Lipid-dependence of target membrane stability during influenza viral fusion.

Although influenza kills about a half million people each year, even after excluding pandemics, there is only one set of antiviral drugs: neuraminidase inhibitors. By using a new approach utilizing giant unilamellar vesicles and infectious X-31 influenza virus, and testing for the newly identified pore intermediate of membrane fusion, we observed ∼30-87% poration, depending upon lipid composition. Testing the hypothesis that spontaneous curvature (SC) of the lipid monolayer controls membrane poration, our Poisson model and Boltzmann energetic considerations suggest a transition from a leaky to a non-leaky fusion pathway depending on the SC of the target membrane. When the target membrane SC is below approximately -0.20 nm-1 fusion between influenza virus and target membrane is predominantly non-leaky while above that fusion is predominantly leaky, suggesting that influenza hemagglutinin (HA)-catalyzed topological conversion of target membranes during fusion is associated with a loss of membrane integrity.

Subcutaneous adipose tissue imaging of human obesity reveals two types of adipocyte membranes: Insulin-responsive and -nonresponsive.

In adipose tissue, resistance to insulin's ability to increase glucose uptake can be induced by multiple factors, including obesity. Impaired insulin action may take place at different spatial loci at the cellular or subcellular level. To begin to understand the spatial response to insulin in human subcutaneous adipose tissue (hSAT), we developed a quantitative imaging method for activation of a major signaling node in the glucoregulatory insulin signaling pathway. After treatment with insulin or control media, biopsied tissues were immunostained for Akt phosphorylation at Thr-308/9 (pAkt) and then imaged by confocal fluorescence microscopy automated to collect a large grid of high resolution fields. In hSAT from 40 men and women with obesity, substantial heterogeneity of pAkt densities in adipocyte membranes were quantified in each image mosaic using a spatial unit of at least twice the size of the point spread function. Statistical analysis of the distribution of pAkt spatial units was best fit as the weighted sum of two separate distributions, corresponding to either a low or high pAkt density. A "high pAkt fraction" metric was calculated from the fraction of high pAkt distributed units over the total units. Importantly, upon insulin stimulation, tissues from the same biopsy showed either a minimal or a substantial change in the high pAkt fraction. Further supporting a two-state response to insulin stimulation, subjects with similar insulin sensitivity indices are also segregated into either of two clusters identified by the amount of membrane-localized pAkt.

Measuring affinities of fission yeast spindle pole body proteins in live cells across the cell cycle.

Characterizing protein-protein interactions is essential for understanding molecular mechanisms, although reproducing cellular conditions in vitro is challenging and some proteins are difficult to purify. We developed a method to measure binding to cellular structures using fission yeast cells as reaction vessels. We varied the concentrations of Sid2p and Mob1p (proteins of the septation initiation network) and measured their binding to spindle pole bodies (SPBs), the centrosome equivalent of yeast. From our measurements we infer that Sid2p and Mob1p both exist as monomeric, heterodimeric, and homodimeric species throughout the cell cycle. During interphase these species have widely different affinities for their common receptor Cdc11p on the SPB. The data support a model with a subset of Cdc11p binding the heterodimeric species with a Kd < 0.1 µM when Sid2p binds Mob1p-Cdc11p and Kd in the micromolar range when Mob1p binds Sid2p-Cdc11p. During mitosis an additional species presumed to be the phosphorylated Sid2p-Mob1p heterodimer binds SPBs with a lower affinity. Homodimers of Sid2p or Mob1p bind to the rest of Cdc11p at SPBs with lower affinity: Kds > 10 µM during interphase and somewhat stronger during mitosis. These measurements allowed us to account for the fluctuations in Sid2p binding to SPBs throughout the cell cycle.

Fission yeast Myo2: Molecular organization and diffusion in the cytoplasm.

Myosin-II is required for the assembly and constriction of cytokinetic contractile rings in fungi and animals. We used electron microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS) to characterize the physical properties of Myo2 from fission yeast Schizosaccharomyces pombe. By electron microscopy, Myo2 has two heads and a coiled-coiled tail like myosin-II from other species. The first 65 nm of the tail is a stiff rod, followed by a flexible, less-ordered region up to 30 nm long. Myo2 sediments as a 7 S molecule in high salt, but aggregates rather than forming minifilaments at lower salt concentrations; this is unaffected by heavy chain phosphorylation. We used FRAP and FCS to observe the dynamics of Myo2 in live S. pombe cells and in cell extracts at different salt concentrations; both show that Myo2 with an N-terminal mEGFP tag has a diffusion coefficient of ∼ 3 µm2 s-1 in the cytoplasm of live cells during interphase and mitosis. Photon counting histogram analysis of the FCS data confirmed that Myo2 diffuses as doubled-headed molecules in the cytoplasm. FCS measurements on diluted cell extracts showed that mEGFP-Myo2 has a diffusion coefficient of ∼ 30 µm2 s-1 in 50 to 400 mM KCl concentrations.

Chapter 9: Counting proteins in living cells by quantitative fluorescence microscopy with internal standards.

This chapter describes how a confocal microscope can be treated as a spectrophotometer to measure the absolute number of fluorescent molecules in live cells (Wu and Pollard, 2005).(1) The method provides dynamic range of over three orders of magnitude for counting the number of molecules in a single cell. We present a step-by-step guide to measure concentrations in vivo, explaining many of the practical considerations for using this technique. This chapter is meant as a resource for cell biologists, biochemists, and biophysicists interested in quantifying macromolecules involved in their favorite molecular pathways in live cells.

Cerebral vasospasm and ischemia after orbital decompression for graves ophthalmopathy.

PURPOSE: We describe the first reported cases of cerebral vasospasm, ischemia, and infarct after orbital decompression surgery for Graves disease-related ophthalmopathy. METHODS: We present two patients who had inadvertent penetration of the dura mater during orbital decompression surgery. One case was recognized at surgery; the other was not. All patient data were gathered and relayed in accordance with state and U.S. laws. RESULTS: Postoperative cerebrospinal fluid leak, radiographic evidence of subarachnoid hemorrhage, and severe neurologic morbidity occurred in each case. Cerebral ischemia and infarction resulting from subarachnoid hemorrhage-induced cerebral vasospasm occurred in each case. CONCLUSIONS: The risk of dural penetration, cerebral vasospasm, and ischemia should be considered during preoperative evaluation for orbital decompression surgery. The diagnosis, treatment, and prevention of this previously unreported serious complication of this procedure is paramount.