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Robert H Singer

Publications and source records attributed to Robert H Singer.

32 records · Page 2Linked to original sources
PubMed2003

RNA asymmetric distribution and daughter/mother differentiation in yeast.

Active transport and localized translation of the ASH1 mRNA at the bud tip of the budding yeast Saccharomyces cerevisiae is an essential process that is required for the regulation of the mating type switching. ASH1 mRNA localization has been extensively studied over the past few years and the core components of the translocation machinery have been identified. It is composed of four localization elements (zipcodes), within the ASH1 mRNA, and at least three proteins, She1p/Myo4p, She2p and She3p. Whereas the movement of the RNA can be attributed to direct interaction with myosin, the regulation of the RNA expression is less well understood. Recent insights have revealed a role for translation that might have a key function in the regulation of Ash1 protein sorting.

Biological Transport↗
PubMed2003

Gene expression and the myth of the average cell.

We all know that gene expression occurs within cells, yet we do not think of expression in terms of its fundamental unit -- a single cell. Instead, we understand the expression of genes in terms of a cell population as all of our information comes from samples containing millions of cells. From a complex mixture of cells, we attempt to infer the probable state of an average cell in the population. In truth, what we obtain is an averaged cell, a contrivance for representing biological knowledge beyond the limits of detection. We never know the variation among the members of the population that our methods average into a mean. Recent technological advances allow the precise measurement of single-cell transcriptional states to study this variability more rigorously. How genes are expressed in the population is strikingly different to what we have assumed from extrapolating to an average cell. Does the average cell actually exist? As we discuss, it is becoming increasingly clear that it doesn't.

Animals↗
PubMed2003

Asymmetric distribution of nuclear pore complexes and the cytoplasmic localization of beta2-tubulin mRNA in Chlamydomonas reinhardtii.

Although it is generally accepted that nuclear architecture is an important determinant of nuclear activity, it is not clear whether cytoplasmic events, such as transcript localization and cell polarity, are affected by this architecture. Characterization of the nuclear architecture of the single-cell alga Chlamydomonas reinhardtii revealed a polarized nucleus, with nuclear pore complexes preferentially concentrated at the posterior side of the nucleus. Nuclear asymmetry was greatly exaggerated during the upregulation of genes encoding flagellar proteins, when nuclear pore complexes (NPCs) were observed to hyperpolarize to the posterior side of the nucleus while heterochromatin polarized to the anterior side. Interestingly, prior to deflagellation, the beta2-tubulin gene was preferentially located in the posterior region of the nucleus, and following deflagellation, beta2-tubulin transcripts accumulated posteriorly in polysome-rich cytoplasmic regions adjacent to the highest concentration of NPCs, suggesting a connection between nuclear architecture and cytoplasmic transcript localization.

Animals↗
PubMed2003

Role of the parafusin orthologue, PRP1, in microneme exocytosis and cell invasion in Toxoplasma gondii.

The association of PRP1, a Paramecium parafusin orthologue, with Toxoplasma gondii micronemes, now confirmed by immunoelectron microscopy, has here been studied in relation to exocytosis and cell invasion. PRP1 becomes labelled in vivo by inorganic 32P and is dephosphorylated when ethanol is used to stimulate Ca2+-dependent exocytosis of the micronemes. The ethanol Ca2+-stimulated exocytosis is accompanied by translocation of PRP1 and microneme content protein (MIC3) from the apical end of the parasite. Immunoblotting showed that PRP1 is redistributed inside the parasite, while microneme content is secreted. To study whether similar changes occur during cell invasion, quantitative microscopy was performed during secretion, invasion and exit (egress) from the host cell. Time-course experiments showed that fluorescence intensities of PRP1 and MIC3 immediately after invasion were reduced 10-fold compared to preinvasion levels, indicating that PRP1 translocation and microneme secretion accompanies invasion. MIC3 regained fluorescence intensity and apical distribution after 15 min, while PRP1 recovered after 1 h. Intensity of both proteins then increased throughout the parasite division period until host cell lysis, suggesting the need to secrete microneme proteins to egress. These studies suggest that PRP1 associated with the secretory vesicle scaffold serves an important role in Ca2+-regulated exocytosis and cell invasion.

Animals↗
PubMed2002

Two ZBP1 KH domains facilitate beta-actin mRNA localization, granule formation, and cytoskeletal attachment.

Chicken embryo fibroblasts (CEFs) localize beta-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of beta-actin mRNA requires a cis localization element (zipcode) and involves zipcode binding protein 1 (ZBP1), a protein that specifically binds to the zipcode. Both localize to the lamellipodia of polarized CEFs. ZBP1 and its homologues contain two NH2-terminal RNA recognition motifs (RRMs) and four COOH-terminal hnRNP K homology (KH) domains. By using ZBP1 truncations fused to GFP in conjunction with in situ hybridization analysis, we have determined that KH domains three and four were responsible for granule formation and cytoskeletal association. When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination. RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of beta-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized beta-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both beta-actin mRNA localization and cell motility.

Actins↗
PubMed2002

Novel detection and differential utilization of a c-myc transcriptional block in colon cancer chemoprevention.

Mutations in the adenomatous polyposis coli (APC) gene, which initiate almost all human colon cancers, directly target the proto-oncogene, c-myc, by elevating beta-catenin/T-cell factor (TCF) signaling. We have shown that agents ascribed chemopreventive activity for colon cancer in fact also stimulate beta-catenin/TCF activity in vitro. Their effects on c-myc transcription were assayed using a novel variant of fluorescence in situ hybridization that detects c-myc transcription sites in intact nuclei. Increased transcriptional initiation of c-myc induced by the short-chain fatty acid, butyrate, consistent with elevated beta-catenin/TCF activity, was efficiently abrogated by a block to transcriptional elongation, resulting in decreased c-myc expression. 1alpha,25-Dihydroxyvitamin D(3) also induced transcriptional blockage. In contrast, the nonsteroidal anti-inflammatory drug, sulindac, increased c-myc expression, an effect attributable at least in part to its failure to induce transcriptional blockage. We have described a novel approach for evaluating the effects of chemopreventive agents on the expression of a gene critical in colonic tumorigenesis.

Anticarcinogenic Agents↗
PubMed2002

Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling.

We have developed animal models of breast cancer that allow the direct examination of the behavior of individual green fluorescent protein-expressing carcinoma cells in live nonmetastatic and metastatic primary tumors in situ. We have combined this model with multiphoton microscopy to image differences in cell behavior within the primary tumor. Differences in cell behavior between nonmetastatic and metastatic cells in culture and within live primary tumors were correlated with results from cDNA microarray analyses to identify potentially important genetic determinants for breast cancer invasion and metastasis. Using multiphoton microscopy, we found five major differences in carcinoma cell behavior between the nonmetastatic and metastatic primary breast tumors involving extracellular matrix, cell motility, and chemotaxis. Behavioral differences were correlated with seven categories of molecules that were differentially expressed and related to these behaviors. We have found that extracellular matrix composition, actin nucleation factors, molecules involved in mechanical stability and survival, and cell polarity and chemotaxis showed large and consistent differences in gene expression. 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.

Adenocarcinoma↗
PubMed2002

Single-cell gene expression profiling.

A key goal of biology is to relate the expression of specific genes to a particular cellular phenotype. However, current assays for gene expression destroy the structural context. By combining advances in computational fluorescence microscopy with multiplex probe design, we devised technology in which the expression of many genes can be visualized simultaneously inside single cells with high spatial and temporal resolution. Analysis of 11 genes in serum-stimulated cultured cells revealed unique patterns of gene expression within individual cells. Using the nucleus as the substrate for parallel gene analysis, we provide a platform for the fusion of genomics and cell biology: "cellular genomics."

Adenocarcinoma↗
PubMed2002

A predominantly nuclear protein affecting cytoplasmic localization of beta-actin mRNA in fibroblasts and neurons.

The localization of beta-actin mRNA to the leading lamellae of chicken fibroblasts and neurite growth cones of developing neurons requires a 54-nt localization signal (the zipcode) within the 3' untranslated region. In this study we have identified and isolated five proteins binding to the zipcode. One of these we previously identified as zipcode binding protein (ZBP)1, a 4-KH domain protein. A second is now investigated in detail: a 92-kD protein, ZBP2, that is especially abundant in extracts from embryonic brain. We show that ZBP2 is a homologue of the human hnRNP protein, KSRP, that appears to mediate pre-mRNA splicing. However, ZBP2 has a 47-amino acid (aa) sequence not present in KSRP. Various portions of ZBP2 fused to GFP indicate that the protein most likely shuttles between the nucleus and the cytoplasm, and that the 47-aa insert promotes the nuclear localization. Expression of a truncated ZBP2 inhibits the localization of beta-actin mRNA in both fibroblast and neurons. These data suggest that ZBP2, although predominantly a nuclear protein, has a role in the cytoplasmic localization of beta-actin mRNA.

Actins↗
PubMed2002

The nuclear connection in RNA transport and localization.

RNA-binding proteins are involved in various aspects of RNA metabolism such as processing, translational control, stabilization, localization and transport. Many of these proteins bind several RNA targets and have multiple functions. In this review we focus on RNA-binding proteins that are implicated in RNA transport and localization and that also have a role in other aspects of RNA metabolism. These proteins might link nuclear events, such as RNA splicing, with the subsequent cytoplasmic localization of specific transcripts.

Active Transport, Cell Nucleus↗
PubMed2002

Asymmetric sorting of ash1p in yeast results from inhibition of translation by localization elements in the mRNA.

ASH1 mRNA localizes at the bud tip of late-anaphase yeast, resulting in accumulation of Ash1p in the daughter nucleus. We show that disruption of the secondary structure, but not the protein coding, of all four ASH1 localization elements resulted in RNA and protein delocalization. Localization of both was incrementally restored by replacement of each of the four elements. However, transposition of the elements to the 3'UTR reinstated the RNA, but not the protein, localization. Interestingly, the mutant ASH1 mRNA was translated more efficiently, suggesting that asymmetry of Ash1p resulted from translational inhibition by the localization elements. In support of this, Ash1p asymmetry could be rescued by slowing its translation.

5' Untranslated Regions↗
PubMed2002

An active precursor in assembly of yeast nuclear ribonuclease P.

The RNA-protein subunit assembly of nuclear RNase P was investigated by specific isolation and characterization of the precursor and mature forms of RNase P using an RNA affinity ligand. Pre-RNase P was as active in pre-tRNA cleavage as mature RNase P, although it contained only seven of the nine proteins found in mature RNase P. Pop3p and Rpr2p were not required for maturation of the RPR1 RNA subunit and virtually absent from pre-RNase P, implying that they are dispensable for pre-tRNA substrate recognition and cleavage. The RNase P subunit assembly is likely to occur in the nucleolus, where both precursor and mature forms of RNase P RNA are primarily localized. The results provide insight into assembly of nuclear RNase P, and suggest pre-tRNA substrate recognition is largely determined by the RNA subunit.

Amino Acid Sequence↗
PubMed2002

Interactions of elongation factor 1alpha with F-actin and beta-actin mRNA: implications for anchoring mRNA in cell protrusions.

The targeting of mRNA and local protein synthesis is important for the generation and maintenance of cell polarity. As part of the translational machinery as well as an actin/microtubule-binding protein, elongation factor 1alpha (EF1alpha) is a candidate linker between the protein translation apparatus and the cytoskeleton. We demonstrate in this work that EF1alpha colocalizes with beta-actin mRNA and F-actin in protrusions of chicken embryo fibroblasts and binds directly to F-actin and beta-actin mRNA simultaneously in vitro in actin cosedimentation and enzyme-linked immunosorbent assays. To investigate the role of EF1alpha in mRNA targeting, we mapped the two actin-binding sites on EF1alpha at high resolution and defined one site at the N-terminal 49 residues of domain I and the other at the C-terminal 54 residues of domain III. In vitro actin-binding assays and localization in vivo of recombinant full-length EF1alpha and its various truncates demonstrated that the C terminus of domain III was the dominant actin-binding site both in vitro and in vivo. We propose that the EF1alpha-F-actin complex is the scaffold that is important for beta-actin mRNA anchoring. Disruption of this complex would lead to delocalization of the mRNA. This hypothesis was tested by using two dominant negative polypeptides: the actin-binding domain III of EF1alpha and the EF1alpha-binding site of yeast Bni1p, a protein that inhibits EF1alpha binding to F-actin and also is required for yeast mRNA localization. We demonstrate that either domain III of EF1alpha or the EF1alpha-binding site of Bni1p inhibits EF1alpha binding to beta-actin mRNA in vitro and causes delocalization of beta-actin mRNA in chicken embryo fibroblasts. Taken together, these results implicate EF1alpha in the anchoring of beta-actin mRNA to the protrusion in crawling cells.

Actins↗