ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.

Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.

The Oxysterol-Binding Protein Cycle: Burning Off PI(4)P to Transport Cholesterol.

To maintain an asymmetric distribution of ions across membranes, protein pumps displace ions against their concentration gradient by using chemical energy. Here, we describe a functionally analogous but topologically opposite process that applies to the lipid transfer protein (LTP) oxysterol-binding protein (OSBP). This multidomain protein exchanges cholesterol for the phosphoinositide phosphatidylinositol 4-phosphate [PI(4)P] between two apposed membranes. Because of the subsequent hydrolysis of PI(4)P, this counterexchange is irreversible and contributes to the establishment of a cholesterol gradient along organelles of the secretory pathway. The facts that some natural anti-cancer molecules block OSBP and that many viruses hijack the OSBP cycle for the formation of intracellular replication organelles highlight the importance and potency of OSBP-mediated lipid exchange. The architecture of some LTPs is similar to that of OSBP, suggesting that the principles of the OSBP cycle-burning PI(4)P for the vectorial transfer of another lipid-might be general.

PI3K isoforms in cell signalling and vesicle trafficking.

PI3Ks are a family of lipid kinases that phosphorylate intracellular inositol lipids to regulate signalling and intracellular vesicular traffic. Mammals have eight isoforms of PI3K, divided into three classes. The class I PI3Ks generate 3-phosphoinositide lipids, which directly activate signal transduction pathways. In addition to being frequently genetically activated in cancer, similar mutations in class I PI3Ks have now also been found in a human non-malignant overgrowth syndrome and a primary immune disorder that predisposes to lymphoma. The class II and class III PI3Ks are regulators of membrane traffic along the endocytic route, in endosomal recycling and autophagy, with an often indirect effect on cell signalling. Here, we summarize current knowledge of the different PI3K classes and isoforms, focusing on recently uncovered biological functions and the mechanisms by which these kinases are activated. Deeper insight into the PI3K isoforms will undoubtedly continue to contribute to a better understanding of fundamental cell biological processes and, ultimately, of human disease.

Shedding light on the cell biology of extracellular vesicles.

Extracellular vesicles are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles, which originate from the endosomal system or which are shed from the plasma membrane, respectively. They are present in biological fluids and are involved in multiple physiological and pathological processes. Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material. Knowledge of the cellular processes that govern extracellular vesicle biology is essential to shed light on the physiological and pathological functions of these vesicles as well as on clinical applications involving their use and/or analysis. However, in this expanding field, much remains unknown regarding the origin, biogenesis, secretion, targeting and fate of these vesicles.

Sphingolipids and their metabolism in physiology and disease.

Studies of bioactive lipids in general and sphingolipids in particular have intensified over the past several years, revealing an unprecedented and unanticipated complexity of the lipidome and its many functions, which rivals, if not exceeds, that of the genome or proteome. These results highlight critical roles for bioactive sphingolipids in most, if not all, major cell biological responses, including all major cell signalling pathways, and they link sphingolipid metabolism to key human diseases. Nevertheless, the fairly nascent field of bioactive sphingolipids still faces challenges in its biochemical and molecular underpinnings, including defining the molecular mechanisms of pathway and enzyme regulation, the study of lipid-protein interactions and the development of cellular probes, suitable biomarkers and therapeutic approaches.

Ion and lipid orchestration of secondary active transport.

Transporting small molecules across cell membranes is an essential process in cell physiology. Many structurally diverse, secondary active transporters harness transmembrane electrochemical gradients of ions to power the uptake or efflux of nutrients, signalling molecules, drugs and other ions across cell membranes. Transporters reside in lipid bilayers on the interface between two aqueous compartments, where they are energized and regulated by symported, antiported and allosteric ions on both sides of the membrane and the membrane bilayer itself. Here we outline the mechanisms by which transporters couple ion and solute fluxes and discuss how structural and mechanistic variations enable them to meet specific physiological needs and adapt to environmental conditions. We then consider how general bilayer properties and specific lipid binding modulate transporter activity. Together, ion gradients and lipid properties ensure the effective transport, regulation and distribution of small molecules across cell membranes.

Integrating mitochondrial translation into the cellular context.

Mitochondrial-encoded subunits of the oxidative phosphorylation system assemble with nuclear-encoded subunits into enzymatic complexes. Recent findings showed that mitochondrial translation is linked to other mitochondrial functions, as well as to cellular processes. The supply of mitochondrial-encoded proteins is coordinated by the coupling of mitochondrial protein synthesis with assembly of respiratory chain complexes. MicroRNAs imported from the cytoplasm into mitochondria were, surprisingly, found to act as regulators of mitochondrial translation. In turn, translation in mitochondria controls cellular proliferation, and mitochondrial ribosomal subunits contribute to the cytoplasmic stress response. Thus, translation in mitochondria is apparently integrated into cellular processes.

Transmembrane transport in inorganic colloidal cell-mimics.

A key aspect of living cells is their ability to harvest energy from the environment and use it to pump specific atomic and molecular species in and out of their system-typically against an unfavourable concentration gradient1. Active transport allows cells to store metabolic energy, extract waste and supply organelles with basic building blocks at the submicrometre scale. Unlike living cells, abiotic systems do not have the delicate biochemical machinery that can be specifically activated to precisely control biological matter2-5. Here we report the creation of microcapsules that can be brought out of equilibrium by simple global variables (illumination and pH), to capture, concentrate, store and deliver generic microscopic payloads. Borrowing no materials from biology, our design uses hollow colloids serving as spherical cell-membrane mimics, with a well-defined single micropore. Precisely tunable monodisperse capsules are the result of a synthetic self-inflation mechanism and can be produced in bulk quantities. Inside the hollow unit, a photoswitchable catalyst6 produces a chemical gradient that propagates to the exterior through the membrane's micropore and pumps target objects into the cell, acting as a phoretic tractor beam7. An entropic energy barrier8,9 brought about by the micropore's geometry retains the cargo even when the catalyst is switched off. Delivery is accomplished on demand by reversing the sign of the phoretic interaction. Our findings provide a blueprint for developing the next generation of smart materials, autonomous micromachinery and artificial cell-mimics.

Bidirectional cargo transport: moving beyond tug of war.

Vesicles, organelles and other intracellular cargo are transported by kinesin and dynein motors, which move in opposite directions along microtubules. This bidirectional cargo movement is frequently described as a 'tug of war' between oppositely directed molecular motors attached to the same cargo. However, although many experimental and modelling studies support the tug-of-war paradigm, numerous knockout and inhibition studies in various systems have found that inhibiting one motor leads to diminished motility in both directions, which is a 'paradox of co-dependence' that challenges the paradigm. In an effort to resolve this paradox, three classes of bidirectional transport models--microtubule tethering, mechanical activation and steric disinhibition--are proposed, and a general mathematical modelling framework for bidirectional cargo transport is put forward to guide future experiments.

Mitochondrial Pyruvate Import Promotes Long-Term Survival of Antibody-Secreting Plasma Cells.

Durable antibody production after vaccination or infection is mediated by long-lived plasma cells (LLPCs). Pathways that specifically allow LLPCs to persist remain unknown. Through bioenergetic profiling, we found that human and mouse LLPCs could robustly engage pyruvate-dependent respiration, whereas their short-lived counterparts could not. LLPCs took up more glucose than did short-lived plasma cells (SLPCs) in vivo, and this glucose was essential for the generation of pyruvate. Glucose was primarily used to glycosylate antibodies, but glycolysis could be promoted by stimuli such as low ATP levels and the resultant pyruvate used for respiration by LLPCs. Deletion of Mpc2, which encodes an essential component of the mitochondrial pyruvate carrier, led to a progressive loss of LLPCs and of vaccine-specific antibodies in vivo. Thus, glucose uptake and mitochondrial pyruvate import prevent bioenergetic crises and allow LLPCs to persist. Immunizations that maximize these plasma cell metabolic properties might thus provide enduring antibody-mediated immunity.

Organization of the ER-Golgi interface for membrane traffic control.

Coat protein complex I (COPI) and COPII are required for bidirectional membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. While these core coat machineries and other transport factors are highly conserved across species, high-resolution imaging studies indicate that the organization of the ER-Golgi interface is varied in eukaryotic cells. Regulation of COPII assembly, in some cases to manage distinct cellular cargo, is emerging as one important component in determining this structure. Comparison of the ER-Golgi interface across different systems, particularly mammalian and plant cells, reveals fundamental elements and distinct organization of this interface. A better understanding of how these interfaces are regulated to meet varying cellular secretory demands should provide key insights into the mechanisms that control efficient trafficking of proteins and lipids through the secretory pathway.

The ArsQ permease and transport of the antibiotic arsinothricin.

The pentavalent organoarsenical arsinothricin (AST) is a natural product synthesized by the rhizosphere bacterium Burkholderia gladioli GSRB05. AST is a broad-spectrum antibiotic effective against human pathogens such as carbapenem-resistant Enterobacter cloacae. It is a non-proteogenic amino acid and glutamate mimetic that inhibits bacterial glutamine synthetase. The AST biosynthetic pathway is composed of a three-gene cluster, arsQML. ArsL catalyzes synthesis of reduced trivalent hydroxyarsinothricin (R-AST-OH), which is methylated by ArsM to the reduced trivalent form of AST (R-AST). In the culture medium of B. gladioli, both trivalent species appear as the corresponding pentavalent arsenicals, likely due to oxidation in air. ArsQ is an efflux permease that is proposed to transport AST or related species out of the cells, but the chemical nature of the actual transport substrate is unclear. In this study, B. gladioli arsQ was expressed in Escherichia coli and shown to confer resistance to AST and its derivatives. Cells of E. coli accumulate R-AST, and exponentially growing cells expressing arsQ take up less R-AST. The cells exhibit little transport of their pentavalent forms. Transport was independent of cellular energy and appears to be equilibrative. A homology model of ArsQ suggests that Ser320 is in the substrate binding site. A S320A mutant exhibits reduced R-AST-OH transport, suggesting that it plays a role in ArsQ function. The ArsQ permease is proposed to be an energy-independent uniporter responsible for downhill transport of the trivalent form of AST out of cells, which is oxidized extracellularly to the active form of the antibiotic.

AP-3 vesicle uncoating occurs after HOPS-dependent vacuole tethering.

Heterotetrameric adapter (AP) complexes cooperate with the small GTPase Arf1 or lipids in cargo selection, vesicle formation, and budding at endomembranes in eukaryotic cells. While most AP complexes also require clathrin as the outer vesicle shell, formation of AP-3-coated vesicles involved in Golgi-to-vacuole transport in yeast has been postulated to depend on Vps41, a subunit of the vacuolar HOPS tethering complex. HOPS has also been identified as the tether of AP-3 vesicles on vacuoles. To unravel this conundrum of a dual Vps41 function, we anchored Vps41 stably to the mitochondrial outer membrane. By monitoring AP-3 recruitment, we now show that Vps41 can tether AP-3 vesicles to mitochondria, yet AP-3 vesicles can form in the absence of Vps41 or clathrin. By proximity labeling and mass spectrometry, we identify the Arf1 GTPase-activating protein (GAP) Age2 at the AP-3 coat and show that tethering, but not fusion at the vacuole can occur without complete uncoating. We conclude that AP-3 vesicles retain their coat after budding and that their complete uncoating occurs only after tethering at the vacuole.

The trophoblast clock controls transport across placenta in mice.

In mammals, 24-h rhythms of physiology and behavior are organized by a body-wide network of clock genes and proteins. Despite the well-known function of the adult circadian system, the roles of maternal, fetal and placental clocks during pregnancy are poorly defined. In the mature mouse placenta, the labyrinth zone (LZ) is of fetal origin and key for selective nutrient and waste exchange. Recently, clock gene expression has been detected in LZ and other fetal tissues; however, there is no evidence of a placental function controlled by the LZ clock. Here, we demonstrate that specifically the trophoblast layer of the LZ harbors an already functional clock by late gestation, able to regulate in a circadian manner the expression and activity of the xenobiotic efflux pump, ATP-binding cassette sub-family B member 1 (ABCB1), likely gating the fetal exposure to drugs from the maternal circulation to certain times of the day. As more than 300 endogenous and exogenous compounds are substrates of ABCB1, our results might have implications in choosing the maternal treatment time when aiming either maximal/minimal drug availability to the fetus/mother.

Getting active: protein sorting in endocytic recycling.

Endocytic recycling returns proteins to the plasma membrane in many physiological contexts. Studies of these events have helped to elucidate fundamental mechanisms that underlie recycling. Recycling was for some time considered to be the exception to a general mechanism of active cargo sorting in multiple intracellular pathways. In recent years, studies have begun to reconcile this seeming disparity and also suggest explanations for why early recycling studies did not detect active sorting. Further articulation of this emerging trend has far-reaching implications for a deeper understanding of many physiological and pathological events that require recycling.

Dynamics of hydraulic and contractile wave-mediated fluid transport during Drosophila oogenesis.

From insects to mice, oocytes develop within cysts alongside nurse-like sister germ cells. Prior to fertilization, the nurse cells' cytoplasmic contents are transported into the oocyte, which grows as its sister cells regress and die. Although critical for fertility, the biological and physical mechanisms underlying this transport process are poorly understood. Here, we combined live imaging of germline cysts, genetic perturbations, and mathematical modeling to investigate the dynamics and mechanisms that enable directional and complete cytoplasmic transport in Drosophila melanogaster egg chambers. We discovered that during "nurse cell (NC) dumping" most cytoplasm is transported into the oocyte independently of changes in myosin-II contractility, with dynamics instead explained by an effective Young-Laplace law, suggesting hydraulic transport induced by baseline cell-surface tension. A minimal flow-network model inspired by the famous two-balloon experiment and motivated by genetic analysis of a myosin mutant correctly predicts the directionality, intercellular pattern, and time scale of transport. Long thought to trigger transport through "squeezing," changes in actomyosin contractility are required only once NC volume has become comparable to nuclear volume, in the form of surface contractile waves that drive NC dumping to completion. Our work thus demonstrates how biological and physical mechanisms cooperate to enable a critical developmental process that, until now, was thought to be mainly biochemically regulated.

On and off controls within dynein-dynactin on native cargoes.

The dynein-dynactin nanomachine transports cargoes along microtubules in cells. Why dynactin interacts separately with the dynein motor and also with microtubules is hotly debated. Here we disrupted these interactions in a targeted manner on phagosomes extracted from cells, followed by optical trapping to interrogate native dynein-dynactin teams on single phagosomes. Perturbing the dynactin-dynein interaction reduced dynein's on rate to microtubules. In contrast, perturbing the dynactin-microtubule interaction increased dynein's off rate markedly when dynein was generating force against the optical trap. The dynactin-microtubule link is therefore required for persistence against load, a finding of importance because disease-relevant mutations in dynein-dynactin are known to interfere with "high-load" functions of dynein in cells. Our findings call attention to a less studied property of dynein-dynactin, namely, its detachment against load, in understanding dynein dysfunction.

Naphthylphthalamic acid associates with and inhibits PIN auxin transporters.

N-1-naphthylphthalamic acid (NPA) is a key inhibitor of directional (polar) transport of the hormone auxin in plants. For decades, it has been a pivotal tool in elucidating the unique polar auxin transport-based processes underlying plant growth and development. Its exact mode of action has long been sought after and is still being debated, with prevailing mechanistic schemes describing only indirect connections between NPA and the main transporters responsible for directional transport, namely PIN auxin exporters. Here we present data supporting a model in which NPA associates with PINs in a more direct manner than hitherto postulated. We show that NPA inhibits PIN activity in a heterologous oocyte system and that expression of NPA-sensitive PINs in plant, yeast, and oocyte membranes leads to specific saturable NPA binding. We thus propose that PINs are a bona fide NPA target. This offers a straightforward molecular basis for NPA inhibition of PIN-dependent auxin transport and a logical parsimonious explanation for the known physiological effects of NPA on plant growth, as well as an alternative hypothesis to interpret past and future results. We also introduce PIN dimerization and describe an effect of NPA on this, suggesting that NPA binding could be exploited to gain insights into structural aspects of PINs related to their transport mechanism.

Miro proteins connect mitochondrial function and intercellular transport.

Mitochondria are organelles present in most eukaryotic cells, where they play major and multifaceted roles. The classical notion of the main mitochondrial function as the powerhouse of the cell per se has been complemented by recent discoveries pointing to mitochondria as organelles affecting a number of other auxiliary processes. They go beyond the classical energy provision via acting as a relay point of many catabolic and anabolic processes, to signaling pathways critically affecting cell growth by their implication in de novo pyrimidine synthesis. These additional roles further underscore the importance of mitochondrial homeostasis in various tissues, where its deregulation promotes a number of pathologies. While it has long been known that mitochondria can move within a cell to sites where they are needed, recent research has uncovered that mitochondria can also move between cells. While this intriguing field of research is only emerging, it is clear that mobilization of mitochondria requires a complex apparatus that critically involves mitochondrial proteins of the Miro family, whose role goes beyond the mitochondrial transfer, as will be covered in this review.