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Robert H Singer

Publications and source records attributed to Robert H Singer.

At least 37 records · Page 2Linked to original sources
PubMed2005

How and why does beta-actin mRNA target?

beta-Actin mRNA is localized near the leading edge in several cell types where actin polymerization is actively promoting forward protrusion. The localization of the beta-actin mRNA near the leading edge is facilitated by a short sequence in the 3'UTR (untranslated region), the 'zipcode'. Localization of the mRNA at this region is important physiologically. Treatment of chicken embryo fibroblasts with antisense oligonucleotides complementary to the localization sequence (zipcode) in the 3'UTR leads to delocalization of beta-actin mRNA, alteration of cell phenotype and a decrease in cell motility. The dynamic image analysis system (DIAS) used to quantify movement of cells in the presence of sense and antisense oligonucleotides to the zipcode showed that net pathlength and average speed of antisense-treated cells were significantly lower than in sense-treated cells. This suggests that a decrease in persistence of direction of movement and not in velocity results from treatment of cells with zipcode-directed antisense oligonucleotides. We postulate that delocalization of beta-actin mRNA results in delocalization of nucleation sites and beta-actin protein from the leading edge followed by loss of cell polarity and directional movement. Hence the physiological consequences of beta-actin mRNA delocalization affect the stability of the cell phenotype.

3' Untranslated Regions↗
PubMed2005

The great escape: when cancer cells hijack the genes for chemotaxis and motility.

The combined use of the new technologies of multiphoton-based intravital imaging, the chemotaxis-mediated collection of invasive cells, and high sensitivity expression profiling has allowed the correlation of the behavior of invasive tumor cells in vivo with their gene expression patterns. New insights have resulted including a gene expression signature for invasive cells and the tumor microenvironment invasion model. This model proposes that tumor invasion and metastasis can be studied as a problem resembling normal morphogenesis. 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.

Animals↗
PubMed2004

Identification and testing of a gene expression signature of invasive carcinoma cells within primary mammary tumors.

We subjected cells collected using an in vivo invasion assay to cDNA microarray analysis to identify the gene expression profile of invasive carcinoma cells in primary mammary tumors. Expression of genes involved in cell division, survival, and cell motility were most dramatically changed in invasive cells indicating a population that is neither dividing nor apoptotic but intensely motile. In particular, the genes coding for the minimum motility machine that regulates beta-actin polymerization at the leading edge and, therefore, the motility and chemotaxis of carcinoma cells, were dramatically up-regulated. However, ZBP1, which restricts the localization of beta-actin, the substrate for the minimum motility machine, was down-regulated. This pattern of expression implicated ZBP1 as a suppressor of invasion. Reexpression of ZBP1 in metastatic cells with otherwise low levels of ZBP1 reestablished normal patterns of beta-actin mRNA targeting and suppressed chemotaxis and invasion in primary tumors. 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.

Actins↗
PubMed2004

She2p is a novel RNA binding protein with a basic helical hairpin motif.

Selective transport of mRNAs in ribonucleoprotein particles (mRNP) ensures asymmetric distribution of information within and among eukaryotic cells. Actin-dependent transport of ASH1 mRNA in yeast represents one of the best-characterized examples of mRNP translocation. Formation of the ASH1 mRNP requires recognition of zip code elements by the RNA binding protein She2p. We determined the X-ray structure of She2p at 1.95 A resolution. She2p is a member of a previously unknown class of nucleic acid binding proteins, composed of a single globular domain with a five alpha helix bundle that forms a symmetric homodimer. After demonstrating potent, dimer-dependent RNA binding in vitro, we mapped the RNA binding surface of She2p to a basic helical hairpin in vitro and in vivo and present a mechanism for mRNA-dependent initiation of ASH1 mRNP complex assembly.

Amino Acid Sequence↗
PubMed2004

Dynamics of single mRNPs in nuclei of living cells.

Understanding gene expression requires the ability to follow the fate of individual molecules. Here we use a cellular system for monitoring messenger RNA (mRNA)expression to characterize the movement in real time of single mRNA-protein complexes (mRNPs) in the nucleus of living mammalian cells. This mobility was not directed but was governed by simple diffusion. Some mRNPs were partially corralled throughout the nonhomogenous nuclear environment, but no accumulation at subnuclear domains was observed. Following energy deprivation, energy-independent motion of mRNPs was observed in a highly ATP-dependent nuclear environment; movements were constrained to chromatin-poor domains and excluded by newly formed chromatin barriers. This observation resolves a controversy, showing that the energetic requirements of nuclear mRNP trafficking are consistent with a diffusional model.

Active Transport, Cell Nucleus↗
PubMed2004

A new yeast PUF family protein, Puf6p, represses ASH1 mRNA translation and is required for its localization.

In yeast Saccharomyces cerevisiae, Ash1p, a protein determinant for mating-type switching, is segregated within the daughter cell nucleus to establish asymmetry of HO expression. The accumulation of Ash1p results from ASH1 mRNA that is sorted as a ribonucleoprotein particle (mRNP or locasome) to the distal tip of the bud where translation occurs. To study the mechanism regulating ASH1 mRNA translation, we isolated the ASH1 locasome and characterized the associated proteins by MALDI-TOF. One of these proteins was Puf6p, a new member of the PUF family of highly conserved RNA-binding proteins such as Pumilio in Drosophila, responsible for translational repression, usually to effect asymmetric expression. Puf6p-bound PUF consensus sequences in the 3'UTR of ASH1 mRNA and repressed the translation of ASH1 mRNA both in vivo and in vitro. In the puf6 Delta strain, asymmetric localization of both Ash1p and ASH1 mRNA were significantly reduced. 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.

3' Untranslated Regions↗
PubMed2004

Imaging sites of N-wasp activity in lamellipodia and invadopodia of carcinoma cells.

Cell migration is crucial for many biological and pathological processes such as chemotaxis of immune cells, fibroblast migration during wound healing, and tumor cell invasion and metastasis. Cells migrate forward by extending membrane protrusions. The formation of these protrusions is driven by assembly of actin filaments at the leading edge. Neural Wiskott-Aldrich syndrome protein (N-WASP), a ubiquitous member of the WASP family, induces actin polymerization by activating Arp2/3 complex and is thought to regulate the formation of membrane protrusions. However, it is totally unclear how N-WASP activity is spatially and temporally regulated inside migrating cells. To detect and image sites of N-WASP activity during cell motility and invasion in carcinoma cells, we designed an N-WASP fluorescence resonance energy transfer (FRET) biosensor that distinguishes between the active and inactive conformations and mimics the function of endogenous N-WASP. Our data show that N-WASP is involved in lamellipodia extension, where it is activated at the leading edge, as well as in invadopodia formation of invasive carcinoma cells, where it is activated at the base. 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.

Animals↗
PubMed2004

From silencing to gene expression: real-time analysis in single cells.

We have developed an inducible system to visualize gene expression at the levels of DNA, RNA and protein in living cells. The system is composed of a 200 copy transgene array integrated into a euchromatic region of chromosome 1 in human U2OS cells. The condensed array is heterochromatic as it is associated with HP1, histone H3 methylated at lysine 9, and several histone methyltransferases. Upon transcriptional induction, HP1alpha is depleted from the locus and the histone variant H3.3 is deposited suggesting that histone exchange is a mechanism through which heterochromatin is transformed into a transcriptionally active state. RNA levels at the transcription site increase immediately after the induction of transcription and the rate of synthesis slows over time. 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.

Animals↗
PubMed2004

Measurement of barbed ends, actin polymerization, and motility in live carcinoma cells after growth factor stimulation.

Motility is associated with the ability to extend F-actin-rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live-cell time-lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP-beta-actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F-actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F-actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti-cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured.

Actin Depolymerizing Factors↗
PubMed2004

Imaging gene expression in single living cells.

Technical advances in the field of live-cell imaging have introduced the cell biologist to a new, dynamic, subcellular world. The static world of molecules in fixed cells has now been extended to the time dimension. 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.

Animals↗
PubMed2003

The PTB interacting protein raver1 regulates alpha-tropomyosin alternative splicing.

Regulated switching of the mutually exclusive exons 2 and 3 of alpha-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. Here we show that raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induced very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor.

Alternative Splicing↗
PubMed2003

Localization of a beta-actin messenger ribonucleoprotein complex with zipcode-binding protein modulates the density of dendritic filopodia and filopodial synapses.

The dendritic transport and local translation of mRNA may be an essential mechanism to regulate synaptic growth and plasticity. We investigated the molecular mechanism and function of beta-actin mRNA localization in dendrites of cultured hippocampal neurons. Previous studies have shown that beta-actin mRNA localization to the leading edge of fibroblasts or the growth cones of developing neurites involved a specific interaction between a zipcode sequence in the 3' untranslated region and the mRNA-binding protein zipcode-binding protein-1 (ZBP1). Here, we show that ZBP1 is required for the localization of beta-actin mRNA to dendrites. Knock-down of ZBP1 using morpholino antisense oligonucleotides reduced dendritic levels of ZBP1 and beta-actin mRNA and impaired growth of dendritic filopodia in response to BDNF treatment. Transfection of an enhanced green fluorescent protein (EGFP)-beta-actin construct, which contained the zipcode, increased the density of dendritic filopodia and filopodial synapses. Transfection of an EGFP construct, also with the zipcode, resulted in recruitment of endogenous ZBP1 and beta-actin mRNA into dendrites and similarly increased the density of dendritic filopodia. However, the beta-actin zipcode did not affect filopodial length or the density of mature spines. 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.

Actins↗
PubMed2003

RNA localization: visualization in real-time.

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.

Drosophila Proteins↗
PubMed2003

Active transport of the survival motor neuron protein and the role of exon-7 in cytoplasmic localization.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by deletion and/or mutation of the survival motor neuron protein Gene (SMN1) that results in the expression of a truncated protein lacking the C terminal exon-7. Whereas SMN has been shown to be an important component of diverse ribonucleoprotein (RNP) complexes, its function in neurons is unknown. We hypothesize that the active transport of SMN may be important for neurite outgrowth and that disruption of exon-7 could impair its normal intracellular trafficking. SMN was localized in granules that were associated with cytoskeletal filament systems and distributed throughout neurites and growth cones. Live cell imaging of enhanced green fluorescent protein (EGFP)-SMN granules revealed rapid, bidirectional and cytoskeletal-dependent movements. Exon-7 was necessary for localization of SMN into the cytoplasm but was not sufficient for granule formation and transport. A cytoplasmic targeting signal within exon-7 was identified that could completely redistribute the nuclear protein D-box binding factor 1 into the cytoplasm. Neurons transfected with SMN lacking exon-7 had significantly shorter neurites, a defect that could be rescued by redirecting the exon-7 deletion mutant into neurites by a targeting sequence from growth-associated protein-43. These findings provide the first demonstration of cytoskeletal-based active transport of SMN in neuronal processes and the function of exon-7 in cytoplasmic localization. Such observations provide motivation to investigate possible transport defects or inefficiency of SMN associated RNPs in motor neuron axons in SMA.

Actin Cytoskeleton↗
PubMed2003

Fluorescence in situ hybridization: past, present and future.

Fluorescence in situ hybridization (FISH), the assay of choice for localization of specific nucleic acids sequences in native context, is a 20-year-old technology that has developed continuously. Over its maturation, various methodologies and modifications have been introduced to optimize the detection of DNA and RNA. The pervasiveness of this technique is largely because of its wide variety of applications and the relative ease of implementation and performance of in situ studies. Although the basic principles of FISH have remained unchanged, high-sensitivity detection, simultaneous assay of multiple species, and automated data collection and analysis have advanced the field significantly. The introduction of FISH surpassed previously available technology to become a foremost biological assay. Key methodological advances have allowed facile preparation of low-noise hybridization probes, and technological breakthroughs now permit multi-target visualization and quantitative analysis - both factors that have made FISH accessible to all and applicable to any investigation of nucleic acids. In the future, this technique is likely to have significant further impact on live-cell imaging and on medical diagnostics.

Animals↗
PubMed2003

Activity-dependent trafficking and dynamic localization of zipcode binding protein 1 and beta-actin mRNA in dendrites and spines of hippocampal neurons.

RNA binding proteins may be important mediators of the activity-dependent transport of mRNAs to dendritic spines of activated synapses. We used fluorescence microscopy and digital imaging techniques applied to both fixed and live cultured hippocampal neurons to visualize the localization of the mRNA binding protein, zipcode binding protein 1 (ZBP1), and its dynamic movements in response to KCl-induced depolarization at high spatial and temporal resolution. With the use of immunofluorescence, image deconvolution, and three-dimensional reconstruction, ZBP1 was localized in the form of granules that were distributed in dendrites, spines, and subsynaptic sites. KCl depolarization increased the dendritic localization of ZBP1 that was not attributed to an increase in ZBP1 expression. Live cell imaging of single cells before and after perfusion of KCl revealed the rapid and directed efflux of ZBP1 granules from the cell body into dendrites in a proximo-distal gradient. High-speed imaging of enhanced green fluorescence protein-ZBP1 granules revealed rapid anterograde and retrograde movements in dendrites as well as dynamic movements in dendritic spines. A population of ZBP1 granules colocalized with beta-actin mRNA, and their spatial association in dendrites was increased by KCl depolarization. The NMDA receptor antagonist AP-5 impaired the dendritic localization of ZBP1 and beta-actin mRNA and inhibited the KCl-induced transport of ZBP1. 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.

Actins↗
PubMed2003

Real-time visualization of ZBP1 association with beta-actin mRNA during transcription and localization.

BACKGROUND: mRNA localization in somatic cells is an important mechanism for gene expression regulation. In fibroblasts, the protein ZBP1 associates with the sequence that localizes beta-actin mRNA to the leading edge of fibroblasts, augmenting motility. beta-actin mRNA localizes in a cytoskeleton-dependent manner, depending on intact actin and myosin ATP-hydrolysis, and is largely bound to the actin cytoskeleton. 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. RESULTS: Using high-speed imaging, we show here the targeting of this RNA binding protein to beta-actin pre-mRNA transcripts in the nuclei of living cells and measure the residence time of the RNA-protein complex before it leaves the transcription site. Then, the RNA-protein particle is exported to the cytoplasm, where it localizes at velocities of 0.6 microm/s by using actin filaments and/or microtubules. This RNA-ZBP1 complex is required for cytoplasmic localization in fibroblasts; mislocalizing the protein also mislocalizes the RNA, and expressing the protein in a ZBP1-deficient cell line induces beta-actin mRNA localization. CONCLUSIONS: This work demonstrates that the RNA-protein association, essential for cytoplasmic localization, begins as soon as the RNA is transcribed. The ZBP1 then forms a ribonucleoprotein particle and moves in a myosin-dependent fashion by using the cytoskeleton for directional transport.

Actins↗