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‘Coincidence-detecting’ phosphoinositide sensors are used to study changes in the phosphoinositide lipid species found in membranes during the development and maturation of endocytic clathrin-coated vesicles. Changing composition of cell membranes The traffic within cells is busy. At any given time, many vesicles bud off the membrane of one organelle and travel to fuse with another membrane elsewhere. Which characteristics identify the donor and acceptor membranes is an intriguing question. The answer seems to be the lipid and protein composition of the membranes, specifically the presence and relative abundance of the seven species of phosphoinositide lipids, as well as GTP-bound GTPases. Tom Kirchhausen and colleagues describe a new generation of phosphoinositide sensors. They used these sensors to study the phosphoinositide composition of clathrin-associated membranes, which are involved in the process of endocytosis. The findings provide information on how the composition of the membrane changes from the time it is coated with clathrin to form pits, to when the pits close into vesicles, and, once the vesicles bud off, to when they lose their clathrin coating and fuse with endosomes. Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes 1 , 2 . Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments 2 , 3 . It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe ‘coincidence-detecting’ sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.

Key Points The topology of an integral membrane protein describes the number and approximate locations in the sequence of the transmembrane segments, as well as the overall orientation of the protein in a membrane. Topology is controlled primarily by the hydrophobicity and length of transmembrane helices as well as the distribution of positively charged residues in the loops that connect the helices. In most cases, topology is determined co-translationally during the translocon-mediated insertion of a polypeptide into a membrane. Topologies in which both the N terminus and the C terminus of a protein are in the cytoplasm are predominant in both prokaryotic and eukaryotic cells. Membrane proteins evolve primarily by gene duplication and gene fusion. Many membrane proteins form dimers in which the two homologous chains have the same topology (parallel dimer) or opposite topologies (antiparallel dimer). Gene fusions create internally duplicated structures in which the two halves of a protein are orientated either in a parallel or an antiparallel manner. The concept of membrane-protein topology is at least 30-years old. However, proteome-wide data on topology, increasing numbers of high-resolution structures and detailed studies on individual proteins are now showing us how topology is determined by the amino-acid sequence. In the world of membrane proteins, topology defines an important halfway house between the amino-acid sequence and the fully folded three-dimensional structure. Although the concept of membrane-protein topology dates back at least 30 years, recent advances in the field of translocon-mediated membrane-protein assembly, proteome-wide studies of membrane-protein topology and an exponentially growing number of high-resolution membrane-protein structures have given us a deeper understanding of how topology is determined and of how it evolves.

One challenge in cell biology is to decipher the biophysical mechanisms governing protein enrichment on curved membranes and the resulting membrane deformation. The ERM protein ezrin is abundant and associated with cellular membranes that are flat, positively or negatively curved. Using in vitro and cell biology approaches, we assess mechanisms of ezrin’s enrichment on curved membranes. We evidence that wild-type ezrin (ezrinWT) and its phosphomimetic mutant T567D (ezrinTD) do not deform membranes but self-assemble anti-parallelly, zipping adjacent membranes. EzrinTD’s specific conformation reduces intermolecular interactions, allows binding to actin filaments, which reduces membrane tethering, and promotes ezrin binding to positively-curved membranes. While neither ezrinTD nor ezrinWT senses negative curvature alone, we demonstrate that interacting with curvature-sensing I-BAR-domain proteins facilitates ezrin enrichment in negatively-curved membrane protrusions. Overall, our work demonstrates that ezrin can tether membranes, or be targeted to curved membranes, depending on conformations and interactions with actin and curvature-sensing binding partners.

Dynamin, the paradigmatic membrane fission catalyst, assembles as helical scaffolds that hydrolyse GTP to sever the tubular necks of clathrin-coated pits. Using a facile assay system of supported membrane tubes (SMrT) engineered to mimic the dimensions of necks of clathrin-coated pits, we monitor the dynamics of a dynamin-catalysed tube-severing reaction in real time using fluorescence microscopy. We find that GTP hydrolysis by an intact helical scaffold causes progressive constriction of the underlying membrane tube. On reaching a critical dimension of 7.3 nm in radius, the tube undergoes scission and concomitant splitting of the scaffold. In a constant GTP turnover scenario, scaffold assembly and GTP hydrolysis-induced tube constriction are kinetically inseparable events leading to tube-severing reactions occurring at timescales similar to the characteristic fission times seen in vivo . We anticipate SMrT templates to allow dynamic fluorescence-based detection of conformational changes occurring in self-assembling proteins that remodel membranes. Pucadyil and colleagues develop an in vitro technique to analyse the conformational dynamics of dynamin during membrane fission events in a real-time, high-throughput manner, using fluorescence microscopy.

The protein complement of cellular membranes is notoriously resistant to standard proteomic analysis and structural studies. As a result, membrane proteomes remain ill-defined. Here, we report a global topology analysis of the Escherichia coli inner membrane proteome. Using C-terminal tagging with the alkaline phosphatase and green fluorescent protein, we established the periplasmic or cytoplasmic locations of the C termini for 601 inner membrane proteins. By constraining a topology prediction algorithm with this data, we derived high-quality topology models for the 601 proteins, providing a firm foundation for future functional studies of this and other membrane proteomes. We also estimated the overexpression potential for 397 green fluorescent protein fusions; the results suggest that a large fraction of all inner membrane proteins can be produced in sufficient quantities for biochemical and structural work.

The mechanism of voltage gating in K+ channels is controversial. The paddle model posits that highly charged voltage-sensor domains move relatively freely across the lipid bilayer in response to membrane depolarization; competing models picture the charged S4 voltage-sensor helix as being shielded from lipid contact by other parts of the protein. We measured the apparent free energy of membrane insertion of a K+-channel S4 helix into the endoplasmic reticulum membrane and conclude that S4 is poised very near the threshold of efficient bilayer insertion. Our results suggest that the paddle model is not inconsistent with the high charge content of S4.

The cell biology of caveolae is a rapidly growing area of biomedical research. Caveolae are known primarily for their ability to transport molecules across endothelial cells, but modern cellular techniques have dramatically extended our view of caveolae. They form a unique endocytic and exocytic compartment at the surface of most cells and are capable of importing molecules and delivering them to specific locations within the cell, exporting molecules to extracellular space, and compartmentalizing a variety of signaling activities. They are not simply an endocytic device with a peculiar membrane shape but constitute an entire membrane system with multiple functions essential for the cell. Specific diseases attack this system: Pathogens have been identified that use it as a means of gaining entrance to the cell. Trying to understand the full range of functions of caveolae challenges our basic instincts about the cell.

Key Points Cellular membranes are laterally heterogeneous and consist of transient and dynamic domains with varying properties, which prominently include ordered lipid-driven domains that are referred to as lipid (or membrane) rafts. Membrane domains can be induced and regulated by a variety of interactions, which include specific lipid–lipid and lipid–protein interactions, bulk membrane properties, and interactions between membrane components and the underlying cytoskeleton. Advanced microscopy and biochemistry techniques facilitate the study of membrane domains; however, these domains still elude direct in vivo visualization. The multiplicity of possible organizational states and their context-dependent nature most likely account for experimental inconsistencies. Membrane rafts potentially have crucial physiological roles across cell types that range from immune cells to cancer cells. Membrane domains are conserved throughout the domains of life, which supports their functional importance in biological systems. Lipid rafts are relatively ordered membrane domains that are enriched in cholesterol and saturated lipids, and selectively recruit other lipids and proteins. They are dynamic and heterogeneous in composition and are thus challenging to visualize in vivo . New technologies are providing novel insights into the formation, organization and functions of these membrane domains. Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large number of studies have focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed (or ordered) membrane domains that selectively recruit certain lipids and proteins. Recent studies have yielded new insights into this mechanism and its relevance in vivo , owing primarily to the development of improved biochemical and biophysical technologies.

Key Points The endoplasmic reticulum (ER) forms tight membrane contact sites (MCSs) with several organelles in animal cells and yeast. The function of MCSs between the ER and mitochondria and endosomes are summarized in this Review. Electron microscopy studies reveal that although MCSs are less than 30 nm apart, the membranes do not fuse and each organelle maintains its identity. Ribosomes are excluded from the ER membrane at MCSs, and the distance between the ER and other membranes is close enough to suggest that the two organelles are tethered together by other proteins located on apposing membranes. Live-cell fluorescence microscopy reveals that ER-organelle MCSs can remain stable while both organelles traffic through the cell on the cytoskeleton. Recently identified factors have been shown to regulate organelle trafficking through MCS formation. ER–organelle MCSs regulate the lipid environment of the organelle membrane apposed to the ER. Lipid-synthesis proteins on the ER can modify lipids on the membrane of another organelle or on protein complexes. ER MCS may also transfer lipids between membranes. ER–organelle MCSs are sites of dynamic Ca 2+ crosstalk. Organelles can sequester Ca 2+ released from the ER, which can regulate processes in these organelles. Additionally, ER Ca 2+ release may regulate protein complexes at ER MCS. Mitochondria and endosomes undergo fission and fusion to, respectively, maintain their integrity and properly sort signalling receptors in the cell. ER–organelle MCSs define the position of fission for both mitochondria and endosomes, and the ER could have a variety of roles at those specific MCSs that are destined for fission. Endoplasmic reticulum (ER) is typically associated with protein biogenesis. However, recent studies suggest that it additionally synchronizes and regulates a plethora of intracellular events owing to its ability to form tight membrane associations, so-called membrane contact sites (MCSs), with other organelles. The endoplasmic reticulum (ER) is the largest organelle in the cell, and its functions have been studied for decades. The past several years have provided novel insights into the existence of distinct domains between the ER and other organelles, known as membrane contact sites (MCSs). At these contact sites, organelle membranes are closely apposed and tethered, but do not fuse. Here, various protein complexes can work in concert to perform specialized functions such as binding, sensing and transferring molecules, as well as engaging in organelle biogenesis and dynamics. This Review describes the structure and functions of MCSs, primarily focusing on contacts of the ER with mitochondria and endosomes.

Cholesterol is an integral component of eukaryotic cell membranes and a key molecule in controlling membrane fluidity, organization, and other physicochemical parameters. It also plays a regulatory function in antibiotic drug resistance and the immune response of cells against viruses, by stabilizing the membrane against structural damage. While it iswell understood that, structurally, cholesterol exhibits a densification effect on fluid lipid membranes, its effects on membrane bending rigidity are assumed to be nonuniversal; i.e., cholesterol stiffens saturated lipid membranes, but has no stiffening effect on membranes populated by unsaturated lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). This observation presents a clear challenge to structure–property relationships and to our understanding of cholesterol-mediated biological functions. Here, using a comprehensive approach—combining neutron spin-echo (NSE) spectroscopy, solid-state deuterium NMR (²H NMR) spectroscopy, and molecular dynamics (MD) simulations—we report that cholesterol locally increases the bending rigidity of DOPC membranes, similar to saturated membranes, by increasing the bilayer’s packing density. All three techniques, inherently sensitive to mesoscale bending fluctuations, show up to a threefold increase in effective bending rigidity with increasing cholesterol content approaching a mole fraction of 50%. Our observations are in good agreement with the known effects of cholesterol on the area-compressibility modulus and membrane structure, reaffirming membrane structure–property relationships. The current findings point to a scale-dependent manifestation of membrane properties, highlighting the need to reassess cholesterol’s role in controlling membrane bending rigidity over mesoscopic length and time scales of important biological functions, such as viral budding and lipid–protein interactions.