Tumor protein D54 binds intracellular nanovesicles via an amphipathic lipid packing sensor (ALPS) motif

Tumor Protein D54 (TPD54) is an abundant cytosolic protein that belongs to the TPD52 family, a family of four proteins (TPD52, 53, 54 and 55) that are overexpressed in several cancer cells. Even though the functions of these proteins remain elusive, recent investigations indicate that TPD54 binds to very small cytosolic vesicles with a diameter of ca. 30 nm, half the size of classical transport vesicles (e.g. COPI and COPII). Here, we investigated the mechanism of intracellular nanovesicle capture by TPD54. Bioinformatical analysis suggests that TPD54 contains a small coiled-coil followed by several amphipathic helices, which could fold upon binding to lipid membranes. One of these helices has the physicochemical features of an Amphipathic Lipid Packing Sensor (ALPS) motif, which, in other proteins, enables membrane binding in a curvature-dependent manner. Limited proteolysis, CD spectroscopy, tryptophan fluorescence and cysteine mutagenesis coupled to covalent binding of a membrane sensitive probe show that binding of TPD54 to small liposomes is accompanied by large structural changes in the amphipathic helix region. TPD54 binding to artificial liposomes is very sensitive to liposome size and to lipid unsaturation but is poorly dependent on lipid charge. Cellular investigations confirmed the key role of the ALPS motif in vesicle targeting. Surprisingly, the vesicles selected by TPD54 poorly overlap with those captured by the golgin GMAP-210, a long vesicle tether at the Golgi apparatus, which displays a dimeric coiled-coil architecture and an N-terminal ALPS motif. We propose that TPD54 recognizes nanovesicles through a combination of ALPS-dependent and -independent mechanisms.

CurD: A Tool for Diffusion Analyses on Curved Membranes

Curved membranes are abundant and functionally relevant in living matter, yet they have eluded computational studies due to methodological limitations. With multiple approaches available for setting up simulations on such curved membranes, there is a growing need for efficient and versatile tools to analyze their outcomes. Here, we present CurD, a freely available tool for analyzing the diffusion of membrane constituents along curved surfaces. The tool efficiently uses the Vertex-oriented Triangle Propagation algorithm to compute geodesic distances significantly faster than conventional implementations of path-finding algorithms, while providing a friendly command-line interface. With CurD, we resolve the effects of curvature and lipid packing densities on the diffusion of lipids on two curved membranes---one with only mean curvature and another with both Mean and Gaussian curvature. We find that Gaussian curvature plays a surprisingly small role, whereas mean curvature or packing of lipid headgroups largely dictates their mobility.

Lipoprotein-Induced Increases in Cholesterol and 7-Ketocholesterol Result in Opposite Molecular-Scale Biophysical Effects on Membrane Structure

Under hypercholesterolemic conditions, exposure of cells to lipoproteins results in a subtle membrane increase in the levels of cholesterol and 7-ketocholesterol, as compared to normal conditions. The effect of these physiologically relevant concentration increases on multicomponent bilayer membranes was investigated using coarse-grained molecular dynamics simulations. Significant changes in the structural and dynamic properties of the bilayer membranes resulted from these subtle increases in sterol levels, with both sterol species inducing decreases in the lateral area and inhibiting lateral diffusion to varying extents. Cholesterol and 7-ketocholesterol, however, exhibited opposite effects on lipid packing and orientation. The results from this study indicate that the subtle increases in membrane sterol levels induced by exposure to lipoproteins result in molecular-scale biophysical perturbation of membrane structure.

Imaging non-classical mechanical responses of lipid membranes using molecular rotors

Lipid packing in cellular membranes has a direct effect on membrane tension and microviscosity, and plays a central role in cellular adaptation, homeostasis and disease. According to conventional mechanical descriptions,...

Effect of Cholesterol on Membrane Partitioning Dynamics of Hepatitis A Virus-2B peptide

ABSTRACTUnderstanding the viral peptide detection, partitioning and subsequent host membrane composition-based response is required for gaining insights into viral mechanism. Here, we probe the crucial role of presence of membrane lipid packing defects, depending on the membrane composition, in allowing the viral peptide belonging to C-terminal Hepatitis A Virus-2B (HAV-2B) to detect, attach and subsequently partition into the host cell membrane mimics. We conclusively show that the hydrophobic residues in the viral peptide detect the transiently present lipid packing defects, insert themselves into such defects, form anchor points and facilitate the partitioning of the peptide. We also show that the presence of cholesterol significantly alters such lipid packing defects, both in size and in number, thus mitigating the partitioning of the membrane active viral peptide into cholesterol-rich membranes. These results show differential ways in which presence and absence of cholesterol can alter the permeability of the host membranes to the membrane active viral peptide component of HAV-2B virus, via lipid packing defects, and can possibly be a part of general membrane detection mechanism for the viroporin class of viruses.

Probing nanoscale diffusional heterogeneities in cellular membranes through multidimensional single-molecule and super-resolution microscopy

ABSTRACTDiffusion properties notably determine the behavior of biomembranes. Here we report the concurrent nanoscale fine-mapping of membrane topography, diffusivity, and packing order in live mammalian cells through a synergy of single-molecule and super-resolution methods. By identifying a bright, lipophilic fluorescence turn-on probe that enables sustained single-molecule imaging of cellular membranes under stroboscopic excitation, we accumulate the positions and transient displacements of >106 probe molecules to achieve super-resolution topography and diffusivity mapping. We thus determine a trend that the membrane diffusivity drops with increased lipid packing order when comparing the endoplasmic reticulum (ER) membrane, plasma membrane, and nanodomains induced by cholera toxin B. Utilizing our nanoscale mapping capability, we further unveil reduced diffusivity in the ER membrane at ER-plasma membrane contact sites. By next integrating spectrally resolved single-molecule imaging, we show this localized diffusion slowdown is not due to altered lipid packing order, but may instead be attributed to local protein crowding. Our integrated multidimensional single-molecule approach thus unveils and differentiates between nanoscale diffusional heterogeneities of different origins in live-cell membranes.