Simon Walker

Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum

Autophagy is the engulfment of cytosol and organelles by double-membrane vesicles termed autophagosomes. Autophagosome formation is known to require phosphatidylinositol 3-phosphate (PI(3)P) and occurs near the endoplasmic reticulum (ER), but the exact mechanisms are unknown. We show that double FYVE domain–containing protein 1, a PI(3)P-binding protein with unusual localization on ER and Golgi membranes, translocates in response to amino acid starvation to a punctate compartment partially colocalized with autophagosomal proteins. Translocation is dependent on Vps34 and beclin function. Other PI(3)P-binding probes targeted to the ER show the same starvation-induced translocation that is dependent on PI(3)P formation and recognition. Live imaging experiments show that this punctate compartment forms near Vps34-containing vesicles, is in dynamic equilibrium with the ER, and provides a membrane platform for accumulation of autophagosomal proteins, expansion of autophagosomal membranes, and emergence of fully formed autophagosomes. This PI(3)P-enriched compartment may be involved in autophagosome biogenesis. Its dynamic relationship with the ER is consistent with the idea that the ER may provide important components for autophagosome formation.We have recently proposed that some autophagosomes are formed within omegasomes, membrane sites connected to the endoplasmic reticulum and enriched in phosphatidylinositol 3-phosphate. In order to understand if there is any biological advantage to having such a precursor in autophagosome biogenesis, we generated a simple computer program that simulates omegasome and autophagosome formation under a variety of conditions. We concluded from running this simulation that having a transient precursor permits a bigger dynamic range of the autophagic response and allows a more efficient approach to steady state after autophagy stimulation.

High sensitivity cardiac troponin and the under-diagnosis of myocardial infarction in women: prospective cohort study.

Objective To evaluate the diagnosis of myocardial infarction using a high sensitivity troponin I assay and sex specific diagnostic thresholds in men and women with suspected acute coronary syndrome. Design Prospective cohort study. Setting Regional cardiac centre, United Kingdom. Participants Consecutive patients with suspected acute coronary syndrome (n=1126, 46% women). Two cardiologists independently adjudicated the diagnosis of myocardial infarction by using a high sensitivity troponin I assay with sex specific diagnostic thresholds (men 34 ng/L, women 16 ng/L) and compared with current practice where a contemporary assay (50 ng/L, single threshold) was used to guide care. Main outcome measure Diagnosis of myocardial infarction. Results The high sensitivity troponin I assay noticeably increased the diagnosis of myocardial infarction in women (from 11% to 22%; P<0.001) but had a minimal effect in men (from 19% to 21%, P=0.002). Women were less likely than men to be referred to a cardiologist or undergo coronary revascularisation (P<0.05 for both). At 12 months, women with undisclosed increases in troponin concentration (17-49 ng/L) and those with myocardial infarction (≥50 ng/L) had the highest rate of death or reinfarction compared with women without (≤16 ng/L) myocardial infarction (25%, 24%, and 4%, respectively; P<0.001). Conclusions Although having little effect in men, a high sensitivity troponin assay with sex specific diagnostic thresholds may double the diagnosis of myocardial infarction in women and identify those at high risk of reinfarction and death. Whether use of sex specific diagnostic thresholds will improve outcomes and tackle inequalities in the treatment of women with suspected acute coronary syndrome requires urgent attention.

High-sensitivity troponin I concentrations are a marker of an advanced hypertrophic response and adverse outcomes in patients with aortic stenosis.

Aims High-sensitivity cardiac troponin I (cTnI) assays hold promise in detecting the transition from hypertrophy to heart failure in aortic stenosis. We sought to investigate the mechanism for troponin release in patients with aortic stenosis and whether plasma cTnI concentrations are associated with long-term outcome. Methods and results Plasma cTnI concentrations were measured in two patient cohorts using a high-sensitivity assay. First, in the Mechanism Cohort, 122 patients with aortic stenosis (median age 71, 67% male, aortic valve area 1.0 ± 0.4 cm2) underwent cardiovascular magnetic resonance and echocardiography to assess left ventricular (LV) myocardial mass, function, and fibrosis. The indexed LV mass and measures of replacement fibrosis (late gadolinium enhancement) were associated with cTnI concentrations independent of age, sex, coronary artery disease, aortic stenosis severity, and diastolic function. In the separate Outcome Cohort, 131 patients originally recruited into the Scottish Aortic Stenosis and Lipid Lowering Trial, Impact of REgression (SALTIRE) study, had long-term follow-up for the occurrence of aortic valve replacement (AVR) and cardiovascular deaths. Over a median follow-up of 10.6 years (1178 patient-years), 24 patients died from a cardiovascular cause and 60 patients had an AVR. Plasma cTnI concentrations were associated with AVR or cardiovascular death HR 1.77 (95% CI, 1.22 to 2.55) independent of age, sex, systolic ejection fraction, and aortic stenosis severity. Conclusions In patients with aortic stenosis, plasma cTnI concentration is associated with advanced hypertrophy and replacement myocardial fibrosis as well as AVR or cardiovascular death.

P-Rex2, a new guanine-nucleotide exchange factor for Rac

We have identified a new guanine‐nucleotide exchange factor, P‐Rex2, and cloned it from human skeletal muscle and brain libraries. It has widespread tissue distribution but is not expressed in neutrophils. P‐Rex2 is a 183 kDa protein that activates the small GTPase Rac and is regulated by phosphatidylinositol (3,4,5)‐trisphosphate and the βγ subunits of heterotrimeric G proteins in vitro and in vivo. P‐Rex2 has structure, activity and regulatory properties similar to P‐Rex1 but has divergent tissue distribution, as P‐Rex1 is mainly expressed in neutrophils. Together, they form an enzyme family capable of mediating Rac signalling downstream of G protein‐coupled receptors and phosphoinositide 3‐kinase.

Dynamic association of the ULK1 complex with omegasomes during autophagy induction

Summary Induction of autophagy requires the ULK1 protein kinase complex and the Vps34 lipid kinase complex. PtdIns3P synthesised by Vps34 accumulates in omegasomes, membrane extensions of the ER within which some autophagosomes form. The ULK1 complex is thought to target autophagosomes independently of PtdIns3P, and its functional relationship to omegasomes is unclear. Here we show that the ULK1 complex colocalises with omegasomes in a PtdIns3P-dependent way. Live-cell imaging of Atg13 (a ULK1 complex component), omegasomes and LC3 establishes and annotates for the first time a complete sequence of steps leading to autophagosome formation, as follows. Upon starvation, the ULK1 complex forms puncta associated with the ER and sporadically with mitochondria. If PtdIns3P is available, these puncta become omegasomes. Subsequently, the ULK1 complex exits omegasomes and autophagosomes bud off. If PtdIns3P is unavailable, ULK1 puncta are greatly reduced in number and duration. Atg13 contains a region with affinity for acidic phospholipids, required for translocation to punctate structures and autophagy progression.

GAP1 Family Members Constitute Bifunctional Ras and Rap GTPase-activating Proteins

GAP1IP4BP is a member of the GAP1 family of Ras GTPase-activating proteins (Ras GAPs) that includes GAP1m, CAPRI, and RASAL. Composed of a central Ras GAP domain, surrounded by amino-terminal C2 domains and a carboxyl-terminal pleckstrin homology/Brutons tyrosine kinase domain, GAP1IP4BP has previously been shown to possess an unexpected GAP activity on the Ras-related protein Rap, besides the predicted Ras GAP activity (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527–530). Here we have shown that GAP1IP4BP is indeed an efficient Ras/Rap GAP, having Kms of 213 and 42 μm and estimated kcats of 48 and 16 s–1 for Ras and Rap, respectively. For this dual activity, regions outside the Ras GAP domain are required, as the isolated domain (residues 291–569) retains a pronounced Ras GAP activity yet has very low activity toward Rap. Interestingly, mutagenesis of the Ras GAP arginine finger, and surrounding residues important in Ras binding, inhibit both Ras and Rap GAP activity of GAP1IP4BP. Although the precise details by which GAP1IP4BP can function as a Rap GAP remain to be determined, these data are consistent with Rap associating with GAP1IP4BP through the Ras-binding site within the Ras GAP domain. Finally, we have established that such dual Ras/Rap GAP activity is not restricted to GAP1IP4BP. Although GAP1m appears to constitute a specific Ras GAP, CAPRI and RASAL display dual activity. For CAPRI, its Rap GAP activity is modulated upon its Ca2+-induced association with the plasma membrane.

CAPRI and RASAL impose different modes of information processing on Ras due to contrasting temporal filtering of Ca2

The versatility of Ca2+ as a second messenger lies in the complex manner in which Ca2+ signals are generated. How information contained within the Ca2+ code is interpreted underlies cell function. Recently, we identified CAPRI and RASAL as related Ca2+-triggered Ras GTPase-activating proteins. RASAL tracks agonist-stimulated Ca2+ oscillations by repetitively associating with the plasma membrane, yet CAPRI displays a long-lasting Ca2+-triggered translocation that is refractory to cytosolic Ca2+ oscillations. CAPRI behavior is Ca2+- and C2 domain–dependent but sustained recruitment is predominantly Ca2+ independent, necessitating integration of Ca2+ by the C2 domains with agonist-evoked plasma membrane interaction sites for the pleckstrin homology domain. Using an assay to monitor Ras activity in real time, we correlate the spatial and temporal translocation of CAPRI with the deactivation of H-Ras. CAPRI seems to low-pass filter the Ca2+ signal, converting different intensities of stimulation into different durations of Ras activity in contrast to the preservation of Ca2+ frequency information by RASAL, suggesting sophisticated modes of Ca2+-regulated Ras deactivation.

Sensitive Troponin Assay and the Classification of Myocardial Infarction

Background Lowering the diagnostic threshold for troponin is controversial because it may disproportionately increase the diagnosis of myocardial infarction in patients without acute coronary syndrome. We assessed the impact of lowering the diagnostic threshold of troponin on the incidence, management, and outcome of patients with type 2 myocardial infarction or myocardial injury. Methods Consecutive patients with elevated plasma troponin I concentrations (≥50 ng/L; n = 2929) were classified with type 1 (50%) myocardial infarction, type 2 myocardial infarction or myocardial injury (48%), and type 3 to 5 myocardial infarction (2%) before and after lowering the diagnostic threshold from 200 to 50 ng/L with a sensitive assay. Event-free survival from death and recurrent myocardial infarction was recorded at 1 year. Results Lowering the threshold increased the diagnosis of type 2 myocardial infarction or myocardial injury more than type 1 myocardial infarction (672 vs 257 additional patients, P < .001). Patients with myocardial injury or type 2 myocardial infarction were at higher risk of death compared with those with type 1 myocardial infarction (37% vs 16%; relative risk [RR], 2.31; 95% confidence interval [CI], 1.98-2.69) but had fewer recurrent myocardial infarctions (4% vs 12%; RR, 0.35; 95% CI, 0.26-0.49). In patients with troponin concentrations 50 to 199 ng/L, lowering the diagnostic threshold was associated with increased healthcare resource use (P < .05) that reduced recurrent myocardial infarction and death for patients with type 1 myocardial infarction (31% vs 20%; RR, 0.64; 95% CI, 0.41-0.99), but not type 2 myocardial infarction or myocardial injury (36% vs 33%; RR, 0.93; 95% CI, 0.75-1.15). Conclusions After implementation of a sensitive troponin assay, the incidence of type 2 myocardial infarction or myocardial injury disproportionately increased and is now as frequent as type 1 myocardial infarction. Outcomes of patients with type 2 myocardial infarction or myocardial injury are poor and do not seem to be modifiable after reclassification despite substantial increases in healthcare resource use.

Comparison of the T-tubule system in adult rat ventricular and atrial myocytes, and its role in excitation-contraction coupling and inotropic stimulation

Narrow, tubular, inward projections of the sarcolemma (T-tubules) are an established feature of adult mammalian ventricular myocytes that enables them to generate the whole-cell Ca2+ transients and produce coordinated contraction. Loss of T-tubules can occur during ageing and under pathological conditions, leading to altered cardiac excitation-contraction coupling. In contrast to adult ventricular cells, atrial myocytes do not generally express an extensive T-tubule system at any stage of development, and therefore rely on Ca2+ channels around their periphery for the induction of Ca2+ signalling and excitation-contraction coupling. Consequently, the characteristics of systolic Ca2+ signals in adult ventricular and atrial myocytes are temporally and spatially distinct. However, although atrial myocytes do not have the same regularly spaced convoluted T-tubule structures as adult ventricular cells, it has been suggested that a proportion of adult atrial cells have a more rudimentary tubule system. We examined the structure and function of these atrial tubules, and explored their impact on the initiation and recovery of Ca2+ signalling in electrically paced myocytes. The atrial responses were compared to those in adult ventricular cells that had intact T-tubules, or that had been chemically detubulated. We found that tubular structures were present in a significant minority of adult atrial myocytes, and were unlike the T-tubules in adult ventricular cells. In those cells where they were present, the atrial tubules significantly altered the on-set, amplitude, homogeneity and recovery of Ca2+ transients. The properties of adult atrial myocyte Ca2+ signals were different from those in adult ventricular cells, whether intact or detubulated. Excitation-contraction coupling in detubulated adult ventricular myocytes, therefore, does not approximate to atrial signalling, even though Ca2+ signals are initiated in the periphery of the cells in both of these situations. Furthermore, inotropic responses to endothelin-1 were entirely dependent on T-tubules in adult ventricular myocytes, but not in atrial cells. Our data reveal that that the T-tubules in atrial cells impart significant functional properties, but loss of these tubular membranes does not affect Ca2+ signalling as dramatically as detubulation in ventricular myocytes.

P-Rex2 regulates Purkinje cell dendrite morphology and motor coordination

The small GTPase Rac controls cell morphology, gene expression, and reactive oxygen species formation. Manipulations of Rac activity levels in the cerebellum result in motor coordination defects, but activators of Rac in the cerebellum are unknown. P-Rex family guanine-nucleotide exchange factors activate Rac. We show here that, whereas P-Rex1 expression within the brain is widespread, P-Rex2 is specifically expressed in the Purkinje neurons of the cerebellum. We have generated P-Rex2−/− and P-Rex1−/−/P-Rex2−/− mice, analyzed their Purkinje cell morphology, and assessed their motor functions in behavior tests. The main dendrite is thinned in Purkinje cells of P-Rex2−/− pups and dendrite structure appears disordered in Purkinje cells of adult P-Rex2−/− and P-Rex1−/−/P-Rex2−/− mice. P-Rex2−/− mice show a mild motor coordination defect that progressively worsens with age and is more pronounced in females than in males. P-Rex1−/−/P-Rex2−/− mice are ataxic, with reduced basic motor activity and abnormal posture and gait, as well as impaired motor coordination even at a young age. We conclude that P-Rex1 and P-Rex2 are important regulators of Purkinje cell morphology and cerebellar function.