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A single particle cryo-EM reconstruction of an ~160-kD N-terminal fragment of the lipid transport protein VPS13 reveals an ~160-°A long channel lined with hydrophobic residues suitable for solubilizing multiple lipid fatty acid moieties. The structure suggests that VPS13 and related proteins, like the autophagy protein ATG2, can act as bridges between organelle membranes to allow bulk lipid flow between organelles. [ABSTRACT FROM AUTHOR]

The regulated expansion of membrane contact sites, which mediate the nonvesicular exchange of lipids between organelles, requires the recruitment of additional contact site proteins. Yeast Vps13 dynamically localizes to membrane contacts that connect the ER, mitochondria, endosomes, and vacuoles and is recruited to the prospore membrane in meiosis, but its targeting mechanism is unclear. In this study, we identify the sorting nexin Ypt35 as a novel adaptor that recruits Vps13 to endosomal and vacuolar membranes. We characterize an interaction motif in the Ypt35 N terminus and identify related motifs in the prospore membrane adaptor Spo71 and the mitochondrial membrane protein Mcp1. We find that Mcp1 is a mitochondrial adaptor for Vps13, and the Vps13-Mcp1 interaction, but not Ypt35, is required when ER-mitochondria contacts are lost. All three adaptors compete for binding to a conserved six-repeat region of Vps13 implicated in human disease. Our results support a competition-based model for regulating Vps13 localization at cellular membranes. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) are storage organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer and a set of LD-specific proteins. Most LD components are synthesized in the endoplasmic reticulum (ER), an organelle that is often physically connected with LDs. How LD identity is established while maintaining biochemical and physical connections with the ER is not known. Here, we show that the yeast seipin Fld1, in complex with the ER membrane protein Ldb16, prevents equilibration of ER and LD surface components by stabilizing the contact sites between the two organelles. In the absence of the Fld1/Ldb16 complex, assembly of LDs results in phospholipid packing defects leading to aberrant distribution of lipid-binding proteins and abnormal LDs. We propose that the Fld1/Ldb16 complex facilitates the establishment of LD identity by acting as a diffusion barrier at the ER-LD contact sites. [ABSTRACT FROM AUTHOR]

The scission of biological membranes is facilitated by a variety of protein complexes that bind and manipulate lipid bilayers. ESCRT-III (endosomal sorting complex required for transport III) filaments mediate membrane scission during the ostensibly disparate processes of multivesicular endosome biogenesis, cytokinesis, and retroviral budding. However, mechanisms by which ESCRT-III subunits assemble into a polymer remain unknown. Using cryogenic electron microscopy (cryo-EM), we found that the full-length ESCRT-III subunit Vps32/CHMP4B spontaneously forms single-stranded spiral filaments. The resolution afforded by two-dimensional cryo-EM combined with molecular dynamics simulations revealed that individual Vps32/CHMP4B monomers within a filament are flexible and able to accommodate a range of bending angles. In contrast, the interface between monomers is stable and refractory to changes in conformation. We additionally found that the carboxyl terminus of Vps32/CHMP4B plays a key role in restricting the lateral association of filaments. Our findings highlight new mechanisms by which ESCRT-III filaments assemble to generate a unique polymer capable of membrane remodeling in multiple cellular contexts. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) are ubiquitous dynamic organelles that store and supply lipids in all eukaryotic and some prokaryotic cells for energy metabolism, membrane synthesis, and production of essential lipid-derived molecules. Interest in the organelle's cell biology has exponentially increased over the last decade due to the link between LDs and prevalent human diseases and the discovery of new and unexpected functions of LDs. As a result, there has been significant recent progress toward understanding where and how LDs are formed, and the specific lipid pathways that coordinate LD biogenesis. [ABSTRACT FROM AUTHOR]

Proper brain wiring during development is pivotal for adult brain function. Neurons display a high degree of polarization both morphologically and functionally, and this polarization requires the segregation of mRNA, proteins, and lipids into the axonal or somatodendritic domains. Recent discoveries have provided insight into many aspects of the cell biology of axonal development including axon specification during neuronal polarization, axon growth, and terminal axon branching during synaptogenesis. [ABSTRACT FROM AUTHOR]

Spatial organization of membranes into domains of distinct protein and lipid composition is a fundamental feature of biological systems. The plasma membrane is organized in such domains to efficiently orchestrate the many reactions occurring there simultaneously. Despite the almost universal presence of membrane domains, mechanisms of their formation are often unclear. Yeast cells feature prominent plasma membrane domain organization, which is at least partially mediated by eisosomes. Eisosomes are large protein complexes that are primarily composed of many subunits of two Bin--Amphiphysin--Rvs domain--containing proteins, Pil1 and Lsp1. In this paper, we show that these proteins self-assemble into higher-order structures and bind preferentially to phosphoinositide-containing membranes. Using a combination of electron microscopy approaches, we generate structural models of Pil1 and Lsp1 assemblies, which resemble eisosomes in cells. Our data suggest that the mechanism of membrane organization by eisosomes is mediated by self-assembly of its core components into a membrane-bound protein scaffold with lipid-binding specificity. [ABSTRACT FROM AUTHOR]

The protein and lipid composition of eukaryotic plasma membranes is highly dynamic and regulated according to need. The sphingolipid-responsive Pkh kinases are candidates for mediating parts of this regulation, as they affect a diverse set of plasma membrane functions, such as cortical actin patch organization, efficient endocytosis, and eisosome assembly. Eisosomes are large protein complexes underlying the plasma membrane and help to sort a group of membrane proteins into distinct domains. In this study, we identify Nce102 in a genome-wide screen for genes involved in eisosome organization and Pkh kinase signaling. Nce102 accumulates in membrane domains at eisosomes where Pkh kinases also localize. The relative abundance of Nce102 in these domains compared with the rest of the plasma membrane is dynamically regulated by sphingolipids. Furthermore, Nce102 inhibits Pkh kinase signaling and is required for plasma membrane organization. Therefore, Nce102 might act as a sensor of sphingolipids that regulates plasma membrane function. [ABSTRACT FROM AUTHOR]

TIP47 (tail-interacting protein of 47 kD) was characterized as a cargo selection device for mannose 6-phosphate receptors (MPRs), directing their transport from endosomes to the trans-Golgi network. In contrast, our current analysis shows that cytosolic TIP47 is not recruited to organelles of the biosynthetic and endocytic pathways. Knockdown of TIP47 expression had no effect on MPR distribution or trafficking and did not affect lysosomal enzyme sorting. Therefore, our data argue against a function of TIP47 as a sorting device. Instead, TIP47 is recruited to lipid droplets (LDs) by an amino-terminal sequence comprising 11-mer repeats. We show that TIP47 has apolipoprotein-like properties and reorganizes liposomes into small lipid discs. Suppression of TIP47 blocked LD maturation and decreased the incorporation of triacyl-glycerol into LDs. We conclude that TIP47 functions in the biogenesis of LDs. [ABSTRACT FROM AUTHOR]

Polyphosphoinositol lipids convey spatial information partly by their interactions with cellular proteins within defined domains. However, these interactions are prevented when the lipids' head groups are masked by the recruitment of cytosolic effector proteins, whereas these effectors must also have sufficient mobility to maximize functional interactions. To investigate quantitatively how these conflicting functional needs are optimized, we used different fluorescence recovery after photobleaching techniques to investigate inositol lipid-effector protein kinetics in terms of the real-time dissociation from, and diffusion within, the plasma membrane. We find that the protein--lipid complexes retain a relatively rapid (∼0.1-1 µm²/s) diffusion coefficient in the membrane, likely dominated by protein--protein interactions, but the limited time scale (seconds) of these complexes, dictated principally by lipid--protein interactions, limits their range of action to a few microns. Moreover, our data reveal that GAP1[sup IP4BP], a protein that binds PtdIns(4,5)P[sub 2] and PtdIns(3,4,5)P[sub 3] in vitro with similar affinity, is able to "read" PtdIns(3,4,5)P[sub 3] signals in terms of an elongated residence time at the membrane. [ABSTRACT FROM AUTHOR]

In this study, we investigate whether the stable segregation of proteins and lipids within the yeast plasma membrane serves a particular biological function. We show that 21 proteins cluster within or associate with the ergosterol-rich membrane compartment of Can1 (MCC). However, proteins of the endocytic machinery are excluded from MCC. In a screen, we identified 28 genes affecting MCC appearance and found that genes involved in lipid biosynthesis and vesicle transport are significantly overrepresented. Deletion of Pil1, a component of eisosomes, or of Nce102, an integral membrane protein of MCC, results in the dissipation of all MCC markers. These deletion mutants also show accelerated endocytosis of MCC-resident permeases Can1 and Fur4. Our data suggest that release from MCC makes these proteins accessible to the endocytic machinery. Addition of arginine to wild-type cells leads to a similar redistribution and increased turnover of Can1. Thus, MCC represents a protective area within the plasma membrane to control turnover of transport proteins. [ABSTRACT FROM AUTHOR]

We define the dynamics of spatial and temporal reorganization of the team of proteins and lipids serving peroxisome division. The peroxisome becomes competent for division only after it acquires the complete set of matrix proteins involved in lipid metabolism. Overloading the peroxisome with matrix proteins promotes the relocation of acyl-CoA oxidase (Aox), an enzyme of fatty acid β-oxidation, from the matrix to the membrane. The binding of Aox to Pex16p, a membrane-associated peroxin required for peroxisome biogenesis, initiates the biosynthesis of phosphatidic acid and diacylglycerol (DAG) in the membrane. The formation of these two lipids and the subsequent transbilayer movement of DAG initiate the assembly of a complex between the peroxins Pex10p and Pex19p, the dynamin-like GTPase Vps1p, and several actin cytoskeletal proteins on the peroxisomal surface. This protein team promotes membrane fission, thereby executing the terminal step of peroxisome division. [ABSTRACT FROM AUTHOR]

Despite diverse origins, cellular fusion mechanisms converge at a pathway of phospholipid bilayer fusion. In this mini-review, we discuss how proteins can mediate each of the three major stages in the fusion pathway: contact, hemifusion, and the opening of an expanding fusion pore. [ABSTRACT FROM AUTHOR]

Specific spatial arrangements of proteins and lipids are central to the coordination of many biological processes. Tetraspanins have been proposed to laterally organize cellular membranes via specific associations with each other and with distinct integrins. Here, we reveal the presence of tetraspanin-enriched microdomains (TEMs) containing the tetraspanins CD9, CD63, CD81, and CD82 at the plasma membrane. Fluorescence and immunoelectron microscopic analyses document that the surface of HeLa cells is covered by several hundred TEMs, each extending over a few hundred nanometers and containing predominantly two or more tetraspanins. Further, we reveal that the human immunodeficiency virus type 1 (HIV-1) Gag protein, which directs viral assembly and release, accumulates at surface TEMs together with the HIV-1 envelope glycoprotein. TSG101 and VPS28, components of the mammalian ESCRT1 (endosomal sorting complex required for transport), which is part of the cellular extravesiculation machinery critical for HIV-1 budding, are also recruited to cell surface TEMs upon virus expression, suggesting that HIV-1 egress can be gated through these newly mapped microdomains. [ABSTRACT FROM AUTHOR]

Fusion pore opening and expansion are considered the most energy-demanding steps in viral fusion. Whether this also applies to soluble N-ethyl-maleimide sensitive fusion protein altachment protein receptor (SNARE)- and Rab-dependent fusion events has been unknown. We have addressed the problem by characterizing the effects of lysophosphatidylcholine (LPC) and other late-stage inhibitors on lipid mixing and pore opening during vacuole fusion. LPC inhibits fusion by inducing positive curvature in the bilayer and changing its biophysical properties. The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing. Transition from hemifusion to pore opening was sensitive to guanosine-5′-(γ-thio)triphosphate. It required the vacuolar adenosine triphosphatase V0 sector and coincided with its transformation. Pore opening was rate limiting for the reaction. As with viral fusion, opening the fusion pore may be the most energy-demanding step for intracellular, SNARE-dependent fusion reactions, suggesting that fundamental aspects of lipid mixing and pore opening are related for both systems. [ABSTRACT FROM AUTHOR]

Drosophila melanogaster phototransduction proceeds via a phospholipase C (PLC)-triggered cascade of phosphatidylinositol (PI) lipid modifications, many steps of which remain undefined. We describe the involvement of the lipid phosphatidic acid and the enzyme that generates it, phospholipase D (Pld), in this process. Pldnull flies exhibit decreased light sensitivity as well as a heightened susceptibility to retinal degeneration. Pld overexpression rescues flies lacking PLC from light-induced, metarhodopsin-mediated degeneration and restores visual signaling in flies lacking the PI transfer protein, which is a key player in the replenishment of the PI 4,5-bisphosphate (PIP2) substrate used by PLC to transduce light stimuli into neurological signals. Altogether, these findings suggest that Pld facilitates phototransduction by maintaining adequate levels of PIP2 and by protecting the visual system from metarhodopsin-induced, low light degeneration. [ABSTRACT FROM AUTHOR]

Lipid bodies form autonomous intracellular structures in many model cells and in some cells of specific tissue origins They contain hydrophobic substances, a set of structural proteins such as perilipin or adipose differentiation-related protein, enzymes implicated in lipid metabolism, and proteins that participate in signaling and membrane trafficking. Retinosomes, particles reminiscent of lipid bodies, have been identified in retinal pigment epithelium as distinct structures compartmentalizing a metabolic intermediate involved in regeneration of the visual chromophore. These observations suggest that lipid bodies, including retinosomes, carry out specific functions that go beyond those of mere lipid storage organelles. [ABSTRACT FROM AUTHOR]

Profilaggrin is a large epidermal polyprotein that is proteolytically processed during keratinocyte differentiation to release multiple filaggrin monomer units as well as a calcium-binding regulatory NH[sub2]-terminal filaggrin S-100 protein. We show that epidermal deficiency of the transmembrane serine protease Matriptase/MT-SP1 perturbs lipid matrix formation, cornified envelope morphogenesis, and stratum corneum desquamation. Surprisingly, proteomic analysis of Matriptase/MT-SP1-deficient epidermis revealed the selective loss of both proteolytically processed filaggrin monomer units and the NH[sub2]-terminal filaggrin S-100 regulatory protein. This was associated with a profound accumulation of profilaggrin and aberrant profilaggrin-processing products in the stratum corneum. The data identify keratinocyte Matriptase/MT-SP1 as an essential component of the profilaggrin-processing pathway and a key regulator of terminal epidermal differentiation. [ABSTRACT FROM AUTHOR]

Examines the dynamics and size of lipid rafts in the plasma membrane of living cells by measuring the local diffusion of single membrane proteins. Single particle tracking; Agreement between these measurements with lipid rafts being cholesterol-stabilized complexes; Thermal position fluctuation analysis.

Investigates the role of N-glycans on the apical sorting of glycosyl-phosphatidylinositol (GPI)-anchored proteins. Transport of GPI-anchored and non-associated raft proteins; Selection of information on protein moiety; Association of proteins to lipid rafts; Apical delivery of glycosylated proteins.

A new study in this issue (De Saint-Jean et al. 2011. J. Cell Biol. http://dx.doi.org/jcb.201104062) reveals that the sterol transfer protein Osh4p can also transport the signaling phospholipid phosphatidylinositol 4-phosphate (PI(4)P), which binds to the same site in Osh4p as sterol. This finding helps explain some previously published studies and also indicates that lipid/sterol exchange could contribute to establishing a sterol gradient in cells. [ABSTRACT FROM AUTHOR]

In certain cell types, apoptosis in response to extracellular stimuli like Fas depends on a mitochondrial amplificatory loop: the apical caspase-8 cleaves and activates the BH3-only member of the Bcl-2 family BID. In turn, BID induces the release of cytochrome c from mitochondria to the c toplasm, where it is required to fully activate effector caspases. In this issue of The Journal of Cell Biology, Gonzalvez et al. (see p. 681) show that when caspase-8 activation and production of functional BID is required, it is performed on mitochondrial platforms provided by the mitochondrion-specific lipid cardiolipin. Cardiolipin anchors caspase-8 at contact sites between inner and outer mitochondrial membranes, facilitating its self activation. These findings suggests that like other second messengers such as Ca[sup 2+] and cAMP, production of apoptotic messengers can be compartmentalized in close proximity to their intracellular target. [ABSTRACT FROM AUTHOR]

The article discusses research on lipid raft-like structures in yeast vacuole membrane. It references a study by A. Toulmay and W. A. Prinz published in an online 2013 issue of the "Journal of Cell Biology." The researchers tracked the protein Vph1, part of a pump that acidifies the vacuole interior. Results revealed that membrane domains can form live, unperturbed cells.

The article describes a method developed by Kai Simon and his group in collaboration with Andrej Shevchenko called shotgun mass spectrometry technique, which is intended to analyze the lipid composition of the purified organelles at the trans-Golgi network (TGN). Using the new technique is said to provide an opportunity for the researchers to determine how cells sort proteins at the TGN and package them into vesicles for delivery to the plasma membrane or endocytic system.

The article discusses research on an unexpected role for Spartin, a protein linked to the neuronal disease Troyer syndrome, in regulating lipid droplets (LDs). It references a study by S. W. Eastman et al in this same issue. It states that cells carry excess fats into LDs, where they are stored until needed as sources of energy. According to the researcher, Spartin localizes to LDs and binds to a known LD protein called TIP47.

The article discusses research on the interaction between inositol lipids in the plasma membrane and proteins. It references a study by G. R. Hammond et al published in this issue. The study revealed that inositol lipids in the plasma membrane help cells in trasmitting signals and moving around by sticking with various proteins. It is stated that proteins may risk extending their effects beyond the target area if they remain attached too long.

The article highlights two studies which found that establishing the midpoint as the site of bacterial cell division is the work of three proteins and constricting the cell is the work of just one. A team placed MinD protein on a lipid bilayer and then added MinE protein to test whether the proteins drive the oscillations themselves. Accordingly, the work brings closer a cell-free system for studying the events of cytokinesis.

The article focuses on a study conducted by Richard Anderson and colleagues at the University of Wisconsin in Madison regarding lipid in nuclear regulation. It states that a lipid known for its cytoplasmic duties raises mRNA levels by activating a polyadenylating enzyme. The enzyme's speckle-targeting region was used as bait to find PIPK-α-associated proteins which, the authors reasoned, might be regulatory targets of PtdIns4,5P[sub 2] phosphoinositide.

The article discusses a research conducted by Guangwei Min, et al, regarding tetraspanin. Tetraspanin contains a tightly packed quartet of transmembrane helices, according to a new, high resolution electron microscopy structure deduced by Min, et al. The rigid tetraspanin proteins may thus act as stable pilings in a lipid sea, according to the authors.

This article reports that Adam Douglass and Ronald Vale, researchers from of University of California, San Francisco, California find that interactions between proteins, not lipids, drive the formation of plasma membrane microdomains in signaling T cells. In active I cells, signal transduction proteins cluster within the plasma membrane, probably to enhance signaling by concentrating the interacting proteins. Popular model mechanisms for clustering involve either lipid rafts or actin. Clusters were also maintained in the absence of actin polymerization, although actin was needed for cluster formation.

The article reports that a research team formed with Stanford/Massachussetts Institute of Technology (MIT) collaboration has successfully derived the structure of a signaling network in primary human cells from simultaneous measurements of multiple labeled proteins and lipids. Karen Sachs and Douglas Lauffenburger, computational biologists at MIT wanted to use Bayesian networks to model biological systems. These graphical models have been used to predict transcriptional network structures based on mRNA levels.

The article reports that the Wingless (Wg) and Hedgehog (Hh) morphogens spread beween cells by catching a ride on lipoprotein particles. Getting rid of lipophorin with RNAi caused Wg and Hh to pile up near the cells that synthesize them in wing imagirial discs. The morphogens failed to spread as far as they normally do. All these signaling proteins have lipid anchors that could insert into the glycolipid monolayer that surrounds lipoprotein particles, but how that insertion event might be controlled or promoted is unclear. Lipoproteins may provide the morphogens with a vector that has just the right mobility.

The article cites a study by researchers Aurélien Roux, Patricia Bassereau, Bruno Goud and colleagues, which states that in vitro tube formation is favored from liquid-disordered domains, and phase separation induces fission. Proteins that affect lipid distributions in vivo may thus favor or disfavor tubulation and fission events during protein trafficking. The preferential tubulation from liquid-disordered domains makes sense, as reduced interactions between lipid head groups make these domains more easily deformable.

The article presents a research study which states that functional protein-lipid interaction motifs can be formed when partial motifs from two proteins unite. Many more motifs may be lurking in proteins than previously expected. The researchers also reports that the binding of TRPC3 and PLC-y 1 to each other brings together two halves of a split PH domain, a motif associated with the binding of lipids and other proteins. Mutation of the partial PH domains changes the lipid-binding profile of the TRPC3-PLC-11 complex, and reduces the amount of TRPC3 at the plasma membrane 24 h after stimulation.

This article focuses on a research shown as sorting protein uses the coincidence of a lipid signal and membrane curving to direct its tubulation activity to the correct compartment. Relatives of this sorting nexin, SNX1, may control trafficking to and from a number of intracellular compartments. SNX1 chooses its home via two membrane-binding domains. One targeting domain is the PX domain, which is known to bind to the endosomal phosphoinositide, P13 P. The second is a BAR domain, which was shown to target a fly protein to highly curved membranes and tubulate them.

This article focuses on the origin of adult-onset diabetes which may be a counterproductive stress reaction in the endoplasmic reticulum (ER). Type II or adult-onset diabetes has remained a mystery because the body seemingly acts against its own interests. As caloric intake and obesity rise, the body does not increase insulin responses to pack away the excess energy but instead becomes resistant to insulin's energy-storing signal. This just makes the situation worse. ER stress may arise because extra calories have to be processed by the ER as they get turned into either extra proteins or more lipids.

Tiny but dynamic domains define the elusive lipid raft, based on results from researchers Madan Rao, Satyajit Mayor and colleagues. Describing the structure and components of rafts, membrane domains enriched in specific lipids and proteins, has been a long-standing challenge for cell biologists. About a third of any given GPI-anchored proteins species is found in rafts. The rest remain as monomers. This percentage holds true across multiple expression levels, which is inconsistent with equilibrium-based formation.