Shigella Effector OspB Activates mTORC1 in a Manner That Depends on IQGAP1 and Promotes Cell Proliferation.

The intracellular bacterial pathogen Shigella infects and spreads through the human intestinal epithelium. Effector proteins delivered by Shigella into cells promote infection by modulating diverse host functions. We demonstrate that the effector protein OspB interacts directly with the scaffolding protein IQGAP1, and that the absence of either OspB or IQGAP1 during infection leads to larger areas of S. flexneri spread through cell monolayers. We show that the effect on the area of bacterial spread is due to OspB triggering increased cell proliferation at the periphery of infected foci, thereby replacing some of the cells that die within infected foci and restricting the area of bacterial spread. We demonstrate that OspB enhancement of cell proliferation results from activation of mTORC1, a master regulator of cell growth, and is blocked by the mTORC1-specific inhibitor rapamycin. OspB activation of mTORC1, and its effects on cell proliferation and bacterial spread, depends on IQGAP1. Our results identify OspB as a regulator of mTORC1 and mTORC1-dependent cell proliferation early during S. flexneri infection and establish a role for IQGAP1 in mTORC1 signaling. They also raise the possibility that IQGAP1 serves as a scaffold for the assembly of an OspB-mTORC1 signaling complex.

Influence of the microenvironment on cell fate determination and migration.

Several critical cell functions are influenced not only by internal cellular machinery but also by external mechanical and biochemical cues from the surrounding microenvironment. Slight changes to the microenvironment can result in dramatic changes to the cell's phenotype; for example, a change in the nutrients or pH of a tumor microenvironment can result in increased tumor metastasis. While cellular fate and the regulators of cell fate have been studied in detail for several decades now, our understanding of the extracellular regulators remains qualitative and far from comprehensive. In this review, we discuss the microenvironment influence on cell fate in terms of adhesion, migration, and differentiation and focus on both developments in experimental and computation tools to analyze cellular fate.

Professional Profiles, Technical Preferences, Surgical Opinions, and Management of Clinical Scenarios from a Panel of 63 International Experts in the Field of Chiari I Malformation.

BACKGROUND: Chiari I Malformation (CMI) and the topics concerning it have been the subject of numerous discussions and polarizing controversies over the course of the past 20 years. METHODS: The opinions of 63 recognized international Neurosurgical CMI experts from 4 continents, with a collective surgical experience of more than 15,000 CMI cases, were gathered through a detailed questionnaire, divided in two parts: diagnostic and therapeutic. The therapeutic part was organized into four sections: Professional Profile, Technical Preferences, Surgical Opinions, and Clinical Scenarios. RESULTS: The data reflected a wide spectrum of opinions, approaches, and expertise. The second part of the questionnaire dealt with the surgical aspects of CMI care and painted a more complex picture: • 81% of the surgeons preferred the Intradural technique. • 88% of the experts agreed that CMI surgery is not indicated for minimal non-debilitating symptoms alone, or as prophylaxis. • In the face of given clinical scenarios, a wide spectrum of therapeutic approaches was chosen by the whole group, but the 4 Surgeons with the largest case series expressed the same opinion. • Eight out of 63 Surgeons had a surgical experience above 600 cases, were responsible for more than half of the total 15,000 declared CMI cases, and shared a similar profile in terms of technical surgical choices, therapeutic opinions, and low complication rate, with a marked preference for Intradural techniques and tonsillar manipulation. • Once large individual case series were accumulated, we did not see any differences in the opinions and preferences between Adult and Pediatric Neurosurgeons. CONCLUSION: Surgeons who have focused on CMI have been able to accumulate large surgical series, have chosen in their practices the more aggressive (and intrinsically more effective) CMI surgical techniques, and have achieved a low complication rate which compares favorably with that one of the extradural techniques.

Loss of AND-34/BCAR3 expression in mice results in rupture of the adult lens.

PURPOSE: AND-34/BCAR3 (Breast Cancer Anti-Estrogen Resistance 3) associates with the focal adhesion adaptor protein, p130CAS/BCAR1. Expression of AND-34 regulates epithelial cell growth pattern, motility, and growth factor dependence. We sought to establish the effects of the loss of AND-34 expression in a mammalian organism. METHODS: AND-34(-/-) mice were generated by homologous recombination. Histopathology, in situ hybridization, and western blotting were performed on murine tissues. RESULTS: Western analyses confirmed total loss of expression in AND-34(-/-) splenic lymphocytes. Mice lacking AND-34 are fertile and have normal longevity. While AND-34 is widely expressed in wild type mice, histologic analysis of multiple organs in AND-34(-/-) mice is unremarkable and analyses of lymphocyte development show no overt changes. A small percentage of AND-34(-/-) mice show distinctive small white eye lesions resulting from the migration of ruptured cortical lens tissue into the anterior chamber. Following initial vacuolization and liquefaction of the lens cortex first observed at postnatal day three, posterior lens rupture occurs in all AND-34(-/-) mice, beginning as early as three weeks and seen in all mice at three months. Western blot analysis and in situ hybridization confirmed the presence of AND-34 RNA and protein in lens epithelial cells, particularly at the lens equator. Prior data link AND-34 expression to the activation of Akt signaling. While Akt Ser 473 phosphorylation was readily detectable in AND-34(+/+) lens epithelial cells, it was markedly reduced in the AND-34(-/-) lens epithelium. Basal levels of p130Cas phosphorylation were higher in AND-34(+/+) than in AND-34(-/-) lens epithelium. CONCLUSIONS: These results demonstrate the loss of AND-34 dysregulates focal adhesion complex signaling in lens epithelial cells and suggest that AND-34-mediated signaling is required for maintenance of the structural integrity of the adult ocular lens.

Chiari I Malformation: Opinions on Diagnostic Trends and Controversies from a Panel of 63 International Experts.

BACKGROUND: Chiari I malformation (CMI) and the topics concerning it have been the subject of numerous discussions and polarizing controversies over the course of the last 20 years. METHODS: The opinions of 63 recognized international CMI experts from 4 continents, with a collective surgical experience of >15,000 CMI cases, were gathered through a detailed questionnaire. RESULTS: Three facts emerged from the analysis of the results: 1) Most of the replies showed a high level of consensus on most CMI-related topics. 2) Several topics, which had been considered controversial as recently as 10 years ago, are now more widely accepted. 3) The so-called 5-mm rule was rejected by 88.5% of the CMI experts who responded to the questionnaire. CONCLUSIONS: Sixty three recognized international CMI experts from 4 continents, with a collective surgical experience of >15,000 CMI cases were polled about a number of CMI topics. The results showed a high level of consensus, as well as a paradigm shift.

Correction: Rapid Quantification of 3D Collagen Fiber Alignment and Fiber Intersection Correlations with High Sensitivity.

[This corrects the article DOI: 10.1371/journal.pone.0131814.].

Rapid Quantification of 3D Collagen Fiber Alignment and Fiber Intersection Correlations with High Sensitivity.

Metastatic cancers aggressively reorganize collagen in their microenvironment. For example, radially orientated collagen fibers have been observed surrounding tumor cell clusters in vivo. The degree of fiber alignment, as a consequence of this remodeling, has often been difficult to quantify. In this paper, we present an easy to implement algorithm for accurate detection of collagen fiber orientation in a rapid pixel-wise manner. This algorithm quantifies the alignment of both computer generated and actual collagen fiber networks of varying degrees of alignment within 5°°. We also present an alternative easy method to calculate the alignment index directly from the standard deviation of fiber orientation. Using this quantitative method for determining collagen alignment, we demonstrate that the number of collagen fiber intersections has a negative correlation with the degree of fiber alignment. This decrease in intersections of aligned fibers could explain why cells move more rapidly along aligned fibers than unaligned fibers, as previously reported. Overall, our paper provides an easier, more quantitative and quicker way to quantify fiber orientation and alignment, and presents a platform in studying effects of matrix and cellular properties on fiber alignment in complex 3D environments.

Structural insights into the association between BCAR3 and Cas family members, an atypical complex implicated in anti-oestrogen resistance.

The association between novel Src homology 2-containing protein (NSP) and Crk-associated substrate (Cas) family members contributes to integrin and receptor tyrosine kinase signalling and is involved in conferring anti-oestrogen resistance to human breast carcinomas. The precise role of this association in tumorigenesis remains controversial, and the molecular basis for the complex NSP and Cas protein form is unknown. Here we present a pluridisciplinary approach, including small-angle X-ray scattering, that provides first insights into the structure of the complex formed between breast cancer anti-oestrogen resistance 3 (BCAR3, an NSP family member) and human enhancer of filamentation 1 (HEF1, also named NEDD9 or Cas-L, a Cas family protein). Our analysis corroborates a four-helix bundle structure for the NSP-binding domain of HEF1 and a Cdc25-like guanine nucleotide exchange factor (GEF) fold for the Cas-binding domain of BCAR3. Using residues located on helix 2 of the four-helix bundle, HEF1 binds very tightly to a site on BCAR3 that is remote from the putative guanosine triphosphatase binding site of the GEF domain, but similar to a site implicated in allosteric regulation of the homologous SOS (Son of Sevenless) GEF domain. Thus, the association between NSP and Cas proteins might not only create a very stable link between these molecules, co-localising their cellular functions, but also modulate the function of the NSP GEF domains. Such modulation may explain, at least in part, the controversial results published for NSP GEF function.

AND-34/BCAR3 regulates adhesion-dependent p130Cas serine phosphorylation and breast cancer cell growth pattern.

NSP protein family members associate with p130Cas, a focal adhesion adapter protein best known as a Src substrate that integrates adhesion-related signaling. Over-expression of AND-34/BCAR3/NSP2 (BCAR3), but not NSP1 or NSP3, induces anti-estrogen resistance in human breast cancer cell lines. BCAR3 over-expression in epithelial MCF-7 cells augments levels of a phosphorylated p130Cas species that migrates more slowly on SDS-PAGE while NSP1 and NSP3 induce modest or no phosphorylation, respectively. Conversely, reduction in BCAR3 expression in mesenchymal MDA-231 cells by inducible shRNA results in loss of such p130Cas phosphorylation. Replacement of NSP3's serine/proline-rich domain with that of AND-34/BCAR3 instills the ability to induce p130Cas phosphorylation. Phospho-amino acid analysis demonstrates that BCAR3 induces p130Cas serine phosphorylation. Mass spectrometry identified phosphorylation at p130Cas serines 139, 437 and 639. p130Cas serine phosphorylation accumulates for several hours after adhesion of MDA-231 cells to fibronectin and is dependent upon BCAR3 expression. BCAR3 knockdown alters p130Cas localization and converts MDA-231 growth to an epithelioid pattern characterized by striking cohesiveness and lack of cellular projections at colony borders. These studies demonstrate that BCAR3 regulates p130Cas serine phosphorylation that is adhesion-dependent, temporally distinct from previously well-characterized rapid Fak and Src kinase-mediated p130Cas tyrosine phosphorylation and that correlates with invasive phenotype.