A Nobel Prize for membrane traffic: Vesicles find their journey’s end

Cell biologists everywhere rejoiced when this year’s Nobel Prize in Physiology or Medicine was awarded to James Rothman, Randy Schekman, and Thomas Südhof for their contributions to uncovering the mechanisms governing vesicular transport. In this article, we highlight their achievements and also pay tribute to the pioneering scientists before them who set the stage for their remarkable discoveries.In 1974, nearly 40 years ago, the Nobel Prize in Physiology or Medicine was awarded to George E. Palade, Albert Claude, and Christian de Duve for work that effectively established a new field, cell biology. Collectively, the efforts of these three pioneers not only defined the essential features of cells but also how to study them. Correlating morphological observations by electron microscopy with biochemical analysis enabled not only the identification of nearly every major organelle in the eukaryotic cell (although endosomes were missed at that time) but also what their respective functions were. Palade’s efforts demonstrated the now-canonical pathway of protein secretion: synthesis in the endoplasmic reticulum (ER), oligosaccharide processing in the Golgi complex, concentration in secretory granules, and release at the plasma membrane. Palade understood implicitly that the ER, Golgi, secretory granules, and plasma membrane had to be interconnected by a series of vesicular carriers that carried cargo from one station to the next—dissociative transport. He also appreciated that the process had to be regulated if compartment specificity was to be maintained. The need for specificity defined the next major conceptual challenges: how do proteins intended for secretion traverse the compartments of the secretory pathway, how are transport vesicles formed, how do vesicles recognize their appropriate destinations, how does fusion occur after the appropriate destination is reached, and, finally, how are the components from the originating compartment returned or recycled to their sites of origin after fusion with the destination compartment? Palade may have framed these problems, but it was left to the next generation of cell biologists to solve them.This year’s Nobel Prize in Physiology or Medicine awarded to James Rothman, Randy Schekman, and Thomas Südof recognizes a truly remarkable body of work that provides superb conceptual clarity and mechanistic insight into virtually all of the issues defined by Palade and colleagues. To a large extent, the award also provides a satisfying degree of recognition to the large community of scientists who established the field of “molecular” cell biology. But it was the intellectual leadership, passion, and courage provided by this year’s awardees (Figs. 1 and ​and2)2) that played a major role in driving the spectacular advances of the past three decades. Particularly in the case of Rothman and Schekman, the scientific dynamic they helped to generate gave the field focus and excitement, from which came great things. The elegance of their experiments together with the exceptionally clear and simple logic that they presented in their papers moved the field ahead quickly and drew many new converts into membrane trafficking.Open in a separate windowFigure 1.Randy Schekman and James Rothman (center) with many of their former trainees at the American Society for Biochemistry and Molecular Biology meeting on “Biochemistry of Membrane Traffic: Secretory and Endocytic Pathways,” October 2010.PHOTOGRAPH COURTESY OF THE AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGYOpen in a separate windowFigure 2.Thomas Südhof (top row, center) and his laboratory circa 1993.PHOTOGRAPH COURTESY OF THOMAS SÜDHOFThe first foray into a mechanistic, molecular approach to the cell biological problems defined by Palade was really due to the work of Günter Blobel and his colleagues Peter Walter and Bernhard Dobberstein working at The Rockefeller University. These investigators devised a complex but elegant approach enabling the cell-free reconstitution of the first step of secretion, namely the insertion of newly synthesized proteins into and across the ER membrane. Combined with conventional cold-room biochemistry, Blobel and others were able to provide a detailed understanding of the biochemistry of protein translocation. Blobel was duly awarded the Nobel Prize in Physiology or Medicine for his work in 1999. Influenced by Blobel and also Arthur Kornberg, then chair of the Biochemistry Department at Stanford, Jim Rothman (who was a young faculty member at Stanford in the early 1980s) initiated his courageous effort aimed to reconstitute subsequent steps, namely the transport of secretory and membrane protein cargo to and through the Golgi complex. As is often the case with innovative work that pushes the limits of knowledge, Rothman’s interpretations were on occasion controversial, but there was absolutely no controversy regarding the importance of the various components he and his team identified. These components included soluble factors needed for vesicle formation in the Golgi as well as for vesicle fusion, most notably the COPI coat protein complex, NSF (NEM-sensitive factor) and SNAP (soluble NSF attachment protein). With Richard Scheller, Rothman recognized that the synaptic vesicle–associated proteins cloned and purified by Scheller represented both the docking sites for NSF and SNAP and a key component of the mechanism whereby vesicles recognized and even fused with each other. Indeed, the SNAREs (as these proteins are now called) clearly comprise the core fusion machinery that underlies virtually all membrane fusion events in the cell. SNAREs form a family of proteins that are organelle specific, helping to ensure the specificity of membrane traffic as well as the biochemical and functional identity of individual membrane compartments.If Rothman’s work began as a quintessential biochemical approach, Randy Schekman’s started at the other end of the spectrum: genetics. Again with great courage, Schekman decided to use the yeast Saccharomyces cerevisiae as a genetically tractable eukaryote to dissect the steps and various components associated with the secretory pathway. At the time, few thought that yeast cells were capable of higher-order processes such as secretion or that their activities had anything to do with mechanisms in animal cells. Yet Schekman and his then graduate student Peter Novick designed a deceptively simple screen to identify secretory (or “sec”) mutants. Their approach was to look for cells that could not secrete by reasoning that continued synthesis of secretory cargo would render the mutant cells more dense. The approach worked, and literally dozens of mutants were discovered, a large number of which could be shown to generate intriguing phenotypes and to control key steps in the secretory, or sometimes even the endocytic, pathway. Although the original sec screens done by Schekman and colleagues did not immediately turn up the SNARE proteins, they did reveal the presence of small Ras-related monomeric GTPases of the Rab family that helped enforce the specificity of vesicle interactions. They also uncovered cytoplasmic coat proteins (COPII) and complex cytosolic “tethers” that serve to gather vesicles at their targets before the final fusion step. When an increasing number of sec mutants began to overlap with components identified by Rothman’s independent biochemical purifications of components required for fusion or vesicle budding, it was clear that both groups (and indeed the field) were on the right track and the transport machinery was universal. Through whatever controversies bubbled up over the years, this basic fact remained unchallenged. Schekman too moved toward the same type of functional biochemical analysis championed by Rothman, and the circle was completed.Focused on one of the key problems in neurobiology, Thomas Südhof’s efforts may appear less general but are no less important. The synapse represented a special case in the area of membrane traffic since the realization that neuronal transmission reflected the release of quanta of neurotransmitters due to the action potential–triggered fusion of synaptic vesicles with the presynaptic plasma membrane. The work of Cesare Montecucco and colleagues on bacterial toxins provided an important insight, namely that synaptic vesicle release can be blocked by certain bacterial toxins (e.g., botulinum toxin) that act as specific SNARE proteases. Scheller and Rothman had shown that the SNAREs comprised the basic unit of the fusion machinery, but this insight alone did not explain how secretion in the synapse was coupled so tightly to electrical activity. Thomas Südhof’s remarkable body of work, although not growing out of the molecular cell biology community as much as the neuroscience community, provided the conceptual answer: the synaptotagmins. These proteins were found to associate with SNAREs and serve as Ca²⁺-sensing triggers that temporally linked synaptic vesicle transmission to individual neuronal impulses. In addition, Südhof and colleagues discovered Munc18 in the mouse, which corresponds to the yeast Sec1 protein, and demonstrated that it interacts with the SNARE complex, revealing that Munc18 as well as other members of the Sec1/Munc18-like protein family function as part of the vesicle fusion machinery.Collectively, these are remarkable achievements that provide conceptual and mechanistic understanding of basic cellular processes at the most fundamental level. It is certainly the case that others, for example Scheller and Novick mentioned here, might just as easily have been included in this award. Regrettably only three are permitted, and there can be no doubt but that the three selected are entirely deserving given not only the nature of their findings but also the scientific leadership they contributed in a myriad of intangible ways to the incredible progress we have witnessed in the post-Palade era of cell biology.We congratulate our colleagues and friends Jim, Randy, and Thomas for this well-deserved honor. Mazal tov!     

Rothman and Schekman SNAREd by Lasker for trafficking

This year, the recipients of the Lasker Award for Basic Medical Research are James Rothman and Randy Schekman. This highly anticipated honor highlights their unique contributions to our understanding of the mechanisms of membrane traffic.     

Lelio Orci,the modern master of morphology

Lelio Orci has made seminal contributions to our understanding of pancreatic islet structure and function. He introduced quantitative criteria to structural analysis in the study of endocrine pancreas in a series of works performed in collaboration with Albert Renold, Roger Unger, and Donald Steiner. Orci has moved islet cell morphology from the primitive era of histochemistry and electron microscopy into the modern era of cell biology, applying the most advanced techniques and covering every aspect of normal and pathological structure–function relationships. In collaboration with James Rothman in New York and Randy Schekman in Berkley, Orci discovered that the transport steps from the endoplasmic reticulum to the Golgi complex, and within the Golgi, are mediated by two sets of vesicles coated with protein envelopes different from clathrin.     

Please release me

In this issue of Neuron, Südhof and colleagues determine which of the eight Ca(2+)-binding synaptotagmin isoforms expressed in brain can support synchronous neurotransmitter release at mammalian CNS synapses. Unexpectedly, only three-synaptotagmin-1, -2, and -9-can serve as Ca(2+) sensors for fast transmission. Further characterization reveals the unique ability of each isoform to shape neurotransmission.     

Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins

Exchange of proteins at sorting endosomes is not only critical to numerous signaling pathways but also to receptor-mediated signaling and to pathogen entry into cells; however, how this process is regulated in synaptic vesicle cycling remains unexplored. In this work, we present evidence that loss of function of a single neuronally expressed GTPase activating protein (GAP), Skywalker (Sky) facilitates endosomal trafficking of synaptic vesicles at Drosophila neuromuscular junction boutons, chiefly by controlling Rab35 GTPase activity. Analyses of genetic interactions with the ESCRT machinery as well as chimeric ubiquitinated synaptic vesicle proteins indicate that endosomal trafficking facilitates the replacement of dysfunctional synaptic vesicle components. Consequently, sky mutants harbor a larger readily releasable pool of synaptic vesicles and show a dramatic increase in basal neurotransmitter release. Thus, the trafficking of vesicles via endosomes uncovered using sky mutants provides an elegant mechanism by which neurons may regulate synaptic vesicle rejuvenation and neurotransmitter release.     

Exocytosis of catecholamine (CA)-containing and CA-free granules in chromaffin cells

Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholamine-free granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis.     

Lipid metabolism and vesicle trafficking: more than just greasing the transport machinery.

The movement of lipids from their sites of synthesis to ultimate intracellular destinations must be coordinated with lipid metabolic pathways to ensure overall lipid homeostasis is maintained. Thus, lipids would be predicted to play regulatory roles in the movement of vesicles within cells. Recent work has highlighted how specific lipid metabolic events can affect distinct vesicle trafficking steps and has resulted in our first glimpses of how alterations in lipid metabolism participate in the regulation of intracellular vesicles. Specifically, (i) alterations in sphingolipid metabolism affect the ability of SNAREs to fuse membranes, (ii) sterols are required for efficient endocytosis, (iii) glycerophospholipids and phosphorylated phosphatidylinositols regulate Golgi-mediated vesicle transport, (iv) lipid acylation is required for efficient vesicle transport mediated membrane fission, and (v) the addition of glycosylphosphatidylinositol lipid anchors to proteins orders them into distinct domains that result in their preferential sorting from other vesicle destined protein components in the endoplasmic reticulum. This review describes the experimental evidence that demonstrates a role for lipid metabolism in the regulation of specific vesicle transport events.     

Autophagy in yeast: a review of the molecular machinery

Autophagy is a membrane trafficking mechanism that delivers cytoplasmic cargo to the vacuole/lysosome for degradation and recycling. In addition to non-specific bulk cytosol, selective cargoes, such as peroxisomes, are sorted for autophagic transport under specific physiological conditions. In a nutrient-rich growth environment, many of the autophagic components are recruited for executing a biosynthetic trafficking process, the cytoplasm to vacuole targeting (Cvt) pathway, that transports the resident hydrolases aminopeptidase I and alpha-mannosidase to the vacuole in Saccharomyces cerevisiae. Recent studies have identified pathway-specific components that are necessary to divert a protein kinase and a lipid kinase complex to regulate the conversion between the Cvt pathway and autophagy. Downstream of these proteins, the general machinery for transport vesicle formation involves two novel conjugation systems and a putative membrane protein complex. Completed vesicles are targeted to, and fuse with, the vacuole under the control of machinery shared with other vacuolar trafficking pathways. Inside the vacuole, a potential lipase and several proteases are responsible for the final steps of vesicle breakdown, precursor enzyme processing and substrate turnover. In this review, we discuss the most recent developments in yeast autophagy and point out the challenges we face in the future.     

Vesicle fusion in protein transport through the Golgi in vitro does not involve long-lived prefusion intermediates. A reassessment of the kinetics of transport as measured by glycosylation.

The well-characterized cell-free assay measuring protein transport between compartments of the Golgi [Balch, W. E., Dunphy, W. G., Braell, W. A., & Rothman, J. E. (1984) Cell 39, 405-416] utilizes glycosylation of a glycoprotein to mark movement of that protein from one Golgi compartment to the next. Glycosylation had been thought to occur immediately after vesicles carrying the glycoprotein fuse with their transport target. Therefore, the kinetics of glycosylation were taken to reflect the kinetics of vesicle fusion. We previously isolated and raised monoclonal antibodies against a protein (the prefusion operating protein, POP) which is required in this assay at a step after vesicles have apparently been formed and interacted with the target membranes, but long before glycosylation takes place. This was therefore presumed to be a reaction involving targeted but unfused vesicles. Here we report that POP is identical to uridine monophosphokinase, as revealed by molecular cloning. We show that POP is not active in transport per se but instead enhances the glycosylation used to mark transport. This indicated that, contrary to previous assumptions, glycosylation might lag significantly behind vesicle fusion. We directly show this to be true. This alters the interpretation of several earlier studies. In particular, the previously reported existence of a late, prefusion intermediate, the "NEM-resistant intermediate", can be seen to be due to effects on glycosylation and not indicative of true fusion events.     

How to get to the right place at the right time: Rab/Ypt small GTPases and vesicle transport

Summary Vesicles often must be transported over long distances in a very crowded cytoplasmic environment encumbered by the cytoskeleton and membranes of different origin that provide an important barrier to their free diffusion. In animal cells with specialised tasks, such as neurons or endothelial cells, vesicles that are directed to the cell periphery are linked to the microtubular cytoskeleton tracks via association with motor proteins that allow their vectorial movement. In lower eukaryotes the actin cytoskeleton plays a prominent role in organising vesicle movement during polarised growth and mating. The Ras-like small GTPases of the Rab/Ypt family play an essential role in vesicle trafficking and due to their diversity and specific localisation have long been implicated in the selective delivery of vesicles. Recent evidence has cast doubt on the classical point of view of how this class of proteins acts in vesicle transport and suggests their involvement also in the events that permit vesicle anchoring to the cytoskeleton. Therefore, after a brief review of what is known about how vesicle movement is achieved in mammalian and yeast systems, and how Rab/Ypt proteins regulate the vesicle predocking events, it is discussed how these proteins might participate in the events that lead to vesicle movement through association with the cytoskeleton machinery.     

The t-SNARE syntaxin is sufficient for spontaneous fusion of synaptic vesicles to planar membranes

Vesicular trafficking and exocytosis are directed by the complementary interaction of membrane proteins that together form the SNARE complex. This complex is composed of proteins in the vesicle membrane (v-SNAREs) that intertwine with proteins of the target membrane (t-SNAREs). Here we show that modified synaptic vesicles (mSV), containing v-SNAREs, spontaneously fuse to planar membranes containing the t-SNARE, syntaxin 1A. Fusion was Ca(2+)-independent and did not occur with vesicles lacking v-SNAREs. Therefore, syntaxin alone forms a functional fusion complex with v-SNAREs. Our functional fusion assay uses synaptic vesicles that are modified, so each fusion event results in an observable transient current. The mSV do not fuse with protein-free membranes. Additionally, artificial vesicles lacking v-SNAREs do not fuse with membranes containing syntaxin. This technique can be adapted to measure fusion in other SNARE systems and should enable the identification of proteins critical to vesicle-membrane fusion. This will further our understanding of exocytosis and may improve targeting and delivery of therapeutic agents packaged in vesicles.     

How to get to the right place at the right time: Rab/Ypt small GTPases and vesicle transport

Vesicles often must be transported over long distances in a very crowded cytoplasmic environment encumbered by the cytoskeleton and membranes of different origin that provide an important barrier to their free diffusion. In animal cells with specialised tasks, such as neurons or endothelial cells, vesicles that are directed to the cell periphery are linked to the microtubular cytoskeleton tracks via association with motor proteins that allow their vectorial movement. In lower eukaryotes the actin cytoskeleton plays a prominent role in organising vesicle movement during polarised growth and mating. The Ras-like small GTPases of the Rab/Ypt family play an essential role in vesicle trafficking and due to their diversity and specific localisation have long been implicated in the selective delivery of vesicles. Recent evidence has cast doubt on the classical point of view of how this class of proteins acts in vesicle transport and suggests their involvement also in the events that permit vesicle anchoring to the cytoskeleton. Therefore, after a brief review of what is known about how vesicle movement is achieved in mammalian and yeast systems, and how Rab/Ypt proteins regulate the vesicle predocking events, it is discussed how these proteins might participate in the events that lead to vesicle movement through association with the cytoskeleton machinery.     

Lipid metabolism and regulation of membrane trafficking

The past 20 years have witnessed tremendous progress in our understanding of the molecular machinery that controls protein and membrane transport between organelles (Scheckman R, Orci L. Coat proteins and vesicle budding. Science 1996;271: 1526–1533 and Rothman JE. Mechanisms of intracellular protein transport. Nature 1994;372: 55–63.) The research efforts responsible for these impressive advances have largely focused on the identification and characterization of protein factors that participate in membrane trafficking events. The role of membranes and their lipid constituents has received considerably less attention. Indeed, until rather recently, popular models for mechanisms of membrane trafficking had relegated membrane lipids to the status of a passive platform, subject to deformation by the action of coat proteins whose polymerization and depolymerization govern vesicle budding and fusion reactions. The 1990s, and particularly its last half, has brought fundamental reappraisals of the interface of lipids and lipid metabolism in regulating intracellular membrane trafficking events. Some of the emerging themes are reviewed here.     

Molecular machinery and mechanism of cell secretion

Secretion occurs in all living cells and involves the delivery of intracellular products to the cell exterior. Secretory products are packaged and stored in membranous sacs or vesicles within the cell. When the cell needs to secrete these products, the secretory vesicles containing them dock and fuse at plasma membrane-associated supramolecular structures, called porosomes, to release their contents. Specialized cells for neurotransmission, enzyme secretion, or hormone release use a highly regulated secretory process. Similar to other fundamental cellular processes, cell secretion is precisely regulated. During secretion, swelling of secretory vesicles results in a build-up of intravesicular pressure, allowing expulsion of vesicular contents. The extent of vesicle swelling dictates the amount of vesicular contents expelled. The discovery of the porosome as the universal secretory machinery, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane have been determined. The molecular mechanism of secretory vesicle swelling and the fusion of opposing bilayers, that is, the fusion of secretory vesicle membrane at the base of the porosome membrane, have also been resolved. These findings reveal, for the first time, the universal molecular machinery and mechanism of secretion in cells.     

Kinetic diversity in the fusion of exocytotic vesicles.

The speed at which secretory vesicles fuse with the plasma membrane is a key parameter for neuronal and endocrine functions. We determined the precise time courses for fusion of small clear and large dense-core vesicles in PC12 and chromaffin cells by simultaneously measuring both plasma membrane areas and release of vesicular contents. We found that instantaneous increases in cytosolic Ca2+ concentration evoked vesicle fusion, but with time constants that varied over four orders of magnitude among different types of vesicles and cells. This indicates that the molecular machinery for the final Ca2+-dependent fusion steps of exocytosis is highly variable and is as critical as Ca2+ signalling processes in determining the speed and amount of secretion of neurotransmitters and hormones. Our results suggest a new possibility that the molecules responsible for the final fusion reaction that leads to vesicle fusion are key determinants for neuronal plasticity and hormonal disorders.     

Molecular anatomy of a trafficking organelle

Membrane traffic in eukaryotic cells involves transport of vesicles that bud from a donor compartment and fuse with an acceptor compartment. Common principles of budding and fusion have emerged, and many of the proteins involved in these events are now known. However, a detailed picture of an entire trafficking organelle is not yet available. Using synaptic vesicles as a model, we have now determined the protein and lipid composition; measured vesicle size, density, and mass; calculated the average protein and lipid mass per vesicle; and determined the copy number of more than a dozen major constituents. A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins. Synaptic vesicles are dominated by proteins, possess a surprising diversity of trafficking proteins, and, with the exception of the V-ATPase that is present in only one to two copies, contain numerous copies of proteins essential for membrane traffic and neurotransmitter uptake.     

Molecular engineering of exocytic vesicle traffic enhances the productivity of Chinese hamster ovary cells

A complex vesicle trafficking system manages the precise and regulated distribution of proteins, membranes and other molecular cargo between cellular compartments as well as the secretion of (heterologous) proteins in mammalian cells. Sec1/Munc18 (SM) proteins are key components of the system by regulating membrane fusion. However, it is not clear how SM proteins contribute to the overall exocytosis. Here, functional analysis of the SM protein Sly1 and Munc18c suggested a united, positive impact upon SNARE-based fusion of ER-to-Golgi- and Golgi-to-plasma membrane-addressed exocytic vesicles and increased the secretory capacity of different therapeutic proteins in Chinese hamster ovary cells up to 40 pg/cell/day. Sly1- and Munc18c-based vesicle traffic engineering cooperated with Xbp-1-mediated ER/Golgi organelle engineering. Our study supports a model for united function of SM proteins in stimulating vesicle trafficking machinery and provides a generic secretion engineering strategy to improve biopharmaceutical manufacturing of important protein therapeutics.     

Use of fluorescence-activated vesicle sorting for isolation of Naked2-associated, basolaterally targeted exocytic vesicles for proteomics analysis

By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor-alpha (TGFalpha), Naked2 coats TGFalpha-containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGFalpha-containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na(+)/K(+)-ATPase alpha1 was identified as an additional cargo within these vesicles. As an initial validation step, we confirmed their presence and that of three additional proteins tested (annexin A1, annexin A2, and IQGAP1) in wild-type Naked2 vesicles. To our knowledge, this is the first large scale protein characterization of a population of basolaterally targeted exocytic vesicles and supports the use of fluorescence-activated vesicle sorting as a useful tool for isolation of cellular organelles for comprehensive proteomics analysis.     

Probing intracellular vesicle trafficking and membrane remodelling by cryo-EM

Protein transport between the membranous compartments of the eukaryotic cells is mediated by the constant fission and fusion of the membrane-bounded vesicles from a donor to an acceptor membrane. While there are many membrane remodelling complexes in eukaryotes, COPII, COPI, and clathrin-coated vesicles are the three principal classes of coat protein complexes that participate in vesicle trafficking in the endocytic and secretory pathways. These vesicle-coat proteins perform two key functions: deforming lipid bilayers into vesicles and encasing selective cargoes. The three trafficking complexes share some commonalities in their structural features but differ in their coat structures, mechanisms of cargo sorting, vesicle formation, and scission. While the structures of many of the proteins involved in vesicle formation have been determined in isolation by X-ray crystallography, elucidating the proteins' structures together with the membrane is better suited for cryogenic electron microscopy (cryo-EM). In recent years, advances in cryo-EM have led to solving the structures and mechanisms of several vesicle trafficking complexes and associated proteins.     

Lipid vesicle-cell interactions. II. Induction of cell fusion

The ability of lipid vesicles of simple composition (lecithin, lysolecithin, and stearylamine) to induce cells of various types to fuse has been investigated. One in every three or four cells in monolayer cultures can be induced to fuse with a vesicle dose of about 100 per cell. At such dosages and for exposures of 15 min to 1 h, vesicles have essentially no effect on cell viability. Under anaerobic conditions, these cells lyse rather than fuse. Avian erythrocytes are readily fused with lipid vesicles in the presence of dextran. Fusion indices increase linearly with the zeta potential of the vesicles (increasing stearylamine content), indicating that contact between vesicle and cell membrane is required. Fusion indices increase sublinearly with increasing lysolecithin content. Divalent cations increase fusion indices at high vesicle doses. The data presented are consistent with the hypothesis that cell fusion occurs via simultaneous fusion of a vesicle with two adhering cell membranes.