A functional interaction between liprin-α1 and B56γ regulatory subunit of protein phosphatase 2A supports tumor cell motility.

Scaffold liprin-α1 is required to assemble dynamic plasma membrane-associated platforms (PMAPs) at the front of migrating breast cancer cells, to promote protrusion and invasion. We show that the N-terminal region of liprin-α1 contains an LxxIxE motif interacting with B56 regulatory subunits of serine/threonine protein phosphatase 2A (PP2A). The specific interaction of B56γ with liprin-α1 requires an intact motif, since two point mutations strongly reduce the interaction. B56γ mediates the interaction of liprin-α1 with the heterotrimeric PP2A holoenzyme. Most B56γ protein is recovered in the cytosolic fraction of invasive MDA-MB-231 breast cancer cells, where B56γ is complexed with liprin-α1. While mutation of the short linear motif (SLiM) does not affect localization of liprin-α1 to PMAPs, localization of B56γ at these sites specifically requires liprin-α1. Silencing of B56γ or liprin-α1 inhibits to similar extent cell spreading on extracellular matrix, invasion, motility and lamellipodia dynamics in migrating MDA-MB-231 cells, suggesting that B56γ/PP2A is a novel component of the PMAPs machinery regulating tumor cell motility. In this direction, inhibition of cell spreading by silencing liprin-α1 is not rescued by expression of B56γ binding-defective liprin-α1 mutant. We propose that liprin-α1-mediated recruitment of PP2A via B56γ regulates cell motility by controlling protrusion in migrating MDA-MB-231 cells.

Paxillin: A Hub for Mechano-Transduction from the ß3 Integrin-Talin-Kindlin Axis.

Focal adhesions are specialized integrin-dependent adhesion complexes, which ensure cell anchoring to the extracellular matrix. Focal adhesions also function as mechano-signaling platforms by perceiving and integrating diverse physical and (bio)chemical cues of their microenvironment, and by transducing them into intracellular signaling for the control of cell behavior. The fundamental biological mechanism of creating intracellular signaling in response to changes in tensional forces appears to be tightly linked to paxillin recruitment and binding to focal adhesions. Interestingly, the tension-dependent nature of the paxillin binding to adhesions, combined with its scaffolding function, suggests a major role of this protein in integrating multiple signals from the microenvironment, and accordingly activating diverse molecular responses. This minireview offers an overview of the molecular bases of the mechano-sensitivity and mechano-signaling capacity of core focal adhesion proteins, and highlights the role of paxillin as a key component of the mechano-transducing machinery based on the interaction of cells to substrates activating the ß3 integrin-talin1-kindlin.

Predictive Models for the Functional Recovery of Transplanted Kidney.

BACKGROUND: Renal transplantation is the gold standard treatment for end-stage renal disease, however, in 20% of cases, the graft develops a delayed graft function (DGF) that is associated with both early and late worsening of the outcome. The aim of this study was to examine and validate in a population of transplanted patients the appropriateness of the predictive score systems of DGF available to identify patients who might take advantage of a tailored immunosuppressive therapy. MATERIALS AND METHODS: We conducted a systematic review of the literature to identify articles concerning scoring systems predicting DGF to identify those applicable to the study population and subsequently comparing their appropriateness for defining the most accurate one. RESULTS: From an analysis of the scientific literature, we found 7 scoring systems predicting DGF. Of these, 3 can be calculated for the study population. We enrolled 247 renal transplants in the study. DGF was recorded in 41 cases (15.95%). The Irish score recognized 25 of 41 cases (60.98%), the Jeldres score 41 of 41 cases (100%), and the Chapal score only 7 of 41 (17.07%). Although the Irish score did not identify all cases of DGF, the analysis of data revealed that it is the most accurate, with area under the receiver operating characteristic almost overlapping. CONCLUSIONS: The study resulted in some interesting and promising conclusions about the predictability of DGF, defining the Irish score as the most reliable. This result can be considered the fundamental requirement to develop a custom therapeutic algorithm to be applied to all recipients with higher probability of developing DGF.

Indocyanine Green Angiography for Quality Assessment of the Kidney During Transplantation: An Outcome Predictor Prospective Study.

BACKGROUND: Microvascular damage is the main cause of delayed graft function (DGF) after kidney transplant. Assessing its extent may be helpful in predicting DGF to achieve better postoperative management, especially in terms of an immunosuppressive regimen. Our aim was to explore the capability of intraoperative indocyanine green (ICG) angiography to examine the microvasculature of the kidney. METHODS: We conducted a prospective cohort study on 37 kidney transplant recipients in a high-volume kidney transplant center. During surgery, after graft implant, an ICG angiography was performed through a high-definition Storz camera system (Karl Storz GmbH, Tuttlingen, Germany) with successive quantitative assessment of fluorescence using Icy bioimage analysis. RESULTS: All transplanted kidneys that showed immediate recovery of their function had a fluorescent intensity ≥49.953 with a mean of 96.930 ± 21. The fluorescence intensity for kidneys that showed a delayed recovery of their function never exceeded 55.648, and the mean was 37.718 ± 13. The difference between the 2 groups was statistically significant with a P value < .001. The only kidney that never recovered showed a fluorescence intensity consistently <25.220, the lowest detected. CONCLUSIONS: This study demonstrates that intraoperative ICG angiography may be used to assess the microvasculature of the graft. A statistically significant difference in terms of fluorescent intensity can be highlighted between kidneys that immediately recover their function and those with delayed recovery. Further larger studies are needed to confirm the capability of the technique to predict DGF to optimize the transplanted patients' management.

Structural and functional analysis of LIM domain-dependent recruitment of paxillin to αvß3 integrin-positive focal adhesions.

The LIM domain-dependent localization of the adapter protein paxillin to ß3 integrin-positive focal adhesions (FAs) is not mechanistically understood. Here, by combining molecular biology, photoactivation and FA-isolation experiments, we demonstrate specific contributions of each LIM domain of paxillin and reveal multiple paxillin interactions in adhesion-complexes. Mutation of ß3 integrin at a putative paxillin binding site (ß3VE/YA) leads to rapidly inward-sliding FAs, correlating with actin retrograde flow and enhanced paxillin dissociation kinetics. Induced mechanical coupling of paxillin to ß3VE/YA integrin arrests the FA-sliding, thereby disclosing an essential structural function of paxillin for the maturation of ß3 integrin/talin clusters. Moreover, bimolecular fluorescence complementation unveils the spatial orientation of the paxillin LIM-array, juxtaposing the positive LIM4 to the plasma membrane and the ß3 integrin-tail, while in vitro binding assays point to LIM1 and/or LIM2 interaction with talin-head domain. These data provide structural insights into the molecular organization of ß3 integrin-FAs.

Induction of ligand promiscuity of αVß3 integrin by mechanical force.

αVß3 integrin can bind to multiple extracellular matrix proteins, including vitronectin (Vn) and fibronectin (Fn), which are often presented to cells in culture as homogenous substrates. However, in tissues, cells experience highly complex and changing environments. To better understand integrin ligand selection in such complex environments, we employed binary-choice substrates of Fn and Vn to dissect αVß3 integrin-mediated binding to different ligands on the subcellular scale. Super-resolution imaging revealed that αVß3 integrin preferred binding to Vn under various conditions. In contrast, binding to Fn required higher mechanical load on αVß3 integrin. Integrin mutations, structural analysis and chemical inhibition experiments indicated that the degree of hybrid domain swing-out is relevant for the selection between Fn and Vn; only a force-mediated, full hybrid domain swing-out facilitated αVß3-Fn binding. Thus, force-dependent conformational changes in αVß3 integrin increased the diversity of available ligands for binding and therefore enhanced the ligand promiscuity of this integrin.This article has an associated First Person interview with the first author of the paper.

Cell death induced by 2-phenylethynesulfonamide uncovers a pro-survival function of BAX.

PES (2-phenylethynesulfonamide) was initially identified as an inhibitor of p53 translocation to mitochondria and named Pifithrin-µ. Further studies showed that PES selectively killed tumour cells and was thus a promising anticancer agent. PES-induced cell death was characterised by a non-apoptotic, autophagosome-rich phenotype. We observed this phenotype via electron microscopy in wild type (wt) and double Bax-/- Bak-/- (DKO) mouse embryonic fibroblasts (MEFs) treated with PES. We excluded the involvement of effector caspases, BAX and BAK, in causing PES-triggered cell death. Therefore, apoptosis was ruled out as the lethal mode of action of PES. Surprisingly, MEFs containing BAX were significantly protected from PES treatments. BAX overexpression in Bax-/- MEFs confirmed this pro-survival effect. Moreover, this protective effect required the ability of BAX to localise to mitochondrial membranes. Conversely, mitochondrial fusion induced by treatment with Mdivi-1 conferred increased resistance to MEFs subjected to PES treatment. The involvement of BAX in the regulation of mitochondrial dynamics has been reported. We propose the promotion of mitochondrial fusion by BAX to be the pro-survival function attributed to BAX.