A Gain-of-Function Allele of cbp-1, the Caenorhabditis elegans Ortholog of the Mammalian CBP/p300 Gene, Causes an Increase in Histone Acetyltransferase Activity and Antagonism of Activated Ras

An RTK-Ras-mitogen-activated protein kinase (MAPK) signaling pathway plays a key role in vulval induction in Caenorhabditis elegans. We have previously carried out screens for suppressors of activated Ras to identify factors that play critical roles in the regulation of the pathway. ku258 was isolated as a semidominant allele that suppresses the Multivulva phenotype caused by activated let-60 ras. Our genetic and molecular analyses indicate that ku258 is a gain-of-function allele resulting from two point mutations in the C. elegans homolog of the transcriptional coactivator p300/CBP, cbp-1. Genetic data also suggest that cbp-1 may act downstream of the Ras signaling pathway, but not primarily downstream of the Wnt signaling pathway, to negatively regulate vulval cell fate specification. cbp-1 may function in concert with LIN-1, an Ets transcription factor family member that is one of the targets of MAPK. In vitro histone acetylation assays have revealed that together, the two point mutations cause a sevenfold increase in the histone acetyltransferase (HAT) activity of recombinant CBP-1. To our knowledge, this is the only such HAT activity mutation isolated in a CBP/p300 family protein, and this mutation may define a negative role of the HAT activity in antagonizing Ras function in a specific developmental event

Phosphoinositides in cell architecture

Inositol phospholipids have been implicated in almost all aspects of cellular physiology including spatiotemporal regulation of cellular signaling, acquisition of cellular polarity, specification of membrane identity, cytoskeletal dynamics, and regulation of cellular adhesion, motility, and cytokinesis. In this review, we examine the critical role phosphoinositides play in these processes to execute the establishment and maintenance of cellular architecture. Epithelial tissues perform essential barrier and transport functions in almost all major organs. Key to their development and function is the establishment of epithelial cell polarity.We place a special emphasis on highlighting recent studies demonstrating phosphoinositide regulation of epithelial cell polarity and how individual cells use phosphoinositides to further organize into epithelial tissues

The Caenorhabditis elegans Nuclear Receptor Gene nhr-25 Regulates Epidermal Cell Development

The development of the epidermis of Caenorhabditis elegans involves cell fusion, migration, and differentiation events. To understand the mechanisms underlying these processes, we characterized the roles of NHR-25, a member of the nuclear receptor family of transcription factors. The NHR-25 homologs Ftz-F1 in Drosophila and SF-1 in mammals are involved in various biological processes, including regulation of patterning during development, reproduction, metabolism, metamorphosis, and homeostasis. Impairment of nhr-25 activity leads to severe phenotypes in embryos and many postembryonic tissues. Further analysis has indicated that nhr-25 activity is required for the proper development, including cell-cell fusion, of several epidermal cell types, such as the epidermal syncytial, seam, and Pn.p cells. Our results also suggest that nhr-25 is likely to regulate cell-cell junctions and/or fusion. In a subset of Pn.p cells, called vulval precursor cells, nhr-25 acts collaboratively with the lin-39 Hox gene in regulating vulval cell differentiation. Additionally, our data suggest that nhr-25 may also function with another Hox gene, nob-1, during embryogenesis. Overall, our results indicate that nhr-25 plays an integral role in regulating cellular processes of epidermal cells

Scrib regulates HGF-mediated epithelial morphogenesis and is stabilized by Sgt1-HSP90.

Scribble was originally identified as a Drosophila protein that regulates epithelial polarity and formation of the basolateral surface. The mammalian orthologue, Scrib, is evolutionarily conserved, but does not appear to be necessary for apical-basolateral epithelial polarity. Instead, it is implicated in the regulation of cell survival, protein trafficking, adhesion and migration. A key issue is to understand the molecular pathway by which Scrib participates in these processes. We have investigated Scrib using a three-dimensional epithelial cell culture system. We show a novel association between the leucine-rich repeat domain of Scrib and the co-chaperone Sgt1 and demonstrate that these proteins are necessary for epithelial morphogenesis and tubulogenesis following hepatocyte growth factor (HGF) stimulation. The molecular chaperone HSP90 is also required for Sgt1 association with Scrib, and both Sgt1 and HSP90 are needed to ensure proper Scrib protein levels. Furthermore, reduced Scrib stability, following inhibition of Sgt1-HSP90, lowers the cellular abundance of the Scrib-βPix-PAK complex. Inhibition of any member of this complex, Scrib, βPix or PAK, is sufficient to block HGF-mediated epithelial morphogenesis. The identification of Scrib as an Sgt1-HSP90 client protein required for three-dimensional cell migration suggests that chaperone-mediated regulation of polarity protein stability and homeostasis is an unappreciated mechanism underlying dynamic rearrangements during morphogenesis

Ultrahigh-Throughput Mammalian Single-Cell Reverse-Transcriptase Polymerase Chain Reaction in Microfluidic Drops

The behaviors of complex biological systems are often dictated by the properties of their heterogeneous and sometimes rare cellular constituents. Correspondingly, the analysis of individual cells from a heterogeneous population can reveal information not obtainable by ensemble measurements. Reverse-transcriptase polymerase chain reaction (RT-PCR) is a widely used method that enables transcriptional profiling and sequencing analysis on bulk populations of cells. Major barriers to successfully implementing this technique for mammalian single-cell studies are the labor, cost, and low-throughput associated with current approaches. In this report, we describe a novel droplet-based microfluidic system for performing ∼50000 single-cell RT-PCR reactions in a single experiment while consuming a minimal amount of reagent. Using cell type-specific staining and TaqMan RT-PCR probes, we demonstrate the identification of specific cells from a mixed human cell population. The throughput, robust detection rate and specificity of this method makes it well-suited for characterizing large, heterogeneous populations of cells at the transcriptional level

Digital RT-PCR Taqman assays in microfluidic drops following picoinjection of reverse transcriptase.

<p>(A) Control RT-PCR reactions containing PC3 cell total RNA were emulsified on a T-junction drop maker, thermocycled, and imaged. FAM (green) fluorescence indicates Taqman detection of an EpCAM transcript and Cy5 (red) indicates detection of CD44 transcripts. Brightfield images (BF) of the same drops are shown in the image panel on the far right. The red arrows indicate the direction of emulsion flow in the illustrations. (B) RT-PCR reactions lacking reverse transcriptase were emulsified on a T-junction drop maker and subsequently picoinjected with reverse transcriptase. Picoinjection fluid is pictured as dark gray in the schematic diagram on the left. Brightfield images demonstrate that the drops roughly doubled in size after picoinjection. (C) RT-PCR reactions subjected to picoinjection omitting the reverse transcriptase show no Taqman signal for EpCAM and CD44, demonstrating the specificity of the Taqman assay. Scale bars  = 100 µm.</p

Dual transcript detection analysis indicates minimal cross-contamination during picoinjection.

<p>(A) Scatter plots of FAM and HEX drop intensities for a co-encapsulated control sample (left) and dual population picoinjected sample (right). Using this analysis, large numbers of Taqman multiplexed drops were identified in the co-encapsulated controls that were virtually absent in the dual population picoinjected drops (upper right quadrants of gated scatter plots). (B) A bar graph of different bright drop populations relative to the total bright count for co-encapsulation control and for dual population picoinjection. The data represent the average of three experimental replicates.</p

Comparison of digital RT-PCR detection rates between control drops and drops that were picoinjected with reverse transcriptase.

<p>(A) Scatter plots of FAM and Cy5 drop intensities for a control sample (left) and picoinjected sample (right). The gating thresholds used to label a drop as positive or negative for Taqman signal are demarcated by the lines, and divide the scatter plot into quadrants, double negative drops (–,–), FAM positive (–,+), Cy5 positive (+,–), positive for both FAM and Cy5 (+,+). Numbers of drops in each quadrant are indicated. (B) The bar graph shows the average Taqman positive drop count with picoinjection relative to the normalized count for CD44 and EpCAM Taqman assays for control populations. The control detection rate value is defined as 1 for each replicate. The data represent the average of four independent experimental replicates.</p