Differences in CD80 and CD86 transendocytosis reveal CD86 as a key target for CTLA-4 immune regulation.

CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity.

Membrane fission is promoted by insertion of amphipathic helices and is restricted by crescent BAR domains.

Shallow hydrophobic insertions and crescent-shaped BAR scaffolds promote membrane curvature. Here, we investigate membrane fission by shallow hydrophobic insertions quantitatively and mechanistically. We provide evidence that membrane insertion of the ENTH domain of epsin leads to liposome vesiculation, and that epsin is required for clathrin-coated vesicle budding in cells. We also show that BAR-domain scaffolds from endophilin, amphiphysin, GRAF, and ß2-centaurin limit membrane fission driven by hydrophobic insertions. A quantitative assay for vesiculation reveals an antagonistic relationship between amphipathic helices and scaffolds of N-BAR domains in fission. The extent of vesiculation by these proteins and vesicle size depend on the number and length of amphipathic helices per BAR domain, in accord with theoretical considerations. This fission mechanism gives a new framework for understanding membrane scission in the absence of mechanoenzymes such as dynamin and suggests how Arf and Sar proteins work in vesicle scission.

Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.

How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.

Molecular mechanism and physiological functions of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is the endocytic portal into cells through which cargo is packaged into vesicles with the aid of a clathrin coat. It is fundamental to neurotransmission, signal transduction and the regulation of many plasma membrane activities and is thus essential to higher eukaryotic life. Morphological stages of vesicle formation are mirrored by progression through various protein modules (complexes). The process involves the formation of a putative FCH domain only (FCHO) initiation complex, which matures through adaptor protein 2 (AP2)-dependent cargo selection, and subsequent coat building, dynamin-mediated scission and finally auxilin- and heat shock cognate 70 (HSC70)-dependent uncoating. Some modules can be used in other pathways, and additions or substitutions confer cell specificity and adaptability.

Endophilin marks and controls a clathrin-independent endocytic pathway.

Endocytosis is required for internalization of micronutrients and turnover of membrane components. Endophilin has been assigned as a component of clathrin-mediated endocytosis. Here we show in mammalian cells that endophilin marks and controls a fast-acting tubulovesicular endocytic pathway that is independent of AP2 and clathrin, activated upon ligand binding to cargo receptors, inhibited by inhibitors of dynamin, Rac, phosphatidylinositol-3-OH kinase, PAK1 and actin polymerization, and activated upon Cdc42 inhibition. This pathway is prominent at the leading edges of cells where phosphatidylinositol-3,4-bisphosphate-produced by the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate by SHIP1 and SHIP2-recruits lamellipodin, which in turn engages endophilin. This pathway mediates the ligand-triggered uptake of several G-protein-coupled receptors such as α2a- and ß1-adrenergic, dopaminergic D3 and D4 receptors and muscarinic acetylcholine receptor 4, the receptor tyrosine kinases EGFR, HGFR, VEGFR, PDGFR, NGFR and IGF1R, as well as interleukin-2 receptor. We call this new endocytic route fast endophilin-mediated endocytosis (FEME).

Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis.

During endocytosis, energy is invested to narrow the necks of cargo-containing plasma membrane invaginations to radii at which the opposing segments spontaneously coalesce, thereby leading to the detachment by scission of endocytic uptake carriers. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin. Clathrin-independent endocytic events are often less reliant on dynamin, and whether in these cases BAR domain proteins such as endophilin contribute to scission has remained unexplored. Here we show, in human and other mammalian cell lines, that endophilin-A2 (endoA2) specifically and functionally associates with very early uptake structures that are induced by the bacterial Shiga and cholera toxins, which are both clathrin-independent endocytic cargoes. In controlled in vitro systems, endoA2 reshapes membranes before scission. Furthermore, we demonstrate that endoA2, dynamin and actin contribute in parallel to the scission of Shiga-toxin-induced tubules. Our results establish a novel function of endoA2 in clathrin-independent endocytosis. They document that distinct scission factors operate in an additive manner, and predict that specificity within a given uptake process arises from defined combinations of universal modules. Our findings highlight a previously unnoticed link between membrane scaffolding by endoA2 and pulling-force-driven dynamic scission.

Molecular mechanism of Fast Endophilin-Mediated Endocytosis.

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Clathrin-mediated endocytosis (CME) is the housekeeping pathway in resting cells but additional Clathrin-independent endocytic (CIE) routes, including Fast Endophilin-Mediated Endocytosis (FEME), internalize specific cargoes and support diverse cellular functions. FEME is part of the Dynamin-dependent subgroup of CIE pathways. Here, we review our current understanding of the molecular mechanism of FEME. Key steps are: (i) priming, (ii) cargo selection, (iii) membrane curvature and carrier formation, (iv) membrane scission and (v) cytosolic transport. All steps are controlled by regulatory mechanisms mediated by phosphoinositides and by kinases such as Src, LRRK2, Cdk5 and GSK3ß. A key feature of FEME is that it is not constitutively active but triggered upon the stimulation of selected cell surface receptors by their ligands. In resting cells, there is a priming cycle that concentrates Endophilin into clusters on discrete locations of the plasma membrane. In the absence of receptor activation, the patches quickly abort and new cycles are initiated nearby, constantly priming the plasma membrane for FEME. Upon activation, receptors are swiftly sorted into pre-existing Endophilin clusters, which then bud to form FEME carriers within 10 s. We summarize the hallmarks of FEME and the techniques and assays required to identify it. Next, we review similarities and differences with other CIE pathways and proposed cargoes that may use FEME to enter cells. Finally, we submit pending questions and future milestones and discuss the exciting perspectives that targeting FEME may boost treatments against cancer and neurodegenerative diseases.

Endocytosis in proliferating, quiescent and terminally differentiated cells.

Endocytosis mediates nutrient uptake, receptor internalization and the regulation of cell signaling. It is also hijacked by many bacteria, viruses and toxins to mediate their cellular entry. Several endocytic routes exist in parallel, fulfilling different functions. Most studies on endocytosis have used transformed cells in culture. However, as the majority of cells in an adult body have exited the cell cycle, our understanding is biased towards proliferating cells. Here, we review the evidence for the different pathways of endocytosis not only in dividing, but also in quiescent, senescent and terminally differentiated cells. During mitosis, residual endocytosis is dedicated to the internalization of caveolae and specific receptors. In non-dividing cells, clathrin-mediated endocytosis (CME) functions, but the activity of alternative processes, such as caveolae, macropinocytosis and clathrin-independent routes, vary widely depending on cell types and functions. Endocytosis supports the quiescent state by either upregulating cell cycle arrest pathways or downregulating mitogen-induced signaling, thereby inhibiting cell proliferation. Endocytosis in terminally differentiated cells, such as skeletal muscles, adipocytes, kidney podocytes and neurons, supports tissue-specific functions. Finally, uptake is downregulated in senescent cells, making them insensitive to proliferative stimuli by growth factors. Future studies should reveal the molecular basis for the differences in activities between the different cell states.

Myostatin-like proteins regulate synaptic function and neuronal morphology.

Growth factors of the TGFß superfamily play key roles in regulating neuronal and muscle function. Myostatin (or GDF8) and GDF11 are potent negative regulators of skeletal muscle mass. However, expression of myostatin and its cognate receptors in other tissues, including brain and peripheral nerves, suggests a potential wider biological role. Here, we show that Myoglianin (MYO), the Drosophila homolog of myostatin and GDF11, regulates not only body weight and muscle size, but also inhibits neuromuscular synapse strength and composition in a Smad2-dependent manner. Both myostatin and GDF11 affected synapse formation in isolated rat cortical neuron cultures, suggesting an effect on synaptogenesis beyond neuromuscular junctions. We also show that MYO acts in vivo to inhibit synaptic transmission between neurons in the escape response neural circuit of adult flies. Thus, these anti-myogenic proteins act as important inhibitors of synapse function and neuronal growth.

Chlamydia exploits filopodial capture and a macropinocytosis-like pathway for host cell entry.

Pathogens hijack host endocytic pathways to force their own entry into eukaryotic target cells. Many bacteria either exploit receptor-mediated zippering or inject virulence proteins directly to trigger membrane reorganisation and cytoskeletal rearrangements. By contrast, extracellular C. trachomatis elementary bodies (EBs) apparently employ facets of both the zipper and trigger mechanisms and are only ~400 nm in diameter. Our cryo-electron tomography of C. trachomatis entry revealed an unexpectedly diverse array of host structures in association with invading EBs, suggesting internalisation may progress by multiple, potentially redundant routes or several sequential events within a single pathway. Here we performed quantitative analysis of actin organisation at chlamydial entry foci, highlighting filopodial capture and phagocytic cups as dominant and conserved morphological structures early during internalisation. We applied inhibitor-based screening and employed reporters to systematically assay and visualise the spatio-temporal contribution of diverse endocytic signalling mediators to C. trachomatis entry. In addition to the recognised roles of the Rac1 GTPase and its associated nucleation-promoting factor (NPF) WAVE, our data revealed an additional unrecognised pathway sharing key hallmarks of macropinocytosis: i) amiloride sensitivity, ii) fluid-phase uptake, iii) recruitment and activity of the NPF N-WASP, and iv) the localised generation of phosphoinositide-3-phosphate (PI3P) species. Given their central role in macropinocytosis and affinity for PI3P, we assessed the role of SNX-PX-BAR family proteins. Strikingly, SNX9 was specifically and transiently enriched at C. trachomatis entry foci. SNX9-/- cells exhibited a 20% defect in EB entry, which was enhanced to 60% when the cells were infected without sedimentation-induced EB adhesion, consistent with a defect in initial EB-host interaction. Correspondingly, filopodial capture of C. trachomatis EBs was specifically attenuated in SNX9-/- cells, implicating SNX9 as a central host mediator of filopodial capture early during chlamydial entry. Our findings identify an unanticipated complexity of signalling underpinning cell entry by this major human pathogen, and suggest intriguing parallels with viral entry mechanisms.

Reduced insulin signaling maintains electrical transmission in a neural circuit in aging flies.

Lowered insulin/insulin-like growth factor (IGF) signaling (IIS) can extend healthy lifespan in worms, flies, and mice, but it can also have adverse effects (the "insulin paradox"). Chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber system (GFS), a simple escape response neuronal circuit, by increasing targeting of the gap junctional protein innexin shaking-B to gap junctions (GJs). Endosomal recycling of GJs was also stimulated in cultured human cells when IIS was reduced. Furthermore, increasing the activity of the recycling small guanosine triphosphatases (GTPases) Rab4 or Rab11 was sufficient to maintain GJs upon elevated IIS in cultured human cells and in flies, and to rescue age-related loss of GJs and of GFS function. Lowered IIS thus elevates endosomal recycling of GJs in neurons and other cell types, pointing to a cellular mechanism for therapeutic intervention into aging-related neuronal disorders.

Local actin polymerization during endocytic carrier formation.

Extracellular macromolecules, pathogens and cell surface proteins rely on endocytosis to enter cells. Key steps of endocytic carrier formation are cargo molecule selection, plasma membrane folding and detachment from the cell surface. While dedicated proteins mediate each step, the actin cytoskeleton contributes to all. However, its role can be indirect to the actual molecular events driving endocytosis. Here, we review our understanding of the molecular steps mediating local actin polymerization during the formation of endocytic carriers. Clathrin-mediated endocytosis is the least reliant on local actin polymerization, as it is only engaged to counter forces induced by membrane tension or cytoplasmic pressure. Two opposite situations are coated pit formation in yeast and at the basolateral surface of polarized mammalian cells which are, respectively, dependent and independent on actin polymerization. Conversely, clathrin-independent endocytosis forming both nanometer [CLIC (clathrin-independent carriers)/GEEC (glycosylphosphatidylinositol (GPI)-anchored protein enriched endocytic compartments), caveolae, FEME (fast endophilin-mediated endocytosis) and IL-2ß (interleukin-2ß) uptake] and micrometer carriers (macropinocytosis) are dependent on actin polymerization to power local membrane deformation and carrier budding. A variety of endocytic adaptors can recruit and activate the Cdc42/N-WASP or Rac1/WAVE complexes, which, in turn, engage the Arp2/3 complex, thereby mediating local actin polymerization at the membrane. However, the molecular steps for RhoA and formin-mediated actin bundling during endocytic pit formation remain unclear.

Membrane curvature at a glance.

Membrane curvature is an important parameter in defining the morphology of cells, organelles and local membrane subdomains. Transport intermediates have simpler shapes, being either spheres or tubules. The generation and maintenance of curvature is of central importance for maintaining trafficking and cellular functions. It is possible that local shapes in complex membranes could help to define local subregions. In this Cell Science at a Glance article and accompanying poster, we summarize how generating, sensing and maintaining high local membrane curvature is an active process that is mediated and controlled by specialized proteins using general mechanisms: (i) changes in lipid composition and asymmetry, (ii) partitioning of shaped transmembrane domains of integral membrane proteins or protein or domain crowding, (iii) reversible insertion of hydrophobic protein motifs, (iv) nanoscopic scaffolding by oligomerized hydrophilic protein domains and, finally, (v) macroscopic scaffolding by the cytoskeleton with forces generated by polymerization and by molecular motors. We also summarize some of the discoveries about the functions of membrane curvature, where in addition to providing cell or organelle shape, local curvature can affect processes like membrane scission and fusion as well as protein concentration and enzyme activation on membranes.

Clustered Intracellular Salmonella enterica Serovar Typhimurium Blocks Host Cell Cytokinesis.

Several bacterial pathogens and viruses interfere with the cell cycle of their host cells to enhance virulence. This is especially apparent in bacteria that colonize the gut epithelium, where inhibition of the cell cycle of infected cells enhances the intestinal colonization. We found that intracellular Salmonella enterica serovar Typhimurium induced the binucleation of a large proportion of epithelial cells by 14 h postinvasion and that the effect was dependent on an intact Salmonella pathogenicity island 2 (SPI-2) type 3 secretion system. The SPI-2 effectors SseF and SseG were required to induce binucleation. SseF and SseG are known to maintain microcolonies of Salmonella-containing vacuoles close to the microtubule organizing center of infected epithelial cells. During host cell division, these clustered microcolonies prevented the correct localization of members of the chromosomal passenger complex and mitotic kinesin-like protein 1 and consequently prevented cytokinesis. Tetraploidy, arising from a cytokinesis defect, is known to have a deleterious effect on subsequent cell divisions, resulting in either chromosomal instabilities or cell cycle arrest. In infected mice, proliferation of small intestinal epithelial cells was compromised in an SseF/SseG-dependent manner, suggesting that cytokinesis failure caused by S Typhimurium delays epithelial cell turnover in the intestine.

Preferential invasion of mitotic cells by Salmonella reveals that cell surface cholesterol is maximal during metaphase.

Cell surface-exposed cholesterol is crucial for cell attachment and invasion of many viruses and bacteria, including the bacterium Salmonella, which causes typhoid fever and gastroenteritis. Using flow cytometry and 3D confocal fluorescence microscopy, we found that mitotic cells, although representing only 1-4% of an exponentially growing population, were much more efficiently targeted for invasion by Salmonella. This targeting was not dependent on the spherical shape of mitotic cells, but was instead SipB and cholesterol dependent. Thus, we measured the levels of plasma membrane and cell surface cholesterol throughout the cell cycle using, respectively, brief staining with filipin and a fluorescent ester of polyethylene glycol-cholesterol that cannot flip through the plasma membrane, and found that both were maximal during mitosis. This increase was due not only to the rise in global cell cholesterol levels along the cell cycle but also to a transient loss in cholesterol asymmetry at the plasma membrane during mitosis. We measured that cholesterol, but not phosphatidylserine, changed from a ∼2080 outerinner leaflet repartition during interphase to ∼5050 during metaphase, suggesting this was specific to cholesterol and not due to a broad change of lipid asymmetry during metaphase. This explains the increase in outer surface levels that make dividing cells more susceptible to Salmonella invasion and perhaps to other viruses and bacteria entering cells in a cholesterol-dependent manner. The change in cholesterol partitioning also favoured the recruitment of activated ERM (Ezrin, Radixin, Moesin) proteins at the plasma membrane and thus supported mitotic cell rounding.

Cooperative recruitment of dynamin and BIN/amphiphysin/Rvs (BAR) domain-containing proteins leads to GTP-dependent membrane scission.

Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.

Redistribution of caveolae during mitosis.

Caveolae form a specialized platform within the plasma membrane that is crucial for an array of important biological functions, ranging from signaling to endocytosis. Using total internal reflection fluorescence (TIRF) and 3D fast spinning-disk confocal imaging to follow caveola dynamics for extended periods, and electron microscopy to obtain high resolution snapshots, we found that the vast majority of caveolae are dynamic with lifetimes ranging from a few seconds to several minutes. Use of these methods revealed a change in the dynamics and localization of caveolae during mitosis. During interphase, the equilibrium between the arrival and departure of caveolae from the cell surface maintains the steady-state distribution of caveolin-1 (Cav1) at the plasma membrane. During mitosis, increased dynamics coupled to an imbalance between the arrival and departure of caveolae from the cell surface induces a redistribution of Cav1 from the plasma membrane to intracellular compartments. These changes are reversed during cytokinesis. The observed redistribution of Cav1 was reproduced by treatment of interphase cells with nocodazole, suggesting that microtubule rearrangements during mitosis can mediate caveolin relocalization. This study provides new insights into the dynamics of caveolae and highlights precise regulation of caveola budding and recycling during mitosis.

SKIP, the host target of the Salmonella virulence factor SifA, promotes kinesin-1-dependent vacuolar membrane exchanges.

In Salmonella-infected cells, the bacterial effector SifA forms a functional complex with the eukaryotic protein SKIP (SifA and kinesin-interacting protein). The lack of either partner has important consequences on the intracellular fate and on the virulence of this pathogen. In addition to SifA, SKIP binds the microtubule-based motor kinesin-1. Yet the absence of SifA or SKIP results in an unusual accumulation of kinesin-1 on the bacterial vacuolar membrane. To understand this apparent contradiction, we investigated the interaction between SKIP and kinesin-1 and the function of this complex. We show that the C-terminal RUN (RPIP8, UNC-14 and NESCA) domain of SKIP interacted specifically with the tetratricopeptide repeat (TPR) domain of the kinesin light chain. Overexpression of SKIP induced a microtubule- and kinesin-1-dependent anterograde movement of late endosomal/lysosomal compartments. In infected cells, SifA contributed to the fission of vesicles from the bacterial vacuole and the SifA/SKIP complex was required for the formation and/or the anterograde transport of kinesin-1-enriched vesicles. These observations reflect the role of SKIP as a linker and/or an activator for kinesin-1.

Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis.

Cytotoxic T lymphocytes and natural killer cells destroy target cells via the polarized exocytosis of lytic effector proteins, perforin and granzymes, into the immunologic synapse. How these molecules enter target cells is not fully understood. It is debated whether granzymes enter via perforin pores formed at the plasma membrane or whether perforin and granzymes are first endocytosed and granzymes are then released from endosomes into the cytoplasm. We previously showed that perforin disruption of the plasma membrane induces a transient Ca(2+) flux into the target cell that triggers a wounded membrane repair response in which lysosomes and endosomes donate their membranes to reseal the damaged membrane. Here we show that perforin activates clathrin- and dynamin-dependent endocytosis, which removes perforin and granzymes from the plasma membrane to early endosomes, preserving outer membrane integrity. Inhibiting clathrin- or dynamin-dependent endocytosis shifts death by perforin and granzyme B from apoptosis to necrosis. Thus by activating endocytosis to preserve membrane integrity, perforin facilitates granzyme uptake and avoids the proinflammatory necrotic death of a membrane-damaged cell.

Dynasore, a cell-permeable inhibitor of dynamin.

Dynamin is essential for clathrin-dependent coated vesicle formation. It is required for membrane budding at a late stage during the transition from a fully formed pit to a pinched-off vesicle. Dynamin may also fulfill other roles during earlier stages of vesicle formation. We have screened about 16,000 small molecules and have identified 1, named here dynasore, that interferes in vitro with the GTPase activity of dynamin1, dynamin2, and Drp1, the mitochondrial dynamin, but not of other small GTPases. Dynasore acts as a potent inhibitor of endocytic pathways known to depend on dynamin by rapidly blocking coated vesicle formation within seconds of dynasore addition. Two types of coated pit intermediates accumulate during dynasore treatment, U-shaped, half formed pits and O-shaped, fully formed pits, captured while pinching off. Thus, dynamin acts at two steps during clathrin coat formation; GTP hydrolysis is probably needed at both steps.