Initiation of T cell signaling by CD45 segregation at 'close contacts'.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.The authors thank R.A. Cornall, M.L. Dustin and P.A. van der Merwe for comments on the manuscript and S. Ikemizu for useful discussions about the structure. We also thank W. Lu and T. Walter for technical support with protein expression and crystallization, the staff at Diamond Light Source beamlines I02, I03 and I04-1 (proposal mx10627) and European Synchrotron Radiation Facility beamlines ID23EH1 and ID23EH2 for assistance at the synchrotrons, G. Sutton for assistance with MALS experiments, and M. Fritzsche for advice on the calcium analysis. This work was funded by the Wellcome Trust (098274/Z/12/Z to S.J.D.; 090532/Z/09/Z to R.J.C.G.; 090708/Z/09/Z to D.K.), the UK Medical Research Council (G0700232 to A.R.A.), the Royal Society (UF120277 to S.F.L.) and Cancer Research UK (C20724/A14414 to C.S.; C375/A10976 to E.Y.J.). The Oxford Division of Structural Biology is part of the Wellcome Trust Centre for Human Genetics, Wellcome Trust Core Award Grant Number 090532/Z/09/Z. We acknowledge financial support from Instruct, an ESFRI Landmark Project. The OPIC electron microscopy facility was funded by a Wellcome Trust JIF award (060208/Z/00/Z).This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/ni.339

Size-dependent protein segregation at membrane interfaces

Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane protein organization, such as E-cadherin enrichment in epithelial junctional complexes and CD45 exclusion from the signaling foci of immunological synapses. To isolate the role of protein size in these processes, we reconstituted membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between binding and non-binding proteins can dramatically alter their organization at membrane interfaces in the absence of active contributions from the cytoskeleton, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally-driven membrane height fluctuations that transiently limit access to the interface. This simple, sensitive, and highly effective means of passively segregating proteins has implications for signaling at cell-cell junctions and protein sorting at intracellular contact points between membrane-bound organelles

Diagnosis and management of acute variceal bleeding: Asian Pacific Association for Study of the Liver recommendations

Acute variceal bleeding (AVB) is a medical emergency and associated with a mortality of 20% at 6 weeks. Significant advances have occurred in the recent past and hence there is a need to update the existing consensus guidelines. There is also a need to include the literature from the Eastern and Asian countries where majority of patients with portal hypertension (PHT) live. The expert working party, predominantly from the Asia-Pacific region, reviewed the existing literature and deliberated to develop consensus guidelines. The working party adopted the Oxford system for developing an evidence-based approach. Only those statements that were unanimously approved by the experts were accepted. AVB is defined as a bleed in a known or suspected case of PHT, with the presence of hematemesis within 24 h of presentation, and/or ongoing melena, with last melanic stool within last 24 h. The time frame for the AVB episode is 48 h. AVB is further classified as active or inactive at the time of endoscopy. Combination therapy with vasoactive drugs (< 30 min of hospitalization) and endoscopic variceal ligation (door to scope time < 6 h) is accepted as first-line therapy. Rebleeding (48 h of T (0)) is further sub-classified as very early rebleeding (48 to 120 h from T (0)), early rebleeding (6 to 42 days from T (0)) and late rebleeding (after 42 days from T (0)) to maintain uniformity in clinical trials. Emphasis should be to evaluate the role of adjusted blood requirement index (ABRI), assessment of associated comorbid conditions and poor predictors of non-response to combination therapy, and proposed APASL (Asian Pacific Association for Study of the Liver) Severity Score in assessing these patients. Role of hepatic venous pressure gradient in AVB is considered useful. Antibiotic (cephalosporins) prophylaxis is recommended and search for acute ischemic hepatic injury should be done. New guidelines have been developed for management of variceal bleed in patients with non-cirrhotic PHT and variceal bleed in pediatric patients. Management of acute variceal bleeding in Asia-Pacific region needs special attention for uniformity of treatment and future clinical trials