Bioimaging in the mid-infrared using an organometallic carbonyl tag

Two stable and water-soluble organometallic carbonyl cluster derivatives have been prepared and shown to enter the cell with ease. The CO stretching vibrations afford strong mid-infrared signals which have been demonstrated, for the first time, to be of utility in cell imaging via an IR microscope.     

Pre-spliceosomal binding of U1 small nuclear ribonucleoprotein (RNP) and heterogenous nuclear RNP E1 is associated with suppression of a growth hormone receptor pseudoexon

Pseudoexons occur frequently in the human genome. This paper characterizes a pseudoexon in the GH receptor gene. Inappropriate activation of this pseudoexon causes Laron syndrome. Using in vitro splicing assays, pseudoexon silencing was shown to require a combination of a weak 5' pseudosplice-site and splicing silencing elements within the pseudoexon. Immunoprecipitation experiments showed that specific binding of heterogenous nuclear ribonucleoprotein E1 (hnRNP E1) and U1 small nuclear ribonucleoprotein (snRNP) in the pre-spliceosomal complex was associated with silencing of pseudoexon splicing. The possible role of hnRNP E1 was further supported by RNA interference experiments in cultured cells. Immunoprecipitation experiments with three other pseudoexons suggested that pre-spliceosomal binding of U1 snRNP is a potential general mechanism of suppression of pseudoexons.     

Protrusive waves guide 3D cell migration along nanofibers

In vivo, cells migrate on complex three-dimensional (3D) fibrous matrices, which has made investigation of the key molecular and physical mechanisms that drive cell migration difficult. Using reductionist approaches based on 3D electrospun fibers, we report for various cell types that single-cell migration along fibronectin-coated nanofibers is associated with lateral actin-based waves. These cyclical waves have a fin-like shape and propagate up to several hundred micrometers from the cell body, extending the leading edge and promoting highly persistent directional movement. Cells generate these waves through balanced activation of the Rac1/N-WASP/Arp2/3 and Rho/formins pathways. The waves originate from one major adhesion site at leading end of the cell body, which is linked through actomyosin contractility to another site at the back of the cell, allowing force generation, matrix deformation and cell translocation. By combining experimental and modeling data, we demonstrate that cell migration in a fibrous environment requires the formation and propagation of dynamic, actin based fin-like protrusions.     

Genome-wide RNAi screens identify genes required for Ricin and PE intoxications

Protein toxins such as Ricin and Pseudomonas exotoxin (PE) pose major public health challenges. Both toxins depend on host cell machinery for internalization, retrograde trafficking from endosomes to?the ER, and translocation to cytosol. Although both toxins follow a similar intracellular route, it is unknown how much they rely on the same genes. Here we conducted two genome-wide RNAi screens identifying genes required for intoxication and demonstrating that requirements are strikingly different between PE and Ricin, with only 13% overlap. Yet factors required by both toxins are present from the endosomes to the ER, and, at the morphological level, the toxins colocalize in multiple structures. Interestingly, Ricin, but not PE, depends on Golgi complex integrity and colocalizes significantly with a medial Golgi marker. Our data are consistent with two intertwined pathways converging and diverging at multiple points and reveal the complexity of retrograde membrane trafficking in mammalian cells.     

Soluble periplasmic production of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens

Cost-effective production of soluble recombinant protein in a bacterial system remains problematic with respect to expression levels and quality of the expressed target protein. These constraints have particular meaning today as "biosimilar" versions of innovator protein drugs are entering the clinic and the marketplace. A high throughput, parallel processing approach to expression strain engineering was used to evaluate soluble expression of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens. The human g-csf gene was optimized for expression in P. fluorescens and cloned into a set of periplasmic expression vectors. These plasmids were transformed into a variety of P. fluorescens host strains each having a unique phenotype, to evaluate soluble expression in a 96-well growth and protein expression format. To identify a strain producing high levels of intact, soluble Met-G-CSF product, more than 150 protease defective host strains from the Pfēnex Expression Technology? toolbox were screened in parallel using biolayer interferometry (BLI) to quantify active G-CSF binding to its receptor. A subset of these strains was screened by LC-MS analysis to assess the quality of the expressed G-CSF protein. A single strain with an antibiotic resistance marker insertion in the pfaI gene was identified that produced>99% Met-GCSF. A host with a complete deletion of the autotransporter-coding gene pfaI from the genome was constructed, and expression of soluble, active Met-GSCF in this strain was observed to be 350mg/L at the 1 liter fermentation scale.     

Scoring schemes of palindrome clusters for more sensitive prediction of replication origins in herpesviruses

Many empirical studies show that there are unusual clusters of palindromes, closely spaced direct and inverted repeats around the replication origins of herpesviruses. In this paper, we introduce two new scoring schemes to quantify the spatial abundance of palindromes in a genomic sequence. Based on these scoring schemes, a computational method to predict the locations of replication origins is developed. When our predictions are compared with 39 known or annotated replication origins in 19 herpesviruses, close to 80% of the replication origins are located within 2% of the genome length. A list of predicted locations of replication origins in all the known herpesviruses with complete genome sequences is reported.     

Drebrin E2 is differentially expressed and phosphorylated in parietal cells in the gastric mucosa

Developmentally regulated brain proteins (drebrins) are highly expressed in brain where they may regulate actin filament formation in dendritic spines. Recently, the drebrin E2 isoform was detected in certain epithelial cell types including the gastric parietal cell. In gastric parietal cells, activation of HCl secretion is correlated with actin filament formation and elongation within intracellular canaliculi, which are the sites of acid secretion. The aim of this study was to define the pattern of drebrin expression in gland units in the intact rabbit oxyntic gastric mucosa and to initiate approaches to define the functions of this protein in parietal cells. Drebrin E2 expression was limited entirely or almost entirely to parietal cells and depended upon the localization of parietal cells along the gland axis. Rabbit drebrin E2 was cloned and found to share 86% identity with human drebrin 1a and to possess a number of cross-species conserved protein-protein interaction and phosphorylation consensus sites. Two-dimensional Western blot and phosphoaffinity column analyses confirmed that drebrin is phosphorylated in parietal cells, and several candidate phosphorylation sites were identified by mass spectrometry. Overexpression of epitope-tagged drebrin E2 led to the formation of microspikes and F-actin-rich ring-like structures in cultured parietal cells and suppressed cAMP-dependent acid secretory responses. In Madin-Darby canine kidney cells, coexpression of epitope-tagged drebrin and the Rho family GTPase Cdc42, which induces filopodial extension, produced an additive increase in the length of microspike projections. Coexpression of dominant negative Cdc42 with drebrin E2 did not prevent drebrin-induced microspike formation. These findings suggest that 1) drebrin can induce the formation of F-actin-rich membrane projections by Cdc42-dependent and -independent mechanisms; and that 2) drebrin plays an active role in directing the secretagogue-dependent formation of F-actin-rich filaments on the parietal cell canalicular membrane. Finally, the differential distribution of drebrin in parietal cells along the gland axis suggests that drebrin E2 may be an important marker of parietal cell differentiation and functionality.     

Notch1 antiapoptotic activity is abrogated by caspase cleavage in dying T lymphocytes

Excessive signaling via the Notch1 receptor inhibits apoptosis in T lymphocytes. Since several antiapoptotic proteins are cleaved by caspases during cell death, we investigated whether Notch1 was a caspase substrate. Results demonstrate that the intracellular domain of Notch1 (NICD) is cleaved into six fragments during apoptosis in Jurkat cells or peripheral T lymphocytes. Notch1 cleavage is prevented by the caspase inhibitors DEVD-fmk and VEID-fmk or by Bcl-2 expression. Caspase-3 and caspase-6 cleave the NICD into six fragments using sites located within the NF-kappaB binding domain, the ankyrin repeats and the transactivation domain. Notch1 cleavage correlates with the loss of HES-1 expression in apoptotic T cells. Notch1 fragments cannot inhibit activation-induced cell death in a T-cell hybridoma, confirming the abrogation of Notch1 antiapoptotic activity by caspases. The ability of the NICD but not the fragments to antagonize Nur77 activity supports a role for this factor in Notch1 antiapoptotic function.