Membrane tethering by the atlastin GTPase depends on GTP hydrolysis but not on forming the cross-over configuration.

The membrane-anchored atlastin GTPase couples nucleotide hydrolysis to the catalysis of homotypic membrane fusion to form a branched endoplasmic reticulum network. Trans dimerization between atlastins anchored in opposing membranes, accompanied by a cross-over conformational change, is thought to draw the membranes together for fusion. Previous studies on the conformational coupling of atlastin to its GTP hydrolysis cycle have been carried out largely on atlastins lacking a membrane anchor. Consequently, whether fusion involves a discrete tethering step and, if so, the potential role of GTP hydrolysis and cross-over in tethering remain unknown. In this study, we used membrane-anchored atlastins in assays that separate tethering from fusion to dissect the requirements for each. We found that tethering depended on GTP hydrolysis, but, unlike fusion, it did not depend on cross-over. Thus GTP hydrolysis initiates stable head-domain contact in trans to tether opposing membranes, whereas cross-over formation plays a more pivotal role in powering the lipid rearrangements for fusion.

Doppler ultrasound signal in Achilles tendinopathy reduces immediately after activity.

BACKGROUND: A relationship has been identified between vascularization on Doppler ultrasound (Doppler signal) and Achilles tendon pain. Doppler signal may increase minutes after prolonged activity, but the immediate effect is unknown. The aim of the study was to investigate the immediate effect of short term activity on Achilles tendon Doppler signal. Achilles tendinopathy patients (7 patients, 10 tendons) and asymptomatic controls (6 controls, 12 tendons) performed 2 activity tasks; a 2 minute continuous step task and one minute continuous calf raise task. Doppler signal was measured at rest and within a minute after each activity. The presence of Doppler signal was quantified using both semi quantitative (modified Ohberg scale; 0=no signal, 5 = > 90% of pathological area contains Doppler signal) and quantitative methods (pixel number). Doppler signal was present in 90% of symptomatic individuals and in none of the asymptomatic controls. The modified Ohberg scale and pixel number reduced significantly after both activity tasks and heart rate increased significantly (p < 0.05). Doppler signal in Achilles tendinopathy may decrease immediately after activities that load the calf muscle and increase heart rate, suggesting that this activity should be avoided prior to imaging to avoid false negative results.

An intramolecular salt bridge drives the soluble domain of GTP-bound atlastin into the postfusion conformation.

Endoplasmic reticulum (ER) network branching requires homotypic tethering and fusion of tubules mediated by the atlastin (ATL) guanosine triphosphatase (GTPase). Recent structural studies on the ATL soluble domain reveal two dimeric conformers proposed to correspond to a tethered prefusion state and a postfusion state. How the prefusion conformer transitions to the postfusion conformer is unknown. In this paper, we identify an intramolecular salt bridge mediated by two residues outside the GTPase domain near the point of rotation that converts the prefusion dimer to the postfusion state. Charge reversal of either residue blocked ER network branching, whereas a compensatory charge reversal to reestablish electrostatic attraction restored function. In vitro assays using the soluble domain revealed that the salt bridge was dispensable for GTP binding and hydrolysis but was required for forming the postfusion dimer. Unexpectedly, the postfusion conformation of the soluble domain was achieved when bound to the nonhydrolyzable GTP analogue guanosine 5'-[ß,γ-imido]triphosphate, suggesting that nucleotide hydrolysis might not be required for the prefusion to postfusion conformational change.

Indole acts as an extracellular cue regulating gene expression in Vibrio cholerae.

Indole has been proposed to act as an extracellular signal molecule influencing biofilm formation in a range of bacteria. For this study, the role of indole in Vibrio cholerae biofilm formation was examined. It was shown that indole activates genes involved in vibrio polysaccharide (VPS) production, which is essential for V. cholerae biofilm formation. In addition to activating these genes, it was determined using microarrays that indole influences the expression of many other genes, including those involved in motility, protozoan grazing resistance, iron utilization, and ion transport. A transposon mutagenesis screen revealed additional components of the indole-VPS regulatory circuitry. The indole signaling cascade includes the DksA protein along with known regulators of VPS production, VpsR and CdgA. A working model is presented in which global control of gene expression by indole is coordinated through sigma(54) and associated transcriptional regulators.