Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator

Previous studies have shown that the urokinase-type plasminogen activator receptor (uPAR) is localized to the adherence sites of leukocytes and tumor cells suggesting that pericellular proteolysis may accompany focal activation of adherence. To assess for focused pericellular proteolytic activity, we prepared two-dimensional substrates coated with FITC-casein or Bodipy FL-BSA. These molecules are poorly fluorescent, but become highly fluorescent after proteolytic degradation. Fluorescent peptide products were observed at adherence sites of stationary human neutrophils and at lamellipodia of polarized neutrophils. During cell migration, multiple regions of proteolysis appeared sequentially beneath the cell. Similarly, proteolytic action was restricted to adherence sites of resting HT1080 tumor cells but localized to the invadopodia of active cells. Using an extracellular fluorescence quenching method, we demonstrate that these fluorescent peptide products are extracellular. The uPA/uPAR system played an important role in the observed proteolytic activation. Plasminogen activator inhibitor-1 significantly reduced focal proteolysis. Sites of focal proteolysis matched the membrane distribution of uPAR. When uPA was dissociated from uPAR by acid washing, substantially reduced pericellular proteolysis was found. uPAR-negative T47D tumor cells did not express significant levels of substrate proteolysis. However, transfectant clones expressing uPAR (for example, T47D-26) displayed high levels of fluorescence indicating proteolysis at adherence sites. To provide further evidence for the role of the uPA/uPAR system in pericellular proteolysis, peritoneal macrophages from uPA knock-out (uPA–/–) and control (uPA+/+) mice were studied. Pericellular proteolysis was dramatically reduced in uPA-negative peritoneal macrophages. Thus, we have: (1) developed a novel methodology to detect pericellular proteolytic function, (2) demonstrated focused activation of proteolytic enzymatic activity in several cell types, (3) demonstrated its usefulness in real-time studies of cell migration, and (4) showed that the uPA/uPAR system is an important contributor to focal pericellular proteolysis.     

Moesin contributes an essential structural role in Drosophila photoreceptor morphogenesis

Ezrin-Radixin-Moesin (ERM) family proteins organize heterogeneous sub-plasma membrane protein scaffolds that shape membranes and their physiology. In Drosophila oocytes and imaginal discs, epithelial organization, fundamental to development and physiology, is devastated by the loss of Moesin. Here, we show that Moesin is crucial for Drosophila photoreceptor morphogenesis. Beyond its requirement for retinal epithelium integrity, Moesin is essential for the proper assembly of the apical membrane skeleton that builds the photosensitive membrane, the rhabdomere. Moesin localizes to the rhabdomere base, a dynamic locus of cytoskeletal reorganization and membrane traffic. Downregulation of Moesin through RNAi or genetic loss of function profoundly disrupts the membrane cytoskeleton and apical membrane organization. We find normal levels and distribution of Moesin in photoreceptors of a Moesin mutant previously regarded as protein null, suggesting alternative interpretations for studies using this allele. Our results show an essential structural role for Moesin in photoreceptor morphology.     

Stage-specific markers define early steps of procambium development in Arabidopsis leaves and correlate termination of vein formation with mesophyll differentiation

During leaf development, ground meristem cells along continuous lines undergo coordinated oriented cell divisions and differentiate to form procambial cells, the precursors of all vascular cells. The molecular genetic dissection of early procambial development suffers from the lack of easily identifiable markers, especially of cell states preceding procambium formation. In this study, we have identified and characterized three reporter gene expression markers that reflect three distinct preprocambial stages, as well as one marker whose expression seems to be perfectly congruent with the appearance of procambial cells. All four markers are invariably expressed in continuous domains connected to pre-existing vasculature and their expression profiles reveal a common spatiotemporal pattern of early vein formation. We observed progressive extension of vascular strands at the preprocambial stage, suggesting that veins are initiated as freely ending preprocambial domains and that network formation occurs through subsequent fusion of these domains. Consistent with this interpretation, we demonstrate that veins are generally not programmed to become freely ending or interconnected network elements. Instead, we found that the progressive extension of preprocambial domains can be interrupted experimentally and that this leads to less complex vein patterns consisting of fewer vein orders, in which even lower-order veins become freely ending. Mesophyll differentiation turned out to be strictly correlated with the termination of preprocambial domain extension. These findings suggest that Arabidopsis vein pattern is not inherently determinate, but arises through reiterative initiation of new preprocambial branches until this process becomes terminated by the differentiation of mesophyll.     

Identification of ComW as a new component in the regulation of genetic transformation in Streptococcus pneumoniae

Regulation of competence for genetic transformation in Streptococcus pneumoniae depends on a quorum-sensing system, genes involved in DNA uptake and recombination and a link between these two gene sets. The alternative sigma factor ComX provides this link. ComE, the response regulator of the quorum-sensing system, is required for expression of ComX and other early genes. However, an unknown ComE-dependent regulator is also required for competence when comX is expressed under control of the raffinose-responsive promoter of the aga operon. The gene comW (SP0018) is required for a high level of competence and is regulated by the quorum-sensing system, but its function is unknown. To explore its role further, comW was cloned into the multicopy plasmid pMSP3535, under the control of a nisin-inducible promoter (P(N)), and transformed into pneumococcal strains containing a raffinose-inducible comX gene (P(R)::comX). Further introduction of a comE deletion blocked the endogenous CSP signal transduction pathway. In the resulting strain, competence was independent of CSP but depended on treatment with both nisin and raffinose, showing that coexpression of comW and comX complemented the comE deficiency. ComX protein accumulation and expression of a late competence gene in the above strain support the conclusion that ComW is a new positive factor involved in competence regulation.     

The investigation of cytosolic phospholipase A2 using ELISA

Two studies of the behaviour of cytosolic phospholipase A(2) (cPLA(2)) in the red blood cell (RBC), as measured by ELISA, are described. In the first study we show a significant increase in cPLA(2) in patients with schizophrenia compared to controls and suggest that this measure, if corroborated, could be used as a diagnostic marker. In a second study we found that washing the RBC introduced an unknown confounding variable which led us to reject this study. A subsequent investigation of washing red cells showed that the washing effect may be due to a plasma factor likely to be more than 5kDa MW which can be removed from red cells by washing with buffers. When the cells are washed, the concentration of cPLA(2) in the red cell, as measured by ELISA, significantly increases. We advise against washing the red cell in any study that involves measuring cPLA(2) by ELISA.     

Microarray method to monitor 40 intestinal bacterial species in the study of azo dye reduction

Azo dyes are widely used in dye manufacturing, paper printing, textile industries, and as tattoo pigmentation. Since intestinal and skin bacteria can metabolize certain azo dyes to carcinogenic compounds, many researchers have studied the azoreductases of these bacteria. In this study, we used a microarray method to identify the intestinal bacterial species from cultured fecal samples in Brain Heart Infusion (BHI) broth with or without azo dyes that may be involved in azo dye reduction. The microarray was designed to identify 40 bacterial species that are reported in the literature to be predominant in human feces. Results from this study showed 26-30 species are present in the cultured fecal samples. The representative bacteria were then examined for the azo dye reduction activity.     

Folding of helical membrane proteins: the role of polar, GxxxG-like and proline motifs

Helical integral membrane proteins share several structural determinants that are widely conserved across their universe. The discovery of common motifs has furthered our understanding of the features that are important to stability in the membrane environment, while simultaneously providing clues about proteins that lack high-resolution structures. Motif analysis also helps to target mutagenesis studies, and other experimental and computational work. Three types of transmembrane motifs have recently seen interesting developments: the GxxxG motif and its like; polar and hydrogen bonding motifs; and proline motifs.     

Vasomotor responses in MnSOD-deficient mice

MnSOD is the only mammalian isoform of SOD that is necessary for life. MnSOD(-/-) mice die soon after birth, and MnSOD(+/-) mice are more susceptible to oxidative stress than wild-type (WT) mice. In this study, we examined vasomotor function responses in aortas of MnSOD(+/-) mice under normal conditions and during oxidative stress. Under normal conditions, contractions to serotonin (5-HT) and prostaglandin F2alpha (PGF2alpha), relaxation to ACh, and superoxide levels were similar in aortas of WT and MnSOD(+/-) mice. The mitochondrial inhibitor antimycin A reduced contraction to PGF2alpha and impaired relaxation to ACh to a similar extent in aortas of WT and MnSOD(+/-) mice. The Cu/ZnSOD and extracellular SOD inhibitor diethyldithiocarbamate (DDC) paradoxically enhanced contraction to 5-HT and superoxide more in aortas of WT mice than in MnSOD(+/-) mice. DDC impaired relaxation to ACh and reduced total SOD activity similarly in aortas of both genotypes. Tiron, a scavenger of superoxide, normalized contraction to 5-HT, relaxation to ACh, and superoxide levels in DDC-treated aortas of WT and MnSOD(+/-) mice. Hypoxia, which reportedly increases superoxide, reduced contractions to 5-HT and PGF2alpha similarly in aortas of WT and MnSOD(+/-) mice. The vasomotor response to acute hypoxia was similar in both genotypes. In summary, under normal conditions and during acute oxidative stress, vasomotor function is similar in WT and MnSOD(+/-) mice. We speculate that decreased mitochondrial superoxide production may preserve nitric oxide bioavailability during oxidative stress.