Structural basis of ELKS/Rab6B interaction and its role in vesicle capturing enhanced by liquid-liquid phase separation.

ELKS proteins play a key role in organizing intracellular vesicle trafficking and targeting in both neurons and non-neuronal cells. While it is known that ELKS interacts with the vesicular traffic regulator, the Rab6 GTPase, the molecular basis governing ELKS-mediated trafficking of Rab6-coated vesicles, has remained unclear. In this study, we solved the Rab6B structure in complex with the Rab6-binding domain of ELKS1, revealing that a C-terminal segment of ELKS1 forms a helical hairpin to recognize Rab6B through a unique binding mode. We further showed that liquid-liquid phase separation (LLPS) of ELKS1 allows it to compete with other Rab6 effectors for binding to Rab6B and accumulate Rab6B-coated liposomes to the protein condensate formed by ELKS1. We also found that the ELKS1 condensate recruits Rab6B-coated vesicles to vesicle-releasing sites and promotes vesicle exocytosis. Together, our structural, biochemical, and cellular analyses suggest that ELKS1, via the LLPS-enhanced interaction with Rab6, captures Rab6-coated vesicles from the cargo transport machine for efficient vesicle release at exocytotic sites. These findings shed new light on the understanding of spatiotemporal regulation of vesicle trafficking through the interplay between membranous structures and membraneless condensates.

Capturing the mechanics of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis enables selective uptake of molecules into cells in response to changing cellular needs. It occurs through assembly of coat components around the plasma membrane that determine vesicle contents and facilitate membrane bending to form a clathrin-coated transport vesicle. In this review we discuss recent cryo-electron microscopy structures that have captured a series of events in the life cycle of a clathrin-coated vesicle. Both single particle analysis and tomography approaches have revealed details of the clathrin lattice structure itself, how AP2 may interface with clathrin within a coated vesicle and the importance of PIP2 binding for assembly of the yeast adaptors Sla2 and Ent1 on the membrane. Within cells, cryo-electron tomography of clathrin in flat lattices and high-speed AFM studies provided new insights into how clathrin morphology can adapt during CCV formation. Thus, key mechanical processes driving clathrin-mediated endocytosis have been captured through multiple techniques working in partnership.

Fabrication of CaCO3-Coated Vesicles by Biomineralization and Their Application as Carriers of Drug Delivery Systems.

We fabricated CaCO3-coated vesicles as drug carriers that release their cargo under a weakly acidic condition. We designed and synthesized a peptide lipid containing the Val-His-Val-Glu-Val-Ser sequence as the hydrophilic part, and with two palmitoyl groups at the N-terminal as the anchor groups of the lipid bilayer membrane. Vesicles embedded with the peptide lipids were prepared. The CaCO3 coating of the vesicle surface was performed by the mineralization induced by the embedded peptide lipid. The peptide lipid produced the mineral source, CO32-, for CaCO3 mineralization through the hydrolysis of urea. We investigated the structure of the obtained CaCO3-coated vesicles using transmission electron microscopy (TEM). The vesicles retained the spherical shapes, even in vacuo. Furthermore, the vesicles had inner spaces that acted as the drug cargo, as observed by the TEM tomographic analysis. The thickness of the CaCO3 shell was estimated as ca. 20 nm. CaCO3-coated vesicles containing hydrophobic or hydrophilic drugs were prepared, and the drug release properties were examined under various pH conditions. The mineralized CaCO3 shell of the vesicle surface was dissolved under a weakly acidic condition, pH 6.0, such as in the neighborhood of cancer tissues. The degradation of the CaCO3 shell induced an effective release of the drugs. Such behavior suggests potential of the CaCO3-coated vesicles as carriers for cancer therapies.

Multi-modal adaptor-clathrin contacts drive coated vesicle assembly.

Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the ß2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of ß2 hinge-appendage (ß2HA). We find that the ß2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, ß2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of ß2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage. We propose that ß2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.

Weakly Internalized Receptors Use Coated Vesicle Heterogeneity to Evade Competition during Endocytosis.

The uptake of receptors by clathrin-mediated endocytosis underlies signaling, nutrient import, and recycling of transmembrane proteins and lipids. In the complex, crowded environment of the plasma membrane, receptors are internalized when they bind to components of the clathrin coat, such as the major adaptor protein, AP2. Receptors with higher affinity for AP2 are known to be more strongly internalized compared to receptors with lower affinity. However, it remains unclear how receptors with different affinities compete for space within crowded endocytic structures. To address this question, we constructed receptors with varying affinities for AP2 and allowed them to compete against one another during internalization. As expected, the internalization of a receptor with high affinity for AP2 was reduced when it was coexpressed with a competing receptor of similar affinity. However, receptors of low affinity for AP2 were surprisingly difficult to displace from endocytic structures, even when expressed alongside receptors with much higher affinity. To understand how these low-affinity receptors are protected from competition, we looked at AP2 heterogeneity across clathrin-coated structures. When we examined structures with lower-than-average AP2 content, we found that they were relatively enriched in cargo of low affinity for AP2 and depleted of cargo with high affinity. These findings suggest that the heterogeneity of adaptor protein content across the population of endocytic structures enables the internalization of diverse receptors. Given the critical role that internalization plays in signaling, this effect may help to prevent strongly internalized receptors from interfering with the cell's ability to process signals from weakly internalized receptors.

Targeted gene silencing in vivo by platelet membrane-coated metal-organic framework nanoparticles.

Small interfering RNA (siRNA) is a powerful tool for gene silencing that has been used for a wide range of biomedical applications, but there are many challenges facing its therapeutic use in vivo. Here, we report on a platelet cell membrane-coated metal-organic framework (MOF) nanodelivery platform for the targeted delivery of siRNA in vivo. The MOF core is capable of high loading yields, and its pH sensitivity enables endosomal disruption upon cellular uptake. The cell membrane coating provides a natural means of biointerfacing with disease substrates. It is shown that high silencing efficiency can be achieved in vitro against multiple target genes. Using a murine xenograft model, significant antitumor targeting and therapeutic efficacy are observed. Overall, the biomimetic nanodelivery system presented here provides an effective means of achieving gene silencing in vivo and could be used to expand the applicability of siRNA across a range of disease-relevant applications.

Twenty-five years after coat protein complex II.

In 1994, a convergence of ideas and collaborative research orchestrated by Randy Schekman led to the discovery of the coat protein complex II (COPII). In this Perspective, the chain of events enabling discovery of a new vesicle coat and progress on understanding COPII budding mechanisms are considered.

Interaction of microtubules and actin during the post-fusion phase of exocytosis.

Exocytosis is the intracellular trafficking step where a secretory vesicle fuses with the plasma membrane to release vesicle content. Actin and microtubules both play a role in exocytosis; however, their interplay is not understood. Here we study the interaction of actin and microtubules during exocytosis in lung alveolar type II (ATII) cells that secrete surfactant from large secretory vesicles. Surfactant extrusion is facilitated by an actin coat that forms on the vesicle shortly after fusion pore opening. Actin coat compression allows hydrophobic surfactant to be released from the vesicle. We show that microtubules are localized close to actin coats and stay close to the coats during their compression. Inhibition of microtubule polymerization by colchicine and nocodazole affected the kinetics of actin coat formation and the extent of actin polymerisation on fused vesicles. In addition, microtubule and actin cross-linking protein IQGAP1 localized to fused secretory vesicles and IQGAP1 silencing influenced actin polymerisation after vesicle fusion. This study demonstrates that microtubules can influence actin coat formation and actin polymerization on secretory vesicles during exocytosis.

From the resolution revolution to evolution: structural insights into the evolutionary relationships between vesicle coats and the nuclear pore.

Nuclear pores and coated vesicles are elaborate multi-component protein complexes that oligomerize on membranes, and stabilize or induce membrane curvature. Their components, nucleoporins and coat proteins, respectively, share similar structural folds and some principles of how they interact with membranes. The protocoatomer hypothesis postulates that this is due to divergent evolution from a common ancestor. It therefore has been suggested that nucleoporins and coat proteins have similar higher order architectures. Here, we review recent work that relied on technical advances in cryo-electron microscopy and integrative structural biology to take a fresh look on how these proteins form membrane coats in situ. We discuss the relationship between the architectures of nuclear pores and coated vesicles, and their evolutionary origins.

An Overview of Protein Secretion in Yeast and Animal Cells.

Protein secretion mediated by the secretory transport pathway is an important cellular process in eukaryotic cells. In the conventional secretory transport pathway, newly synthesized proteins pass through several endomembrane compartments en route to their specific destinations. Transport of secretory proteins between different compartments is shuttled by small, membrane-enclosed vesicles. To ensure the fidelity of transport, eukaryotic cells employ elaborate molecular machineries to accurately sort newly synthesized proteins into specific transport vesicles and precisely deliver these transport vesicles to distinct acceptor compartments. In this review, we summarize the molecular machineries that regulate each step of vesicular transport in the secretory transport pathway in yeast and animal cells.

An Overview of Protein Secretion in Plant Cells.

The delivery of proteins to the apoplast or protein secretion is an essential process in plant cells. Proteins are secreted to perform various biological functions such as cell wall modification and defense response. Conserved from yeast to mammals, both conventional and unconventional protein secretion pathways have been demonstrated in plants. In the conventional protein secretion pathway, secretory proteins with an N-terminal signal peptide are transported to the extracellular region via the endoplasmic reticulum-Golgi apparatus and the subsequent endomembrane system. By contrast, multiple unconventional protein secretion pathways are proposed to mediate the secretion of the leaderless secretory proteins. In this review, we summarize the recent findings and provide a comprehensive overview of protein secretion pathways in plant cells.

Land-locked mammalian Golgi reveals cargo transport between stable cisternae.

The Golgi is composed of a stack of cis, medial, trans cisternae that are biochemically distinct. The stable compartments model postulates that permanent cisternae communicate through bi-directional vesicles, while the cisternal maturation model postulates that transient cisternae biochemically mature to ensure anterograde transport. Testing either model has been constrained by the diffraction limit of light microscopy, as the cisternae are only 10-20 nm thick and closely stacked in mammalian cells. We previously described the unstacking of Golgi by the ectopic adhesion of Golgi cisternae to mitochondria. Here, we show that cargo processing and transport continue-even when individual Golgi cisternae are separated and "land-locked" between mitochondria. With the increased spatial separation of cisternae, we show using three-dimensional live imaging that cis-Golgi and trans-Golgi remain stable in their composition and size. Hence, we provide new evidence in support of the stable compartments model in mammalian cells.The different composition of Golgi cisternae gave rise to two different models for intra-Golgi traffic: one where stable cisternae communicate via vesicles and another one where cisternae biochemically mature to ensure anterograde transport. Here, the authors provide evidence in support of the stable compartments model.

Outerwear through the ages: evolutionary cell biology of vesicle coats.

Vesicular transport was key to the evolution of eukaryotes, and is essential for eukaryotic life today. All modern eukaryotes have a set of vesicle coat proteins, which couple cargo selection to vesicle budding in the secretory and endocytic pathways. Although these coats share common features (e.g. recruitment via small GTPases, ß-propeller-α-solenoid proteins acting as scaffolds), the relationships between them are not always clear. Structural studies on the coats themselves, comparative genomics and cell biology in diverse eukaryotes, and the recent discovery of the Asgard archaea and their 'eukaryotic signature proteins' are helping us to piece together how coats may have evolved during the prokaryote-to-eukaryote transition.

The Evolution of Organellar Coat Complexes and Organization of the Eukaryotic Cell.

Eukaryotic cells possess a remarkably diverse range of organelles that provide compartmentalization for distinct cellular functions and are likely responsible for the remarkable success of these organisms. The origins and subsequent elaboration of these compartments represent a key aspect in the transition between prokaryotic and eukaryotic cellular forms. The protein machinery required to build, maintain, and define many membrane-bound compartments is encoded by several paralog families, including small GTPases, coiled-bundle proteins, and proteins with ß-propeller and α-solenoid secondary structures. Together these proteins provide the membrane coats and control systems to structure and coordinate the endomembrane system. Mechanistically and evolutionarily, they unite not only secretory and endocytic organelles but also the flagellum and nucleus. The ancient origins for these families have been revealed by recent findings, providing new perspectives on the deep evolutionary processes and relationships that underlie eukaryotic cell structure.

Functional implication of the common evolutionary origin of nuclear pore complex and endomembrane management systems.

Nuclear pore complexes (NPCs) are the sole gateway between the cytoplasm and the nucleus serving both as stringent permeability barrier and active transporters between the two compartments of eukaryotic cells. Complete mechanistic understanding of how these two functions are implemented within one and the same transport machine has not been attained to date. Based on several lines of structural evidence, a hypothesis was proposed postulating that NPCs shares common evolutionary origin with other intracellular systems responsible for active management of endomembranes. In this review we attempt to summarize the evidence supporting this hypothesis. The structural data obtained so far is evaluated and supplemented with the analysis of the functional evidence. Based on this analysis, a model is proposed which integrates the knowledge from the field of NPC function with that obtained from other endomembrane management systems in an attempt to shed new light on the mechanism of the NPC active transport.

Receptor-mediated sorting of soluble vacuolar proteins: myths, facts, and a new model.

To prevent their being released to the cell exterior, acid hydrolases are recognized by receptors at some point in the secretory pathway and diverted towards the lytic compartment of the cell (lysosome or vacuole). In animal cells, the receptor is called the mannosyl 6-phosphate receptor (MPR) and it binds hydrolase ligands in the trans-Golgi network (TGN). These ligands are then sequestered into clathrin-coated vesicles (CCVs) because of motifs in the cytosolic tail of the MPR which interact first with monomeric adaptors (Golgi-localized, Gamma-ear-containing, ARF-binding proteins, GGAs) and then with tetrameric (adaptin) adaptor complexes. The CCVs then fuse with an early endosome, whose more acidic lumen causes the ligands to dissociate. The MPRs are then recycled back to the TGN via retromer-coated carriers. Plants have vacuolar sorting receptors (VSRs) which were originally identified in CCVs isolated from pea (Pisum sativum L.) cotyledons. It was therefore assumed that VSRs would have an analogous function in plants to MPRs in animals. Although this dogma has enjoyed wide support over the last 20 years there are many inconsistencies. Recently, results have been published which are quite contrary to it. It now emerges that VSRs and their ligands can interact very early in the secretory pathway, and dissociate in the TGN, which, in contrast to its mammalian counterpart, has a pH of 5.5. Multivesicular endosomes in plants lack proton pump complexes and consequently have an almost neutral internal pH, which discounts them as organelles of pH-dependent receptor-ligand dissociation. These data force a critical re-evaluation of the role of CCVs at the TGN, especially considering that vacuolar cargo ligands have never been identified in them. We propose that one population of TGN-derived CCVs participate in retrograde transport of VSRs from the TGN. We also present a new model to explain how secretory and vacuolar cargo proteins are effectively separated after entering the late Golgi/TGN compartments.

A novel tribasic Golgi export signal directs cargo protein interaction with activated Rab11 and AP-1-dependent Golgi-plasma membrane trafficking.

The reovirus fusion-associated small transmembrane (FAST) proteins comprise a unique family of viral membrane fusion proteins dedicated to inducing cell-cell fusion. We recently reported that a polybasic motif (PBM) in the cytosolic tail of reptilian reovirus p14 FAST protein functions as a novel tribasic Golgi export signal. Using coimmunoprecipitation and fluorescence resonance energy transfer (FRET) assays, we now show the PBM directs interaction of p14 with GTP-Rab11. Overexpression of dominant-negative Rab11 and RNA interference knockdown of endogenous Rab11 inhibited p14 plasma membrane trafficking and resulted in p14 accumulation in the Golgi complex. This is the first example of Golgi export to the plasma membrane that is dependent on the interaction of membrane protein cargo with activated Rab11. RNA interference and immunofluorescence microscopy further revealed that p14 Golgi export is dependent on AP-1 (but not AP-3 or AP-4) and that Rab11 and AP-1 both colocalize with p14 at the TGN. Together these results imply the PBM mediates interactions of p14 with activated Rab11 at the TGN, resulting in p14 sorting into AP1-coated vesicles for anterograde TGN-plasma membrane transport.

Distinct sets of Rab6 effectors contribute to ZW10--and COG-dependent Golgi homeostasis.

The organization of the Golgi apparatus is determined in part by the interaction of Rab proteins and their diverse array of effectors. Here, we used multiple approaches to identify and characterize a small subset of effectors that mimicked the effects of Rab6 on Golgi ribbon organization. In a visual-based, candidate protein screen, we found that the individual depletion of any of three Rab6 effectors, myosin IIA (MyoIIA), Kif20A and Bicaudal D (BicD), was sufficient to suppress Golgi ribbon fragmentation/dispersal coupled to retrograde tether proteins in a manner paralleling Rab6. MyoIIA and Kif20A depletions were pathway selective and suppressed ZW10-dependent Golgi ribbon fragmentation/dispersal only whereas BicD depletion, like Rab6, suppressed both ZW10- and COG-dependent Golgi ribbon fragmentation. The MyoIIA effects could be produced in short-term assays by the reversible myosin inhibitor, blebbistatin. At the electron microscope level, the effects of BicD-depletion mimicked many of those of Rab6-depletion: longer and more continuous Golgi cisternae and a pronounced accumulation of coated vesicles. Functionally, BicD-depleted cells were inhibited in transport of newly synthesized VSV-G protein to the cell surface. In summary, our results indicate small, partially overlapping subsets of Rab6 effectors are differentially important to two tether-dependent pathways essential to Golgi organization and function.

Membrane lipid-modulated mechanism of action and non-cytotoxicity of novel fungicide aminoglycoside FG08.

A novel aminoglycoside, FG08, that differs from kanamycin B only by a C8 alkyl chain at the 4″-O position, was previously reported. Unlike kanamycin B, FG08 shows broad-spectrum fungicidal but not anti-bacterial activities. To understand its specificity for fungi, the mechanism of action of FG08 was studied using intact cells of the yeast Saccharomyces cerevisiae and small unilamellar membrane vesicles. With exposure to FG08 (30 µg mL(-1)), 8-fold more cells were stained with fluorescein isothiocyanate, cells had 4 to 6-fold higher K(+) efflux rates, and 18-fold more cells were stained with SYTOX Green in comparison to exposure to kanamycin B (30 µg mL(-1)). Yeast mutants with aberrant membrane sphingolipids (no sphingoid base C4 hydroxyl group, truncated very long fatty acid chain, or lacking the terminal phosphorylinositol group of mannosyl-diinositolphosphorylphytoceramide were 4 to 8-fold less susceptible to growth inhibition with FG08 and showed 2 to 10-fold lower SYTOX Green dye uptake rates than did the isogenic wild-type strain. FG08 caused leakage of pre-loaded calcein from 50% of small unilamellar vesicles with glycerophospholipid and sterol compositions that mimic the compositions of fungal plasma membranes. Less than 5 and 10% of vesicles with glycerophospholipid and sterol compositions that mimic bacterial and mammalian cell plasma membranes, respectively, showed calcein leakage. In tetrazolium dye cytotoxicity tests, mammalian cell lines NIH3T3 and C8161.9 showed FG08 toxicity at concentrations that were 10 to 20-fold higher than fungicidal minimal inhibitory concentrations. It is concluded that FG08's growth inhibitory specificity for fungi lie in plasma membrane permeability changes involving mechanisms that are modulated by membrane lipid composition.