis...

..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...

..Our analyses indicate that NAF1 binds NAP57 and escorts it to the nascent H/ACA RNA and that GAR1 then replaces NAF1 to yield mature H/ACA RNPs in Cajal bodies and nucleoli...

..The requirement for the PTB-Raver1 interaction that we have characterized may serve to bring the additional repressor regions of both proteins into a configuration that allows them to synergistically effect exon skipping...

..This identifies single-cell gene expression patterns by probing multiple, unique nascent RNA transcripts and yields predictive quantitative gene expression signatures...

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..Using this system, we are able to correlate changes in chromatin structure with the progression of transcriptional activation allowing us to obtain a real-time integrative view of gene expression...

..This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor...

..P-body disassembly is delayed in the motor mutant, myo2-66, indicating that Myo2p may facilitate the release of mRNAs from the P-body...

..The results show that the motility and targeting of peripherin mRNPs, their translational control, and the assembly of an IF cytoskeletal system are linked together in a process we have termed dynamic cotranslation...

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..We also describe the efforts being made towards physiological approaches within the context of living organisms...

..In a recent issue of Molecular Cell, Gorski et al. (2008) demonstrate directly that polymerase assembly kinetics regulate Pol I transcription...

..These retain mRNAs in SGs and prevent premature decay in PBs. Hence, mRNA-binding proteins are essential for translational adaptation during cellular stress by modulating mRNA turnover...

..4. To multiplex the probes with different fluorochromes so that we will be able to detect multiple RNA species in the same cell. Ultimately, the approach can be formatted for use in living tissue. ..

..3. Intravitally imaging of this expression in a single cell within tumor tissue. ..

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..4. To multiplex the probes with different fluorochromes so that we will be able to detect multiple RNA species in the same cell. Ultimately, the approach can be formatted for use in living tissue. ..

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..The high speed microscope described in this proposal will fulfill this need for our research groups. ..

..The expression patterns obtained from tumor cells in pathological samples will allow a correlation of specific genes with disease progression. ..

..We will investigate the pathway using high-resolution microscopy combined with FRET to visualize the spatial distribution of these protein interactions in real time. ..

..Using these approaches, we will have the opportunity to identify where in the pathway the components of localization and regulation of protein expression operate, with high spatial and temporal resolution. ..

..at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1-2 Description, ..

..that controls how and where other proteins are made in the cell. We are particularly interested in proteins that cause cancer, oncogenes, and in determining how these proteins can induce cells to produce tumors. ..

..We are investigating how this protein works to localize protein synthesis in specific regions of the cell by using a super-sensitive microscope to watch the process occur in living cells and tissues from genetically engineered mice. ..

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..4. To multiplex the probes with different fluorochromes so that we will be able to detect multiple RNA species in the same cell. Ultimately, the approach can be formatted for use in living tissue. ..

..We are investigating how this protein works to localize protein synthesis in specific regions of the cell by using a super-sensitive microscope to watch the process occur in living cells and tissues from genetically engineered mice. ..

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..In the final specific aim, the mechanism of nuclear export will be assessed to determine points in the pathway where rate-limitations may exist. ..

..abstract_text> ..

..We will investigate the pathway using high-resolution microscopy combined with FRET to visualize the spatial distribution of these protein interactions in real time. ..

..Using these approaches, we will have the opportunity to identify where in the pathway the components of localization and regulation of protein expression operate, with high spatial and temporal resolution. ..