An InCytes from MBC Selection: Filamin A–β1 Integrin Complex Tunes Epithelial Cell Response to Matrix Tension

The physical properties of the extracellular matrix (ECM) regulate the behavior of several cell types; yet, mechanisms by which cells recognize and respond to changes in these properties are not clear. For example, breast epithelial cells undergo ductal morphogenesis only when cultured in a compliant collagen matrix, but not when the tension of the matrix is increased by loading collagen gels or by increasing collagen density. We report that the actin-binding protein filamin A (FLNa) is necessary for cells to contract collagen gels, and pull on collagen fibrils, which leads to collagen remodeling and morphogenesis in compliant, low-density gels. In stiffer, high-density gels, cells are not able to contract and remodel the matrix, and morphogenesis does not occur. However, increased FLNa-β1 integrin interactions rescue gel contraction and remodeling in high-density gels, resulting in branching morphogenesis. These results suggest morphogenesis can be “tuned” by the balance between cell-generated contractility and opposing matrix stiffness. Our findings support a role for FLNa-β1 integrin as a mechanosensitive complex that bidirectionally senses the tension of the matrix and, in turn, regulates cellular contractility and response to this matrix tension.     

Venezuelan Equine Encephalitis in Panama: Fatal Endemic Disease and Genetic Diversity of Etiologic Viral Strains

Venezuelan equine encephalitis (VEE) is a reemerging, mosquito-borne viral disease of the neotropics that is severely debilitating and sometimes fatal to humans. Periodic epidemics mediated by equine amplification have been recognized since the 1920s, but interepidemic disease is rarely recognized. We report here clinical findings and genetic characterization of 42 cases of endemic VEE detected in Panama from 1961–2004. Recent clusters of cases occurred in Darien (eastern Panama) and Panama provinces (central Panama) near rainforest and swamp habitats. Patients ranged from 10 months to 48 years of age, and the more severe cases with neurological complications, including one fatal infection, were observed in children. The VEE virus strains isolated from these cases all belonged to an enzootic, subtype ID lineage known to circulate among sylvatic vectors and rodent reservoir hosts in Panama and Peru. These findings underscore endemic VEE as an important but usually neglected arboviral disease of Latin America.     

Structural and Functional Studies of Truncated Hemolysin A from Proteus mirabilis

In this study we analyzed the structure and function of a truncated form of hemolysin A (HpmA265) from Proteus mirabilis using a series of functional and structural studies. Hemolysin A belongs to the two-partner secretion pathway. The two-partner secretion pathway has been identified as the most common protein secretion pathway among Gram-negative bacteria. Currently, the mechanism of action for the two-partner hemolysin members is not fully understood. In this study, hemolysis experiments revealed a unidirectional, cooperative, biphasic activity profile after full-length, inactive hemolysin A was seeded with truncated hemolysin A. We also solved the first x-ray structure of a TpsA hemolysin. The truncated hemolysin A formed a right-handed parallel β-helix with three adjoining segments of anti-parallel β-sheet. A CXXC disulfide bond, four buried solvent molecules, and a carboxyamide ladder were all located at the third complete β-helix coil. Replacement of the CXXC motif led to decreased activity and stability according to hemolysis and CD studies. Furthermore, the crystal structure revealed a sterically compatible, dry dimeric interface formed via anti-parallel β-sheet interactions between neighboring β-helix monomers. Laser scanning confocal microscopy further supported the unidirectional interconversion of full-length hemolysin A. From these results, a model has been proposed, where cooperative, β-strand interactions between HpmA265 and neighboring full-length hemolysin A molecules, facilitated in part by the highly conserved CXXC pattern, account for the template-assisted hemolysis.Hemolysin A (HpmA)² and B (HpmB) from Proteus mirabilis belong to the Type V_b or two-partner secretion pathway (1), the most widespread of the five porin-type protein translocating systems found within bacterial, fungal, plant, and animal kingdoms (2). Cell surface adhesions, iron-acquisition proteins, and cytolysins/hemolysins all use two-partner secretion pathways (3–5). The A-component of the two-partner secretion in P. mirabilis is a 166-kDa virulence factor capable of mammalian blood cell lysis upon secretion from the cell. This is accomplished by Sec-dependent transport to the periplasm followed by N-terminal proteolytic processing. Extracellular secretion occurs by transport through the B-component, HpmB, which is a 16-stranded β-barrel transmembrane channel (6). In addition to its role in efficient secretion, HpmB is also necessary for activation of the larger exoprotein A-component (HpmA) (7–10).Studies on hemolytic TpsA members report that: 1) a truncated TpsA containing the N-terminal secretion cap (11) complements and restores hemolytic activity within a non-secreted/inactive pool of full-length TpsA (12), 2) the conserved cysteine residues within a CXXC motif are not required for secretion (12), and 3) the first asparagine within a NPNG hemagglutinin motif is required for efficient secretion (13). Other investigations demonstrate significant conformational change within TpsA members during B-component dependent secretion (8, 14–16).Recent x-ray crystal structures for two TpsA adhesion orthologs, hemagglutinin from Bordetella pertussis (FHA) and high molecular weight protein from Haemophilus influenzae (HMW1) adopt a right-handed parallel β-helix similar to pectate lyase (11, 18, 19). The 301-residue N-terminal FHA fragment (Fha30) contains a 37 parallel stranded β-helix. Stabilization of type I β-turns at two highly conserved regions: ⁶⁶NPNL and ¹⁰⁵NPNG is proposed to play a large role in the ability of this N-terminal fragment to rapidly adopt β-helix architecture (11, 20). Despite 21% sequence identity, the 371-residue HMW1 structure (HMW1-PP) is a similar 47 parallel stranded β-helix. A hypothesis arose from these β-helix structures that suggests extracellular secretion through TpsB channels, and the progressive folding of TpsA members is energetically coupled. Full-length TpsA adhesion members have been proposed to have a filamentous appearance built from a right-handed β-helix fold (21). To date, there is little known about the full-length HpmA domain architecture. However, there are two filamentous hemagglutinin type domains. The N-terminal domain is positioned between residues 30 and 167, whereas the C-terminal domain lies between residues 1200 and 1264 and has been proposed to facilitate cellular aggregation.In this work, we investigated the functional and structural role of truncated hemolysin A (HpmA265) during the template-assisted activation of hemolysis. A previous investigation with ShlA, a homologous TpsA member from Serratia marcescens, has shown similar complementation using a 255-amino acid fragment (12). Here, we demonstrate that HmpA265 can cooperatively cross seed an inactive pool of full-length hemolysin A (HpmA*) to form an exotoxin measured by our template-assisted hemolytic assay (TAHA). We also report that the CXXC motif provides structural stability and facilitates reversible re-folding. The structure reveals a right-handed β-helix, similar to those of FHA and HMW1. A number of conserved features found at the putative subunit interface suggest a mechanism by which activation of inactive HpmA* occurs.     

Adventitial delivery of dominant-negative p67phox attenuates neointimal hyperplasia of the rat carotid artery

Several essential components of NADPH oxidase, including p22phox, gp91phox (nox2) and its homologs nox1 and nox4, p47phox, p67phox, and rac1, are present in the vasculature. We previously reported that p67phox is essential for adventitial fibroblast NADPH oxidase O2- production. Thus we postulated that inhibition of adventitial p67phox activity would attenuate angioplasty-induced hyperplasia. To test this hypothesis, we treated the adventitia of carotid arteries with a control adenovirus (Ad-control), a virus expressing dominant-negative p67phox (Ad-p67dn), or a virus expressing a competitive peptide (gp91ds) targeting the p47phox-gp91phox interaction (Ad-gp91ds). Common carotid arteries (CCAs) from male Sprague-Dawley rats were transfected with Ad-control, Ad-p67dn, or Ad-gp91ds in pluronic gel. After 2 days, a 2-F (Fogarty) catheter was used to injure CCAs in vivo. After 14 days, CCAs were perfusion-fixed and analyzed. In 13 experiments, digital morphometry suggested a reduction of neointimal hyperplasia with Ad-p67dn compared with Ad-control; however, the reduction did not reach statistical significance (P = 0.058). In contrast, a significant reduction was achieved with Ad-gp91ds (P = 0.006). No changes in medial area or remodeling were observed with either treatment. Moreover, adventitial fibroblast proliferation in vitro was inhibited by Ad-gp91ds but not by Ad-p67dn, despite confirmation that Ad-p67dn inhibits NADPH oxidase in fibroblasts. These data appear to suggest that a multicomponent vascular NADPH oxidase plays a role in neointimal hyperplasia. However, inhibition of p47phox may be more effective than inhibition of p67phox at attenuating neointimal growth.     

SWI/SNF chromatin remodeling enzyme ATPases promote cell proliferation in normal mammary epithelial cells

The ATPase subunits of the SWI/SNF chromatin remodeling enzymes, Brahma (BRM) and Brahma‐related gene 1 (BRG1), can induce cell cycle arrest in BRM and BRG1 deficient tumor cell lines, and mice heterozygous for Brg1 are pre‐disposed to breast tumors, implicating loss of BRG1 as a mechanism for unregulated cell proliferation. To test the hypothesis that loss of BRG1 can contribute to breast cancer, we utilized RNA interference to reduce the amounts of BRM or BRG1 protein in the nonmalignant mammary epithelial cell line, MCF‐10A. When grown in reconstituted basement membrane (rBM), these cells develop into acini that resemble the lobes of normal breast tissue. Contrary to expectations, knockdown of either BRM or BRG1 resulted in an inhibition of cell proliferation in monolayer cultures. This inhibition was strikingly enhanced in three‐dimensional rBM culture, although some BRM‐depleted cells were later able to resume proliferation. Cells did not arrest in any specific stage of the cell cycle; instead, the cell cycle length increased by approximately 50%. Thus, SWI/SNF ATPases promote cell cycle progression in nonmalignant mammary epithelial cells. J. Cell. Physiol. 223:667–678, 2010. © 2010 Wiley‐Liss, Inc.     

Potential action of copper surfaces on meticillin-resistant Staphylococcus aureus

Aims: Studies to date have shown rapid killing of bacterial cells when exposed to copper surfaces. The mechanistic action of copper on bacterial cells is so far unknown. Methods and Results: To investigate potential mechanisms involved, meticillin‐resistant Staphylococcus aureus (MRSA) cells (10⁷ CFU) were inoculated onto coupons of copper or stainless steel and stained with either the viability fluorophore 5‐cyano‐2,3‐ditolyl tetrazolium (CTC), to detect respiration, or BacLight? (SYTO9/propidium iodide), to determine cell wall integrity. Coupons were then observed in‐situ using epifluorescence microscopy. In addition, DNA from cells inoculated onto either copper or stainless steel surfaces was isolated and analysed by agarose gel electrophoresis. An effect on cellular respiration with CTC reduction was evident but no effect on cell membrane integrity (BacLight?) was observed. Results from the DNA isolation indicated a copper‐induced detrimental effect on MRSA genomic material as no bands were observed after exposure to copper surface. Conclusions: The results indicate that exposure to copper surfaces rapidly kills MRSA by compromising cellular respiration and damaging DNA, with little effect on cell membrane integrity. Significance and Impact of the study: This research provides a mechanistic explanation in support of previous suggestions that although copper surfaces do not affect membrane integrity of cells, there is still a rapid antimicrobial effect.     

Synthesis and SAR of novel, 4-(phenylsulfamoyl)phenylacetamide mGlu4 positive allosteric modulators (PAMs) identified by functional high-throughput screening (HTS)

Herein we disclose the synthesis and SAR of a series of 4-(phenylsulfamoyl)phenylacetamide compounds as mGlu₄ positive allosteric modulators (PAMs) that were identified via a functional HTS. An iterative parallel approach to these compounds culminated in the discovery of VU0364439 (11) which represents the most potent (19.8 nM) mGlu₄ PAM reported to date.     

SPARC Deficiency Results in Improved Surgical Survival in a Novel Mouse Model of Glaucoma Filtration Surgery

Glaucoma is a disease frequently associated with elevated intraocular pressure that can be alleviated by filtration surgery. However, the post-operative subconjunctival scarring response which blocks filtration efficiency is a major hurdle to the achievement of long-term surgical success. Current application of anti-proliferatives to modulate the scarring response is not ideal as these often give rise to sight-threatening complications. SPARC (secreted protein, acidic and rich in cysteine) is a matricellular protein involved in extracellular matrix (ECM) production and organization. In this study, we investigated post-operative surgical wound survival in an experimental glaucoma filtration model in SPARC-null mice. Loss of SPARC resulted in a marked (87.5%) surgical wound survival rate compared to 0% in wild-type (WT) counterparts. The larger SPARC-null wounds implied that aqueous filtration through the subconjunctival space was more efficient in comparison to WT wounds. The pronounced increase in both surgical survival and filtration efficiency was associated with a less collagenous ECM, smaller collagen fibril diameter, and a loosely-organized subconjunctival matrix in the SPARC-null wounds. In contrast, WT wounds exhibited a densely packed collagenous ECM with no evidence of filtration capacity. Immunolocalization assays confirmed the accumulation of ECM proteins in the WT but not in the SPARC-null wounds. The observations in vivo were corroborated by complementary data performed on WT and SPARC-null conjunctival fibroblasts in vitro. These findings indicate that depletion of SPARC bestows an inherent change in post-operative ECM remodeling to favor wound maintenance. The evidence presented in this report is strongly supportive for the targeting of SPARC to increase the success of glaucoma filtration surgery.