Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting

We present an analytical method to quantify clustering in super-resolution localization images of static surfaces in two dimensions. The method also describes how over-counting of labeled molecules contributes to apparent self-clustering and how the effective lateral resolution of an image can be determined. This treatment applies to clustering of proteins and lipids in membranes, where there is significant interest in using super-resolution localization techniques to probe membrane heterogeneity. When images are quantified using pair correlation functions, the magnitude of apparent clustering due to over-counting will vary inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. Over-counting does not yield apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (Fc{\epsilon}RI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM) and scanning electron microscopy (SEM). We find that apparent clustering of labeled IgE bound to Fc{\epsilon}RI detected with both methods arises from over-counting of individual complexes. Thus our results indicate that these receptors are randomly distributed within the resolution and sensitivity limits of these experiments.Comment: 22 pages, 5 figure

Chaperone-assisted translocation of a polymer through a nanopore

Using Langevin dynamics simulations, we investigate the dynamics of chaperone-assisted translocation of a flexible polymer through a nanopore. We find that increasing the binding energy $\epsilon$ between the chaperone and the chain and the chaperone concentration $N_c$ can greatly improve the translocation probability. Particularly, with increasing the chaperone concentration a maximum translocation probability is observed for weak binding. For a fixed chaperone concentration, the histogram of translocation time $\tau$ has a transition from long-tailed distribution to Gaussian distribution with increasing $\epsilon$. $\tau$ rapidly decreases and then almost saturates with increasing binding energy for short chain, however, it has a minimum for longer chains at lower chaperone concentration. We also show that $\tau$ has a minimum as a function of the chaperone concentration. For different $\epsilon$, a nonuniversal dependence of $\tau$ on the chain length $N$ is also observed. These results can be interpreted by characteristic entropic effects for flexible polymers induced by either crowding effect from high chaperone concentration or the intersegmental binding for the high binding energy.Comment: 10 pages, to appear in J. Am. Chem. So

The ROM / UWO Mummy Project: A Microcosm of Progress in Mummy Research

The beginnings of the Royal Ontario Museum can be traced back to the excavations and collections of Charles Trick Currelly, a staff member of the Egyptian Exploration Fund in the early 1900s. Currelly excavated with Sir Flinders Petrie at Abydos and with Edouard Naville at Deir el Bahari. With the assistance of Robert Mond and others, Currelly amassed a rich and diverse collection that became the basis for the ROM, which opened its doors in 1914. Part of that collection included several Egyptian mummies (Currelly 1971) . The Egyptologicalholdings at the ROM include eight mummies: one dating to the Predynastic Period, five from the Pharaonic Period, one from the Roman Period and one without context. Two of these, Nakht and Djedmaatesankh, have been well studied by Peter Lewin and associates, while three more are the subjects of the current investigation. The objectives of this poster are to review the work and accomplishments of the previous research, to describe the preliminary results of the current research project and to outline directions for future work

Stabilization of Peptide Vesicles by Introducing Inter-Peptide Disulfide Bonds

PURPOSE: Previously, we have shown that the amphiphilic oligopeptide SA2 (Ac-Ala-Ala-Val-Val-Leu-Leu-Leu-Trp-Glu-Glu-COOH) spontaneously self-assemble into nano-sized vesicles in aqueous environment. Relative weak individual intermolecular interactions dominate such oligopeptide assemblies. In this study we aimed at improving the stability of such peptide vesicles by covalently crosslinking the oligopeptide vesicles using disulfide bonds. Two and three cysteines were introduced in the SA2 peptide sequence to allow crosslinking (Ac-Ala-Cys-Val-Cys-Leu-(Leu/Cys)-Leu-Trp-Glu-Glu-COOH). RESULTS: Upon disulfide formation the crosslinked vesicles remained stable under conditions that disrupted the non-crosslinked peptide vesicles. The stabilized vesicles were more closely examined in terms of particle size (distribution) using atomic force microscopy, cryogenic electron microscopy, as well as dynamic light scattering analysis, showing an average particle radius in number between 15 and 20 nm. Using entrapment of calcein it was shown that intermolecular crosslinking of peptides within the vesicles did not affect the permeability for calcein. CONCLUSION: Introduction of cysteines into the hydrophobic domain of the SA2 amphiphilic oligopeptides is a feasible strategy for crosslinking the peptide vesicles. Such small crosslinked oligopeptide vesicles may hold promise for drug delivery applications

Radial Sizing of Lipid Nanotubes Using Membrane Displacement Analysis

We report a novel method for the measurement of lipid nanotube radii. Membrane translocation is monitored between two nanotube-connected vesicles, during the expansion of a receiving vesicle, by observing a photobleached region of the nanotube. We elucidate nanotube radii, extracted from SPE vesicles, enabling quantification of membrane composition and lamellarity. Variances of nanotube radii were measured, showing a growth of 40-56 nm, upon increasing cholesterol content from 0 to 20%

Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids?

Background: We recently reported that sphingomyelin (SM) analogs substituted on the alkyl chain by various fluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneously concentrated into micrometric domains. Despite sharing the same fluorescent ceramide backbone, BODIPY-SM domains segregated from similar domains labelled by BODIPY-D-e-lactosylceramide (D-e-LacCer) and depended on endogenous SM. Methodology/Principal Findings. We show here that BODIPY-SM further differed from BODIPY-D-e-LacCer or -glucosylceramide (GlcCer) domains in temperature dependence, propensity to excimer formation, association with a glycosylphosphatidylinositol (GPI)-anchored fluorescent protein reporter, and lateral diffusion by FRAP, thus demonstrating different lipid phases and boundaries. Whereas BODIPY-D-e-LacCer behaved like BODIPY-GlcCer, its artificial stereoisomer, BODIPY-L-t-LacCer, behaved like BODIPY- and NBD-phosphatidylcholine (PC). Surprisingly, these two PC analogs also formed micrometric patches yet preferably at low temperature, did not show excimer, never associated with the GPI reporter and showed major restriction to lateral diffusion when photobleached in large fields. This functional comparison supported a three-phase micrometric compartmentation, of decreasing order: BODIPY-GSLs > -SM > -PC (or artificial L-t-LacCer). Co-existence of three segregated compartments was further supported by double labelling experiments and was confirmed by additive occupancy, up to ~70% cell surface coverage. Specific alterations of BODIPY-analogs domains by manipulation of corresponding endogenous sphingolipids suggested that distinct fluorescent lipid partition might reflect differential intrinsic propensity of endogenous membrane lipids to form large assemblies. Conclusions/Significance. We conclude that fluorescent membrane lipids spontaneously concentrate into distinct micrometric assemblies. We hypothesize that these might reflect preexisting compartmentation of endogenous PM lipids into non-overlapping domains of differential order: GSLs > SM > PC, resulting into differential self-adhesion of the two former, with exclusion of the latter

α1A-Adrenergic Receptor Induces Activation of Extracellular Signal-Regulated Kinase 1/2 through Endocytic Pathway

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α1A-adrenergic receptor (α1A-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α1A-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α1A-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α1A-AR. α1A-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α1A-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31–8220 (a PKC inhibitor) inhibited α1B-AR- but not α1A-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α1A-AR-induced ERK1/2 activation, which is independent of Gq/PLC/PKC signaling

Link between Intestinal CD36 Ligand Binding and Satiety Induced by a High Protein Diet in Mice

CD36 is a ubiquitous membrane glycoprotein that binds long-chain fatty acids. The presence of a functional CD36 is required for the induction of satiety by a lipid load and its role as a lipid receptor driving cellular signal has recently been demonstrated. Our project aimed to further explore the role of intestinal CD36 in the regulation of food intake. Duodenal infusions of vehicle or sulfo-N-succinimidyl-oleate (SSO) was performed prior to acute infusions of saline or Intralipid (IL) in mice. Infusion of minute quantities of IL induced a decrease in food intake (FI) compared to saline. Infusion of SSO had the same effect but no additive inhibitory effect was observed in presence of IL. No IL- or SSO-mediated satiety occurred in CD36-null mice. To determine whether the CD36-mediated hypophagic effect of lipids was maintained in animals fed a satietogen diet, mice were subjected to a High-Protein diet (HPD). Concomitantly with the satiety effect, a rise in intestinal CD36 gene expression was observed. No satiety effect occurred in CD36-null mice. HPD-fed WT mice showed a diminished FI compared to control mice, after saline duodenal infusion. But there was no further decrease after lipid infusion. The lipid-induced decrease in FI observed on control mice was accompanied by a rise in jejunal oleylethanolamide (OEA). Its level was higher in HPD-fed mice than in controls after saline infusion and was not changed by lipids. Overall, we demonstrate that lipid binding to intestinal CD36 is sufficient to produce a satiety effect. Moreover, it could participate in the satiety effect induced by HPD. Intestine can modulate FI by several mechanisms including an increase in OEA production and CD36 gene expression. Furthermore, intestine of mice adapted to HPD have a diminished capacity to modulate their food intake in response to dietary lipids