Changes in endolysosomal organization define a pre-degenerative state in the crumbs mutant Drosophila retina.

Mutations in the epithelial polarity gene crumbs (crb) lead to retinal degeneration in Drosophila and in humans. The overall morphology of the retina and its deterioration in Drosophila crb mutants has been well-characterized, but the cell biological origin of the degeneration is not well understood. Degenerative conditions in the retina and elsewhere in the nervous system often involve defects in degradative intracellular trafficking pathways.

The BEACH Domain Is Critical for Blue Cheese Function in a Spatial and Epistatic Autophagy Hierarchy.

Unlabelled: () encodes a BEACH domain adaptor protein that, like its human homolog ALFY, promotes clearance of aggregated proteins through its interaction with Atg5 and p62. mutations lead to age-dependent accumulation of ubiquitinated inclusions and progressive neurodegeneration in the fly brain, but neither the influence of autophagy on -related degeneration, nor placement in the autophagic hierarchy have been shown. We present epistatic evidence in a well-defined larval motor neuron paradigm that in mutants, synaptic accumulation of ubiquitinated aggregates and neuronal death can be rescued by pharmacologically amplifying autophagic initiation.

Evidence from ITIR-FCS Diffusion Studies that the Amyloid-Beta (Aβ) Peptide Does Not Perturb Plasma Membrane Fluidity in Neuronal Cells.

The amyloid-beta (Aβ) peptide, commonly found in elevated levels in the brains of patients with Alzheimer's disease (AD) and in the cerebrospinal fluid of individuals presenting mild cognitive impairment, is thought to be one of the major factors resulting in the onset of AD. Although observed and studied at the molecular level for several decades, the exact disease pathology of AD is still not totally clear. One way in which Aβ is thought to affect neurons is by influencing cell membrane fluidity, which could result in abnormal synaptic or signaling function.

Ceramides And Stress Signalling Intersect With Autophagic Defects In Neurodegenerative Drosophila blue cheese (bchs) Mutants.

Sphingolipid metabolites are involved in the regulation of autophagy, a degradative recycling process that is required to prevent neuronal degeneration. Drosophila blue cheese mutants neurodegenerate due to perturbations in autophagic flux, and consequent accumulation of ubiquitinated aggregates. Here, we demonstrate that blue cheese mutant brains exhibit an elevation in total ceramide levels; surprisingly, however, degeneration is ameliorated when the pool of available ceramides is further increased, and exacerbated when ceramide levels are decreased by altering sphingolipid catabolism or blocking de novo synthesis.

Aβ1-25-Derived Sphingolipid-Domain Tracer Peptide SBD Interacts with Membrane Ganglioside Clusters via a Coil-Helix-Coil Motif.

The Amyloid-β (Aβ)-derived, sphingolipid binding domain (SBD) peptide is a fluorescently tagged probe used to trace the diffusion behavior of sphingolipid-containing microdomains in cell membranes through binding to a constellation of glycosphingolipids, sphingomyelin, and cholesterol. However, the molecular details of the binding mechanism between SBD and plasma membrane domains remain unclear. Here, to investigate how the peptide recognizes the lipid surface at an atomically detailed level, SBD peptides in the environment of raft-like bilayers were examined in micro-seconds-long molecular dynamics simulations.

Increased Microbial Butanol Tolerance by Exogenous Membrane Insertion Molecules.

Butanol is an ideal biofuel, although poor titers lead to high recovery costs by distillation. Fluidization of microbial membranes by butanol is one of the major factors limiting titers in butanol-producing bioprocesses. Starting with the hypothesis that certain membrane insertion molecules would stabilize the lipid bilayer in the presence of butanol, we applied a combination of in vivo and in vitro techniques within an in silico framework to describe a new approach to achieve solvent tolerance in bacteria.

Targeting Cell Membrane Lipid Rafts by Stoichiometric Functionalization of Gold Nanoparticles With a Sphingolipid-Binding Domain Peptide.

A non-membrane protein-based nanoparticle agent for the tracking of lipid rafts on live cells is produced by stoichiometric functionalization of gold nanoparticles with a previously characterized sphingolipid- and cell membrane microdomain-binding domain peptide (SBD). The SBD peptide is inserted in a self-assembled monolayer of peptidol and alkane thiol ethylene glycol, on gold nanoparticles surface. The stoichiometric functionalization of nanoparticles with the SBD peptide, essential for single molecule tracking, is achieved by means of non-affinity nanoparticle purification.

Oscillatory phase separation in giant lipid vesicles induced by transmembrane osmotic differentials.

Giant lipid vesicles are closed compartments consisting of semi-permeable shells, which isolate femto- to pico-liter quantities of aqueous core from the bulk. Although water permeates readily across vesicular walls, passive permeation of solutes is hindered. In this study, we show that, when subject to a hypotonic bath, giant vesicles consisting of phase separating lipid mixtures undergo osmotic relaxation exhibiting damped oscillations in phase behavior, which is synchronized with swell-burst lytic cycles: in the swelled state, osmotic pressure and elevated membrane tension due to the influx of water promote domain formation.

Preparation of mica supported lipid bilayers for high resolution optical microscopy imaging.

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM).

TrackArt: the user friendly interface for single molecule tracking data analysis and simulation applied to complex diffusion in mica supported lipid bilayers.

Background: Single molecule tracking (SMT) analysis of fluorescently tagged lipid and protein probes is an attractive alternative to ensemble averaged methods such as fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP) for measuring diffusion in artificial and plasma membranes. The meaningful estimation of diffusion coefficients and their errors is however not straightforward, and is heavily dependent on sample type, acquisition method, and equipment used. Many approaches require advanced computing and programming skills for their implementation.

Lipidomic "deep profiling": an enhanced workflow to reveal new molecular species of signaling lipids.

Current mass spectrometry-based lipidomics aims to comprehensively cover wide ranges of lipid classes. We introduce a strategy to capture phospho-monoester lipids and improve the detection of long-chain base phosphates (LCB-Ps, e.g.

Transient pearling and vesiculation of membrane tubes under osmotic gradients.

We report the experimental observation of osmotically induced transient pearling instabilities in vesicular membranes. Giant phospholipid vesicles subjected to negative osmotic gradient, which drives the influx of water in to the vesicular interior, produces transient cylindrical protrusions. These protrusions exhibit a remarkable pearling intermediate, which facilitates their subsequent retraction.

Accuracy and precision in camera-based fluorescence correlation spectroscopy measurements.

Imaging fluorescence correlation spectroscopy (FCS) performed using array detectors has been successfully used to quantify the number, mobility, and organization of biomolecules in cells and organisms. However, there have not been any systematic studies on the errors in these estimates that are introduced due to instrumental and experimental factors. State-of-the-art array detectors are still restricted in the number of frames that can be recorded per unit time, sensitivity and noise characteristics, and the total number of frames that can be realistically recorded.

Weak glycolipid binding of a microdomain-tracer peptide correlates with aggregation and slow diffusion on cell membranes.

Organized assembly or aggregation of sphingolipid-binding ligands, such as certain toxins and pathogens, has been suggested to increase binding affinity of the ligand to the cell membrane and cause membrane reorganization or distortion. Here we show that the diffusion behavior of the fluorescently tagged sphingolipid-interacting peptide probe SBD (Sphingolipid Binding Domain) is altered by modifications in the construction of the peptide sequence that both result in a reduction in binding to ganglioside-containing supported lipid membranes, and at the same time increase aggregation on the cell plasma membrane, but that do not change relative amounts of secondary structural features. We tested the effects of modifying the overall charge and construction of the SBD probe on its binding and diffusion behavior, by Surface Plasmon Resonance (SPR; Biacore) analysis on lipid surfaces, and by Fluorescence Correlation Spectroscopy (FCS) on live cells, respectively.

Calibration and limits of camera-based fluorescence correlation spectroscopy: a supported lipid bilayer study.

Camera-based fluorescence correlation spectroscopy (FCS) approaches allow the measurement of thousands of contiguous points yielding excellent statistics and details of sample structure. Imaging total internal reflection FCS (ITIR-FCS) provides these measurements on lipid membranes. Herein, we determine the influence of the point spread function (PSF) of the optical system, the laser power used, and the time resolution of the camera on the accuracy of diffusion coefficient and concentration measurements.

Osmotic gradients induce bio-reminiscent morphological transformations in giant unilamellar vesicles.

We report observations of large-scale, in-plane and out-of-plane membrane deformations in giant uni- and multilamellar vesicles composed of binary and ternary lipid mixtures in the presence of net transvesicular osmotic gradients. The lipid mixtures we examined consisted of binary mixtures of DOPC and DPPC lipids and ternary mixtures comprising POPC, sphingomyelin and cholesterol over a range of compositions - both of which produce co-existing phases for selected ranges of compositions at room temperature under thermodynamic equilibrium. In the presence of net osmotic gradients, we find that the in-plane phase separation potential of these mixtures is non-trivially altered and a variety of out-of-plane morphological remodeling events occur.

Fluorescence correlation methods for imaging cellular behavior of sphingolipid-interacting probes.

For cell biologists interested in the properties of cell membranes, their composition, and dynamics, the realization that sphingolipids and cholesterol have the capacity to self-organize into ordered domains has given rise to a need to visualize these lipids in actual living cell membranes. In order to find out how various classes of lipids distribute in the membrane and what their behaviors are, it is extremely useful to apply fluorescent probes that either interact with these lipids, or that themselves behave like naturally occurring lipids. At the same time, imaging modalities to observe their behaviors require the appropriate spatial and temporal resolution, on the milli- or microsecond timescale.

Gebiss: an ImageJ plugin for the specification of ground truth and the performance evaluation of 3D segmentation algorithms.

Background: Image segmentation is a crucial step in quantitative microscopy that helps to define regions of tissues, cells or subcellular compartments. Depending on the degree of user interactions, segmentation methods can be divided into manual, automated or semi-automated approaches. 3D image stacks usually require automated methods due to their large number of optical sections.

Roles of sphingolipids in Drosophila development and disease.

The last 10 years have seen a rebirth of interest in lipid biology in the fields of Drosophila development and neurobiology, and sphingolipids have emerged as controlling many processes that have not previously been studied from the viewpoint of lipid biochemistry. Mutations in sphingolipid regulatory enzymes have been pinpointed as affecting cell survival and growth in tissues ranging from muscle to retina. Specification of cell types are also influenced by sphingolipid regulatory pathways, as genetic interactions of glycosphingolipid biosynthetic enzymes with many well-known signaling receptors such as Notch and epidermal growth factor receptor reveal.

Glycolipid trafficking in Drosophila undergoes pathway switching in response to aberrant cholesterol levels.

In lipid storage diseases, the intracellular trafficking of sphingolipids is altered by conditions of aberrant cholesterol accumulation. Drosophila has been used recently to model lipid storage diseases, but the effects of sterol accumulation on sphingolipid trafficking are not known in the fly, and the trafficking of sphingolipids in general has not been studied in this model organism. Here, we examined the uptake and intracellular distribution of a fluorescent glycolipid analog, BODIPY-lactosyl-ceramide, in Drosophila neurons.

Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy.

Cell membrane organization is dynamic and is assumed to have different characteristic length scales. These length scales, which are influenced by lipid and protein composition as well as by the cytoskeleton, can range from below the optical resolution limit (as with rafts or microdomains) to far above the resolution limit (as with capping phenomena or the formation of lipid "platforms"). The measurement of these membrane features poses a significant problem because membrane dynamics are on the millisecond timescale and are thus beyond the time resolution of conventional imaging approaches.

Probing events with single molecule sensitivity in zebrafish and Drosophila embryos by fluorescence correlation spectroscopy.

Zebrafish and Drosophila are animal models widely used in developmental biology. High-resolution microscopy and live imaging techniques have allowed the investigation of biological processes down to the cellular level in these models. Here, using fluorescence correlation spectroscopy (FCS), we show that even processes on a molecular level can be studied in these embryos.

Alternate raft pathways cooperate to mediate slow diffusion and efficient uptake of a sphingolipid tracer to degradative and recycling compartments.

Several cholesterol-dependent cellular uptake pathways involving microdomain-resident sphingolipids have been characterized, but little is known about what controls the further intracellular trafficking routes of those domains. Here, we present evidence that the uptake and intracellular trafficking of a recently described sphingolipid-binding probe, the sphingolipid binding domain (SBD) peptide, is mediated by two parallel cooperating mechanisms requiring flotillin, dynamin and cdc42, which act in concert to direct a distinct surface behavior and trafficking itinerary. Diffusion measurements of SBD at the cell surface by fluorescence correlation spectroscopy suggest that cdc42- and flotillin-associated uptake sites both correspond to domains of intermediate mobility, but that they can cooperate to form low-mobility, efficiently internalized domains.

The Drosophila BEACH family protein, blue cheese, links lysosomal axon transport with motor neuron degeneration.

Impaired axon transport is one of the earliest pathological manifestations of several neurodegenerative diseases, and mutations in motor proteins can exacerbate or cause degeneration (Williamson and Cleveland, 1999; Gunawardena and Goldstein, 2004; Stokin and Goldstein, 2006). Compromised function in lysosomes and other degradative organelles that intersect with the lysosomal pathway are also strongly implicated in neurodegenerative disease pathology (Nixon and Cataldo, 2006; Rubinsztein, 2006). However, any functional link between these two phenomena has not as yet been recognized.