Biomedical subjects

Dixon J Woodbury

Publications and source records attributed to Dixon J Woodbury.

5 recordsLinked to original sources

PubMed2006

Evidence that nystatin channels form at the boundaries, not the interiors of lipid domains.

Nystatin (nys) is an antifungal agent that preferentially forms ion channels in membranes containing the sterol, ergosterol (erg). The structure of the nystatin channel is not clear, but it is known that multiple nystatin monomers must aggregate to form channels in a sterol-rich membrane. When nys/erg containing vesicles are fused to a sterol-free bilayer, characteristic spikelike changes in membrane conductance are observed. An abrupt increase in conductance is followed by a decay that is generally stepwise linear and the decay time depends strongly on [erg]. These data are inconsistent with the hypothesis that nys channels form uniformly throughout the membrane and decay independently (which would produce exponential decay). We propose that channels are located at the boundaries of lipid superlattices such that diffusion of erg out of the lattice results in correlated channel decay. This was tested using a statistical mechanical analysis and Monte Carlo simulations, which reveal details of the diffusion process and provide insight into conditions at superlattice boundaries during decay. This analysis predicts the linear decay schemes and the dramatic drop in channel decay time observed at erg mol % = 50. This interpretation also explains puzzling data relating conductance spike height to vesicle diameter.

Animals↗

PubMed2006

Reducing liposome size with ultrasound: bimodal size distributions.

Sonication is a simple method for reducing the size of liposomes. We report the size distributions of liposomes as a function of sonication time using three different techniques. Liposomes, mildly sonicated for just 30 sec, had bimodal distributions when surface-weighted with modes at about 140 and 750 nm. With extended sonication, the size distribution remains bimodal but the average diameter of each population decreases and the smaller population becomes more numerous. Independent measurements of liposome size using Dynamic Light Scattering (DLS), transmission electron microscopy (TEM), and the nystatin/ergosterol fusion assay all gave consistent results. The bimodal distribution (even when number-weighted) differs from the Weibull distribution commonly observed for liposomes sonicated at high powers over long periods of time and suggests that a different mechanism may be involved in mild sonication. The observations are consistent with the following mechanism for decreasing liposome size. During ultrasonic irradiation, cavitation, caused by oscillating microbubbles, produces shear fields. Large liposomes that enter these fields form long tube-like appendages that can pinch-off into smaller liposomes. This proposed mechanism is consistent with colloidal theory and the observed behavior of liposomes in shear fields.

Light↗

PubMed2006

SNARE complex regulation by phosphorylation.

SNAREs (soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptors) are ubiquitous proteins that direct vesicular trafficking and exocytosis. In neurons, SNAREs act to mediate release of neurotransmitters, which is a carefully regulated process. Calcium influx has long been shown to be the key trigger of release. However, calcium alone cannot regulate the degree of vesicle content release. For example, only a limited number of docked vesicles releases neurotransmitters when calcium entry occurs; this suggests that exocytosis is regulated by other factors besides calcium influx. Regulation of the degree of release is best explained by looking at the many enzymatic proteins that interact with the SNARE complex. These proteins have been hypothesized to regulate the formation, stability, or disassembly of the SNARE complex and therefore may regulate neurotransmitter release. One group of enzymatic regulators is the protein kinases. These proteins phosphorylate sites on both SNARE proteins and proteins that interact with SNARE proteins. Recent research has identified some of the specific effects that phosphorylation (or dephosphorylation) at these sites can produce. Additionally, palmitoylation of SNAP-25, regulates the localization, and hence activity of this key SNARE protein. This review focuses on the location and effects of phosphorylation on SNARE regulation.

Binding Sites↗

PubMed2004

Determination of proton flux and conductance at pH 6.8 through single FO sectors from Escherichia coli.

We have developed a mathematical model in concert with an assay that allows us to calculate proton (H+) flux and conductance through a single FO of the F1FO ATP synthase. Lipid vesicles reconstituted with just a few functional FO from Escherichia coli were loaded with 250 mM K+ and suspended in a low K+ solution. The pH of the weakly buffered external solution was recorded during sequential treatment with the potassium ionophore valinomycin, the protonophore carbonyl cyanide 3-chlorophenylhydrazone, and HCl. From these pH traces and separate determinations of vesicle size and lipid concentration we calculate the proton conductance through a single FO sector. This methodology is sensitive enough to detect small (15%) conductance changes. We find that wild-type FO has a proton flux of 3100 +/- 500 H+/s/FO at a transmembrane potential of 106 mV (25 degrees C and pH 6.8). This corresponds to a proton conductance of 4.4 fS.

Algorithms↗

PubMed2004

Syntaxin 1A drives fusion of large dense-core neurosecretory granules into a planar lipid bilayer.

The SNARE complex, involved in vesicular trafficking and exocytosis, is composed of proteins in the vesicular membrane (v-SNAREs) that intertwine with proteins of the target membrane (t-SNAREs). Our results show that modified large dense-core neurosecretory granules (NSGs), isolated from the bovine neurohypophysis, spontaneously fuse with a planar lipid membrane containing only the t-SNARE syntaxin 1A. This provides evidence that syntaxin alone is able to form a functional fusion complex with native v-SNAREs of the NSG. The fusion was similar to constitutive, not regulated, exocytosis because changes in free [Ca2+] had no effect on the syntaxin-mediated fusion. Several deletion mutants of syntaxin 1A were also tested. The removal of the regulatory domain did not significantly reduce spontaneous fusion. However, a syntaxin deletion mutant consisting of only the transmembrane domain was incapable of eliciting spontaneous fusion. Finally, a soluble form of syntaxin 1A (lacking its transmembrane domain) was used to saturate the free syntaxin-binding sites of modified NSGs. This treatment blocks spontaneous fusion of these granules to a bilayer containing full-length syntaxin 1A. This method provides an effective model system to study possible regulatory components affecting vesicle fusion.

Amino Acid Motifs↗