An early step in intracellular transport is the selective recognition of a vesicle by its appropriate target membrane, a process regulated by Rab GTPases via the recruitment of tethering effectors1–4. Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs alone5,6,7. Here, we addressed the mechanism whereby a tethered vesicle comes closer towards its target membrane for fusion by reconstituting an endosomal asymmetric tethering machinery consisting of the dimeric coiled-coil protein EEA16,7 recruited to phosphatidylinositol 3-phosphate membranes and binding vesicles harboring Rab5. Surprisingly, structural analysis revealed that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Through dynamic analysis by optical tweezers we confirmed that EEA1 captures a vesicle at a distance corresponding to its extended conformation, and directly measured its flexibility and the forces induced during the tethering reaction. Expression of engineered EEA1 variants defective in the conformational change induced prominent clusters of tethered vesicles in vivo. Our results suggest a new mechanism in which Rab5 induces a change in flexibility of EEA1, generating an entropic collapse force that pulls the captured vesicle toward the target membrane to initiate docking and fusion.

Syntaxin-1A is part of the SNARE complex that forms in membrane fusion in neuronal exocytosis of synaptic vesicles. Together with SNAP-25 the single-span transmembrane protein syntaxin-1A forms the receptor complex on the plasma membrane of neuroendocrine cells. Previous studies have shown that syntaxin-1A occurs in clusters that are different from lipid rafts in neuroendocrine plasma membranes. However, the interactions that promote these clusters have been largely unexplored. Here, we have reconstituted syntaxin-1A into lipid model membranes and we show that syntaxin cluster formation depends on cholesterol in a lipid system that lacks sphingomyelin and therefore does not form liquid-ordered phases that are commonly believed to represent lipid rafts in cell membranes. Rather, the cholesterol-induced clustering of syntaxin is found to be reversed by as little as 1 − 5 mol % of the regulatory lipid phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) and PI-4,5-P2 is shown to bind electrostatically to syntaxin, presumably mediated by the highly positively charged juxtamembrane domain of syntaxin. Possible implications of these results to the regulation of SNARE-mediated membrane fusion are discussed.

BackgroundSpecific genes, such as BCAT1 and IKZF1, are methylated with high frequency in colorectal cancer (CRC) tissue compared to normal colon tissue specimens. Such DNA may leak into blood and be present as cell-free circulating DNA. We have evaluated the accuracy of a novel blood test for these two markers across the spectrum of benign and neoplastic conditions encountered in the colon and rectum.MethodsCirculating DNA was extracted from plasma obtained from volunteers scheduled for colonoscopy for any reason, or for colonic surgery, at Australian and Dutch hospitals. The extracted DNA was bisulphite converted and analysed by methylation specific real-time quantitative PCR (qPCR). A specimen was deemed positive if one or more qPCR replicates were positive for either methylated BCAT1 or IKZF1 DNA. Sensitivity and specificity for CRC were estimated as the primary outcome measures.ResultsPlasma samples were collected from 2105 enrolled volunteers (mean age 62 years, 54 % male), including 26 additional samples taken after surgical removal of cancers. The two-marker blood test was run successfully on 2127 samples. The test identified 85 of 129 CRC cases (sensitivity of 66 %, 95 % CI: 57–74). For CRC stages I-IV, respective positivity rates were 38 % (95 % CI: 21–58), 69 % (95 % CI: 53–82), 73 % (95 % CI: 56–85) and 94 % (95 % CI: 70–100). A positive trend was observed between positivity rate and degree of invasiveness. The colonic location of cancer did not influence assay positivity rates. Gender, age, smoking and family history were not significant predictors of marker positivity. Twelve methylation-positive cancer cases with paired pre- and post-surgery plasma showed reduction in methylation signal after surgery, with complete disappearance of signal in 10 subjects. Sensitivity for advanced adenoma (n = 338) was 6 % (95 % CI: 4–9). Specificity was 94 % (95 % CI: 92–95) in all 838 non-neoplastic pathology cases and 95 % (95 % CI: 92–97) in those with no colonic pathology detected (n = 450).ConclusionsThe sensitivity for cancer of this two-marker blood test justifies prospective evaluation in a true screening population relative to a proven screening test. Given the high rate of marker disappearance after cancer resection, this blood test might also be useful to monitor tumour recurrence.Trial registrationACTRN12611000318987.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1674-2) contains supplementary material, which is available to authorized users.

Recurrence will develop in 30–50% of colorectal cancer (CRC) cases despite apparent clearance following treatment. Carcinoembryonic antigen (CEA) is the only guideline‐recommended blood test for monitoring cases for recurrence, but its sensitivity and specificity are suboptimal. This observational study compared a novel 2‐gene (methylated BCAT1 and IKZF1 DNA) blood test with CEA for detection of recurrent CRC. We conducted a paired comparison of the BCAT1/IKZF1 test with CEA (cut‐off 5 ng/mL) in blood from patients in remission after treatment for primary CRC and undergoing surveillance. Blood collected in the 12 months prior to or 3 months after complete investigational assessment of recurrence status were assayed and the results compared by McNemar's test. Of 397 patients enrolled, 220 underwent satisfactory assessment for recurrence and 122 had blood testing performed within the prescribed period. In 28 cases with recurrent CRC, CEA was positive in 9 (32%; 95% CI 16–52%) compared to 19 (68%; 95% CI 48–84%) positive for methylated BCAT1/IKZF1 (P = 0.002). All samples that were CEA positive were also BCAT1/IKZF1 positive. In 94 patients without clinically detectable recurrence, CEA was positive in 6 (6%, 95% CI 2–13%) and BCAT1/IKZF1 in 12 (13%, 95% CI 7–21%), P = 0.210. The odds ratio of a positive CEA test for recurrence was 6.9 (95% CI 2–22) compared to 14.4 (5–39) for BCAT1/IKZF1. The BCAT1/IKZF1 test was more sensitive for recurrence than CEA and the odds of recurrence given a positive test was twice that of CEA. The BCAT1/IKZF1 test should be further considered for monitoring cases for recurrence.

Objectives:To compare the performance of a new blood test for colorectal cancer (CRC) to an established fecal immunochemical test (FIT) in a study population with the full range of neoplastic and non-neoplastic pathologies encountered in the colon and rectum.Methods:Volunteers were asked to complete a FIT prior to colonoscopy. Blood was collected after bowel preparation but prior to colonoscopy, and plasma was assayed for the presence of methylated BCAT1 and IKZF1 DNA using a multiplex real-time PCR assay. Sensitivity and specificity estimates for the blood test were calculated from true- and false-positive rates for neoplasia and compared with FIT at a range of fecal hemoglobin (Hb) concentration positivity thresholds.Results:In total, 1,381 volunteers (median age 64 years; 49% male) completed both tests prior to colonoscopy. Estimated sensitivity of the BCAT1/IKZF1 blood test for CRC was 62% (41/66; 95% confidence interval 49–74%) with a specificity of 92% (1207/1315; 90–93%). FIT returned the same specificity at a cutoff of 60 μg Hb/g, at which its corresponding sensitivity for cancer was 64% (42/66; 51–75%). In the range of commonly used FIT cutoffs, respective cancer sensitivity and specificity estimates with FIT were: 59% (46–71%) and 93% (92–95%) at 80 μg Hb/g, and 79% (67–88%) and 81% (78–83%) at 10 μg Hb/g. Although estimated sensitivities were not significantly different between the two tests for any stage of cancer, FIT showed a significantly higher sensitivity for advanced adenoma at the lower cutoffs. Specificity of FIT, but not of the BCAT1/IKZF1 blood test, deteriorated substantially in people with overt blood in the feces. When combining FIT (cutoff 10 μg Hb/g) with the BCAT1/IKZF1 blood test, sensitivity for cancer was 89% (79–96%) at 74% (72–77%) specificity.Conclusions:A test based on detection of methylated BCAT1/IKZF1 DNA in blood has comparable sensitivity but better specificity for CRC than FIT at the commonly used positivity threshold of 10 μg Hb/g. Further evaluation of the new test relative to FIT in the population screening context is now required to fully understand the potential advantages and disadvantages of these biomarkers in screening.