Dissecting the mechanism of Ca2+-triggered membrane fusion : probing protein function using thiol reactivity

Ca2+-triggered membrane fusion involves the coordinated actions of both lipids and proteins, but the specific mechanisms remain poorly understood. The urchin cortical vesicle model is a stage-specific native preparation fully enabling the directly coupled functional-molecular analyses necessary to identify critical components of fast triggered membrane fusion. 2. Recent work on lipidic components has established a direct role for cholesterol in the fusion mechanism via local contribution of negative curvature to readily enable the formation of transient lipidic fusion intermediates. In addition, cholesterol- and sphingomyelin-enriched domains regulate the efficiency of fusion by focally organizing other components to ensure an optimized response to the triggering Ca2+ transient. 3. There is less known about the identity of proteins involved in the Ca2+-triggering steps of membrane fusion. Thiol reagents can be used as unbiased tools to probe protein functions. Comparisons of several thiol-reactive reagents have identified different effects on Ca2+ sensitivity and the extent of fusion, suggesting that there are at least two distinct thiol sites that participate in the fusion mechanism: one that regulates the efficiency of Ca2+ sensing/triggering and one that may function during the membrane merger event itself. 4. To identify the proteins that regulate Ca2+ sensitivity, the fluorescent thiol reagent Lucifer yellow iodoacetamide was used to potentiate fusion and simultaneously tag the proteins involved. Ongoing work involves the isolation of cholesterol-enriched membrane fractions to reduce the complexity of the labelled proteome, narrowing the number of candidate proteins

Unbiased Thiol-Labeling and Top-Down Proteomic Analyses Implicate Multiple Proteins in the Late Steps of Regulated Secretion

Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca2+ modulates different stages of exocytosis is still poorly understood. For an unbiased, top-down proteomic approach, select thiol- reactive reagents were used to investigate this process in release-ready native secretory vesicles. We previously characterized a biphasic effect of these reagents on Ca2+-triggered exocytosis: low doses potentiated Ca2+ sensitivity, whereas high doses inhibited Ca2+ sensitivity and extent of vesicle fusion. Capitalizing on this novel potentiating effect, we have now identified fluorescent thiol- reactive reagents producing the same effects: Lucifer yellow iodoacetamide, monobromobimane, and dibromobimane. Top-down proteomic analyses of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions using two-dimensional gel electrophoresis coupled with mass spectrometry identified several candidate targets, some of which have been previously linked to the late steps of regulated exocytosis and some of which are novel. Initial validation studies indicate that Rab proteins are involved in the modulation of Ca2+ sensitivity, and thus the efficiency of membrane fusion, which may, in part, be linked to their previously identified upstream roles in vesicle docking

Identifying critical components of native Ca²⁺-triggered membrane fusion : integrating studies of proteins and lipids

Ca2+-triggered membrane fusion is the defining step of exocytosis. Despite realization that the fusion machinery must include lipids and proteins working in concert, only of late has work in the field focused more equally on both these components. Here we use isolated sea urchin egg cortical vesicles (CV), a stage-specific preparation of Ca2+-sensitive release-ready vesicles that enables the tight coupling of molecular and functional analyses necessary to dissect molecular mechanisms. The stalk-pore hypothesis proposes that bilayermerger proceeds rapidly via transient, high-negative curvature, intermediate membrane structures. Consistent with this, cholesterol, a major component of the CV membrane, contributes to a critical local negative curvature that supports formation of lipidic fusion intermediates. Following cholesterol depletion, structurally dissimilar lipids having intrinsic negative curvature greater than or equal to cholesterol recover the ability of CV to fuse but do not recover fusion efficiency (Ca2+ sensitivity and kinetics). Conversely, cholesterol- and sphingomyelin-enriched microdomains regulate the efficiency of the fusion mechanism, presumably by contributing spatial and functional organization of other critical lipids and proteins at the fusion site. Critical proteins are thought to participate in Ca2+ sensing, initiating membrane deformations, and facilitating fusion pore expansion. Capitalizing on a novel effect of the thiol-reactive reagent iodoacetamide (IA), potentiation of the Ca2+ sensitivity and kinetics, a fluorescently tagged IA has been used to enhance fusion efficiency and simultaneously label the proteins involved. Isolation of cholesterol-enriched CV membrane fractions, using density gradient centrifugation, is being used to narrow the list of protein candidates potentially critical to the mechanism of fast Ca2+-triggered membrane fusion

Secretory vesicle cholesterol : correlating lipid domain organization and Ca2+ triggered fusion

Membrane organization has received substantial research interest since the degree of ordering in membrane regions is relevant in many biological processes. Here we relate the impact of varying cholesterol concentrations on native secretory vesicle fusion and the lateral domain organization of membrane extracts from these vesicles. Membranes of isolated cortical secretory vesicles were either depleted of cholesterol, had cholesterol loaded to excess of native levels, or were depleted of cholesterol but subsequently reloaded to restore native cholesterol levels. Lipid analyses confirmed cholesterol was the only species significantly altered by these treatments. Treated vesicles were characterized for their ability to undergo fusion. Cholesterol depletion resulted in a decrease of Ca2 + sensitivity and the extent of fusion, while cholesterol loading had no effect on fusion parameters. Membrane extracts were characterized in terms of lipid packing by surface pressure-area isotherms whereas the lateral membrane organization was analyzed by Brewster angle microscopy. While no differences in the isotherms were observed, imaging revealed drastic differences in domain size, shape and frequency between the various conditions. Cholesterol depletion induced larger but fewer domains, suggesting that domain coalescence into larger structures may disrupt the native temporal-spatial organization of the fusion machinery and thus inhibit vesicle docking, priming, and fusion. In contrast, adding excess cholesterol, or rescuing with exogenous cholesterol after sterol depletion, resulted in more but smaller domains. Therefore, cholesterol is an important membrane organizer in the process of Ca2 + triggered vesicular fusion, which can be related to specific physical effects on native membrane substructure

Enhancement of the Ca2+-triggering steps of native membrane fusion via thiol-reactivity

Ca2+-triggered membrane fusion is the defining step of exocytosis. Isolated urchin cortical vesicles (CV) provide a stage-specific preparation to study the mechanisms by which Ca2+ triggers the merger of two apposed native membranes. Thiol-reactive reagents that alkylate free sulfhydryl groups on proteins have been consistently shown to inhibit triggered fusion. Here, we characterize a novel effect of the alkylating reagent iodoacetamide (IA). IA was found to enhance the kinetics and Ca2+ sensitivity of both CV-plasma membrane and CV–CV fusion. If Sr2+, a weak Ca2+ mimetic, was used to trigger fusion, the potentiation was even greater than that observed for Ca2+, suggesting that IA acts at the Ca2+-sensing step of triggered fusion. Comparison of IA to other reagents indicates that there are at least two distinct thiol sites involved in the underlying fusion mechanism: one that regulates the efficiency of fusion and one that interferes with fusion competency

Health-status outcomes with invasive or conservative care in coronary disease

BACKGROUND In the ISCHEMIA trial, an invasive strategy with angiographic assessment and revascularization did not reduce clinical events among patients with stable ischemic heart disease and moderate or severe ischemia. A secondary objective of the trial was to assess angina-related health status among these patients. METHODS We assessed angina-related symptoms, function, and quality of life with the Seattle Angina Questionnaire (SAQ) at randomization, at months 1.5, 3, and 6, and every 6 months thereafter in participants who had been randomly assigned to an invasive treatment strategy (2295 participants) or a conservative strategy (2322). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate differences between the treatment groups. The primary outcome of this health-status analysis was the SAQ summary score (scores range from 0 to 100, with higher scores indicating better health status). All analyses were performed in the overall population and according to baseline angina frequency. RESULTS At baseline, 35% of patients reported having no angina in the previous month. SAQ summary scores increased in both treatment groups, with increases at 3, 12, and 36 months that were 4.1 points (95% credible interval, 3.2 to 5.0), 4.2 points (95% credible interval, 3.3 to 5.1), and 2.9 points (95% credible interval, 2.2 to 3.7) higher with the invasive strategy than with the conservative strategy. Differences were larger among participants who had more frequent angina at baseline (8.5 vs. 0.1 points at 3 months and 5.3 vs. 1.2 points at 36 months among participants with daily or weekly angina as compared with no angina). CONCLUSIONS In the overall trial population with moderate or severe ischemia, which included 35% of participants without angina at baseline, patients randomly assigned to the invasive strategy had greater improvement in angina-related health status than those assigned to the conservative strategy. The modest mean differences favoring the invasive strategy in the overall group reflected minimal differences among asymptomatic patients and larger differences among patients who had had angina at baseline

Initial invasive or conservative strategy for stable coronary disease

BACKGROUND Among patients with stable coronary disease and moderate or severe ischemia, whether clinical outcomes are better in those who receive an invasive intervention plus medical therapy than in those who receive medical therapy alone is uncertain. METHODS We randomly assigned 5179 patients with moderate or severe ischemia to an initial invasive strategy (angiography and revascularization when feasible) and medical therapy or to an initial conservative strategy of medical therapy alone and angiography if medical therapy failed. The primary outcome was a composite of death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. A key secondary outcome was death from cardiovascular causes or myocardial infarction. RESULTS Over a median of 3.2 years, 318 primary outcome events occurred in the invasive-strategy group and 352 occurred in the conservative-strategy group. At 6 months, the cumulative event rate was 5.3% in the invasive-strategy group and 3.4% in the conservative-strategy group (difference, 1.9 percentage points; 95% confidence interval [CI], 0.8 to 3.0); at 5 years, the cumulative event rate was 16.4% and 18.2%, respectively (difference, 121.8 percentage points; 95% CI, 124.7 to 1.0). Results were similar with respect to the key secondary outcome. The incidence of the primary outcome was sensitive to the definition of myocardial infarction; a secondary analysis yielded more procedural myocardial infarctions of uncertain clinical importance. There were 145 deaths in the invasive-strategy group and 144 deaths in the conservative-strategy group (hazard ratio, 1.05; 95% CI, 0.83 to 1.32). CONCLUSIONS Among patients with stable coronary disease and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of ischemic cardiovascular events or death from any cause over a median of 3.2 years. The trial findings were sensitive to the definition of myocardial infarction that was used