RESUMO
Dual-layer detectors provide a low-cost solution to improved material decomposition and lesion differentiation in X-ray imaging, while eliminating motion artifacts from multiple exposures. Most designs utilize two indirect detectors with scintillators designed for low-energy and higher-energy detection and separated by a copper filter to harden the beam for high energy detection. To improve the performance of the bottom detector and lower dose requirements, we have previously proposed an alloyed amorphous selenium photodetector to achieve improved resolution and absorption at green wavelengths, better suited to high-performance scintillators such as CsI:Tl. In this work, we demonstrate a baseline prototype for the bottom layer-a continuous, large area 83 µm pixel pitch flat panel indirect detector with well-established amorphous selenium as the photodetector-and verify the architecture's performance and detector design. We characterize lag, noise-power spectrum, detective quantum efficiency, and modular transfer function of the detector, and show resolution up to 6 lp/mm when operated at an applied bias of 150 V. This provides a starting point for evaluating the alloyed selenium materials, and shows promise for this detector in the future dual-layer design.
RESUMO
Primary central nervous system lymphoma (PCNSL) occurs primarily in older patients and has a worse prognosis than other extranodal lymphomas. Contemporary treatment is based on high-dose methotrexate (HD-MTX), which crosses the blood-brain barrier. Secondary CNS lymphoma (SCNSL) can occur concomitantly with systemic lymphoma or later at relapse and generally has a dismal outcome. We reviewed disease characteristics and outcomes of 103 patients (44 PCNSL and 59 SCNSL) treated at our center between 2015 and 2020. Median ages at diagnosis were 64 and 62 years, respectively. In both groups, diffuse large B cell lymphoma (DLBCL) was the major histologic type; in SCNSL, other types were also seen. SCNSL, in contrast with PCNSL, manifested with smaller tumors or cerebrospinal fluid positivity. For SCNSL the mean interval to brain involvement was 18 months (0-138). The overall survival had a trend to worse in SCNSL; median survival 11 months versus 61 months in PCNSL (p = 0.089). Progression-free survival was similar in both groups. A significant proportion of SCNSL patients with poor performance status could not obtain CNS-directed treatments. The strongest predictor of poor outcome was ECOG performance status 2 + at diagnosis for both groups. Charlson comorbidity index was predictive only for the PCNSL cohort. Tumor size was not prognostic for survival. The number of HD-MTX cycles correlated with survival, whereas the regimen itself and average cumulative dose of methotrexate did not play a role. Our study is in line with the recent literature and confirms ongoing challenges. We discuss how the outcomes of CNS lymphomas can be improved.
Assuntos
Neoplasias do Sistema Nervoso Central , Linfoma Difuso de Grandes Células B , Humanos , Idoso , Metotrexato/uso terapêutico , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Neoplasias do Sistema Nervoso Central/epidemiologia , Neoplasias do Sistema Nervoso Central/patologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Comorbidade , Estudos Retrospectivos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêuticoRESUMO
SNAREs provide the specificity and energy for the fusion of vesicles with their target membrane, but how they are sorted into the appropriate vesicles on post-Golgi trafficking pathways is largely unknown. We demonstrate that the clathrin-mediated endocytosis of the SNARE VAMP7 is directly mediated by Hrb, a clathrin adaptor and ArfGAP. Hrb wraps 20 residues of its unstructured C-terminal tail around the folded VAMP7 longin domain, demonstrating that unstructured regions of clathrin adaptors can select cargo. Disrupting this interaction by mutation of the VAMP7 longin domain or depletion of Hrb causes VAMP7 to accumulate on the cell's surface. However, the SNARE helix of VAMP7 binds back onto its longin domain, outcompeting Hrb for binding to the same groove and suggesting that Hrb-mediated endocytosis of VAMP7 occurs only when VAMP7 is incorporated into a cis-SNARE complex. These results elucidate the mechanism of retrieval of a postfusion SNARE complex in clathrin-coated vesicles.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Proteínas R-SNARE/química , Proteínas R-SNARE/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Endocitose , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico , Técnicas do Sistema de Duplo-HíbridoRESUMO
Insulin-stimulated delivery of glucose transporters (GLUT4, also known as SLC2A4) from specialized intracellular GLUT4 storage vesicles (GSVs) to the surface of fat and muscle cells is central to whole-body glucose regulation. This translocation and subsequent internalization of GLUT4 back into intracellular stores transits through numerous small membrane-bound compartments (internal GLUT4-containing vesicles; IGVs) including GSVs, but the function of these different compartments is not clear. Cellugyrin (also known as synaptogyrin-2) and sortilin define distinct populations of IGV; sortilin-positive IGVs represent GSVs, but the function of cellugyrin-containing IGVs is unknown. Here, we demonstrate a role for cellugyrin in intracellular sequestration of GLUT4 in HeLa cells and have used a proximity ligation assay to follow changes in pairwise associations between cellugyrin, sortilin, GLUT4 and membrane trafficking machinery following insulin-stimulation of 3T3-L1 adipoctyes. Our data suggest that insulin stimulates traffic from cellugyrin-containing to sortilin-containing membranes, and that cellugyrin-containing IGVs provide an insulin-sensitive reservoir to replenish GSVs following insulin-stimulated exocytosis of GLUT4. Furthermore, our data support the existence of a pathway from cellugyrin-containing membranes to the surface of 3T3-L1 adipocytes that bypasses GSVs under basal conditions, and that insulin diverts traffic away from this into GSVs.
Assuntos
Membrana Celular/metabolismo , Exocitose/efeitos dos fármacos , Transportador de Glucose Tipo 4/metabolismo , Insulina/farmacologia , Vesículas Secretórias/metabolismo , Células 3T3-L1 , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Membrana Celular/genética , Exocitose/genética , Transportador de Glucose Tipo 4/genética , Camundongos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Vesículas Secretórias/genética , Sinaptogirinas/genética , Sinaptogirinas/metabolismoRESUMO
The delivery of proteins from the plasma membrane to the lysosome for degradation is essential for normal cellular function. There is now a good understanding of the protein complexes involved in sorting proteins at the plasma membrane and into the intralumenal vesicles of the multi-vesicular body. A combination of cell free content mixing assays and live-cell imaging has dissected out the final step in delivery of macromolecules to the lysosome from the multi-vesicular body and provided insights into the molecular mechanisms by which late endosomes and lysosomes exchange lumenal contents. The endocytic pathway has provided a platform with which to understand the autophagic and phagocytic pathways, but the fine details of how traffic through these pathways is regulated remain to be determined.
Assuntos
Endocitose , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Animais , Membrana Celular/metabolismo , Clatrina/metabolismo , Humanos , Transporte ProteicoRESUMO
Lysosomes form part of our innate immunity and are an important line of defence against microbes, viruses and parasites. Although it is more than 50 years since de Duve discovered lysosomes, it is only in more recent years that we are slowly unravelling the molecular mechanisms involved in the delivery of material to the lysosome. However, successful intracellular pathogens often have a better grip on the mechanisms involved in delivery to the lysosome and can manipulate membrane trafficking pathways to create an intracellular environment that is favourable for replication. By studying pathogen effector proteins that are secreted into the host's cytosol, we can learn about both pathogen-survival mechanisms and further regulatory elements involved in trafficking to the lysosome.
Assuntos
Bactérias/imunologia , Imunidade Inata , Lisossomos/imunologia , Vírus/imunologia , Animais , Bactérias/patogenicidade , Membrana Celular/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Vírus/patogenicidadeRESUMO
Salmonella effector protein SseJ is secreted by Salmonella into the host cell cytoplasm where it can then modify host cell processes. Whilst host cell small GTPase RhoA has previously been shown to activate the acyl-transferase activity of SseJ we show here an un-described effect of SseJ protein production upon microtubule dynamism. SseJ prevents microtubule collapse and this is independent of SseJ's acyl-transferase activity. We speculate that the effects of SseJ on microtubules would be mediated via its known interactions with the small GTPases of the Rho family.
Assuntos
Aciltransferases/genética , Proteínas de Bactérias/genética , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Microtúbulos/microbiologia , Salmonella typhimurium/genética , Proteínas rho de Ligação ao GTP/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Linhagem Celular , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica , Ilhas Genômicas , Biblioteca Genômica , Humanos , Imunoprecipitação , Rim/microbiologia , Rim/patologia , Macrófagos/microbiologia , Macrófagos/ultraestrutura , Microtúbulos/ultraestrutura , Ligação Proteica , Ratos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Alinhamento de Sequência , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Rhodococcus equi (R. equi) is an important pulmonary pathogen in foals that often leads to the death of the horse. The bacterium harbors a virulence plasmid that encodes numerous virulence-associated proteins (Vaps) including VapA that is essential for intracellular survival inside macrophages. However, little is known about the precise function of VapA. Here, we demonstrate that VapA causes perturbation to late endocytic organelles with swollen endolysosome organelles having reduced Cathepsin B activity and an accumulation of LBPA, LC3 and Rab7. The data are indicative of a loss of endolysosomal function, which leads cells to upregulate lysosome biogenesis to compensate for the loss of functional endolysosomes. Although there is a high degree of homology of the core region of VapA to other Vap proteins, only the highly conserved core region of VapA, and not VapD of VapG, gives the observed effects on endolysosomes. This is the first demonstration of how VapA works and implies that VapA aids R. equi survival by reducing the impact of lysosomes on phagocytosed bacteria.
Assuntos
Infecções por Actinomycetales/patologia , Proteínas de Bactérias/metabolismo , Broncopneumonia/microbiologia , Catepsina B/metabolismo , Doenças dos Cavalos/patologia , Lisossomos/patologia , Rhodococcus equi/patogenicidade , Infecções por Actinomycetales/microbiologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Regulação Bacteriana da Expressão Gênica , Células HeLa , Doenças dos Cavalos/microbiologia , Cavalos , Humanos , Lisossomos/microbiologia , Macrófagos/microbiologia , Fagocitose , Ratos , Fatores de VirulênciaRESUMO
This protocol describes the generation of a fraction enriched in lysosomes from rat liver. The lysosomes are rapidly isolated using density-gradient centrifugation with gradient media that retain the osmolarity of the lysosomes such that they are functional and can be used in in vitro assays.
Assuntos
Fracionamento Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Hepatócitos , Fígado , Lisossomos , Animais , RatosRESUMO
Lysosomes can be rapidly isolated from tissue culture cells using supraparamagnetic iron oxide particles (SPIONs). In this protocol, colloidal iron dextran (FeDex) particles, a type of SPION, are taken up by cultured mouse macrophage cells via the endocytic pathway. The SPIONs accumulate in lysosomes, the end point of the endocytic pathway, permitting the lysosomes to be isolated magnetically. The purified lysosomes are suitable for in vitro fusion assays or for proteomic analysis.
Assuntos
Fracionamento Celular/métodos , Endocitose , Compostos Férricos/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Nanopartículas/metabolismo , Animais , Linhagem Celular , Magnetismo , CamundongosRESUMO
In the late endocytic pathway, it has been proposed that endocytosed macromolecules are delivered to a proteolytic environment by 'kiss-and-run' events or direct fusion between late endosomes and lysosomes. To test whether the fusion hypothesis accounts for delivery to lysosomes in living cells, we have used confocal microscopy to examine content mixing between lysosomes loaded with rhodamine-dextran and endosomes subsequently loaded with Oregon-Green-dextran. Both kissing and explosive fusion events were recorded. Data from cell-free content-mixing assays have suggested that fusion is initiated by tethering, which leads to formation of a trans-SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) protein complex and then release of lumenal Ca(2+), followed by membrane bilayer fusion. We have shown that the R-SNARE (arginine-containing SNARE) protein VAMP (vesicle-associated membrane protein) 7 is necessary for heterotypic fusion between late endosomes and lysosomes, whereas a different R-SNARE, VAMP 8 is required for homotypic fusion of late endosomes. After fusion of lysosomes with late endosomes, lysosomes are re-formed from the resultant hybrid organelles, a process requiring condensation of content and the removal/recycling of some membrane proteins.
Assuntos
Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Animais , Transporte Biológico Ativo , Endocitose , Endossomos/metabolismo , Técnicas In Vitro , Fusão de Membrana , Modelos Biológicos , RatosRESUMO
Tissue and whole organ regeneration is a dramatic biological response to injury that occurs across different plant and animal phyla. It frequently requires the dedifferentiation of mature cells to a condensed mesenchymal blastema, from which replacement tissues develop. Human somatic cells cannot regenerate in this way and differentiation is considered irreversible under normal developmental conditions. Here, we sought to establish in vitro conditions to mimic blastema formation by generating different three-dimensional (3D) condensates of human mesenchymal stromal cells (MSCs). We identified specific 3D growth environments that were sufficient to dedifferentiate aged human MSCs to an early mesendoderm-like state with reversal of age-associated cell hypertrophy and restoration of organized tissue regenerating capacity in vivo. An optimal auophagic response was required to promote cytoplasmic remodeling, mitochondrial regression, and a bioenergetic shift from oxidative phosphorylation to anaerobic metabolism. Our evidence suggests that human cell dedifferentiation can be achieved through autonomously controlled autophagic flux.
Assuntos
Autofagia , Desdiferenciação Celular , Células-Tronco Mesenquimais/citologia , Idoso , Técnicas de Cultura de Células , Citoplasma/metabolismo , Endoderma/citologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , RegeneraçãoRESUMO
The emergence of influenza A virus (IAV) in domestic avian species and associated transmissions to mammals is unpredictable. In the Americas, the H7 IAVs are of particular concern, and there have been four separate outbreaks of highly pathogenic (HP) H7N3 in domestic poultry in North and South America between 2002 and 2012, with occasional spillover into humans. Here, we use long-term IAV surveillance in North American shorebirds at Delaware Bay, USA, from 1985 to 2012 and in ducks in Alberta, Canada, from 1976 to 2012 to determine which hemagglutinin (HA)-neuraminidase (NA) combinations predominated in Anseriformes (ducks) and Charadriiformes (shorebirds) and whether there is concordance between peaks of H7 prevalence and transmission in wild aquatic birds and the emergence of H7 IAVs in poultry and humans. Whole-genome sequencing supported phylogenetic and genomic constellation analyses to determine whether HP IAVs emerge in the context of specific internal gene segment sequences. Phylogenetic analysis of whole-genome sequences of the H7N3 influenza viruses from wild birds and HP H7N3 outbreaks in the Americas indicate that each HP outbreak was an independent emergence event and that the low pathogenic (LP) avian influenza precursors were most likely from dabbling ducks. The different polybasic cleavage sites in the four HP outbreaks support independent origins. At the 95% nucleotide percent identity-level phylogenetic analysis showed that the wild duck HA, PB1, and M sequences clustered with the poultry and human outbreak sequences. The genomic constellation analysis strongly suggests that gene segments/virus flow from wild birds to domestic poultry.
Assuntos
Doenças das Aves/epidemiologia , Surtos de Doenças/veterinária , Vírus da Influenza A Subtipo H7N3/isolamento & purificação , Influenza Aviária/epidemiologia , Doenças das Aves Domésticas/epidemiologia , Alberta/epidemiologia , Animais , Animais Domésticos , Animais Selvagens , Anseriformes , Aves , Charadriiformes , Delaware/epidemiologia , Patos , Monitoramento Epidemiológico , Humanos , Vírus da Influenza A Subtipo H7N3/genética , Filogenia , Análise de Sequência de DNARESUMO
Phagocytosis is the process by which receptors at the plasma membrane are used to engulf a particle such as a bacterium, parasite, or dead cell. Phagosomes can be isolated from tissue culture cells by various centrifugation methods, including the use of differential density gradients or sucrose step gradients, but these methods are time-consuming or otherwise difficult. We describe here a protocol that avoids centrifugation and relies instead on the uptake of magnetic beads to rapidly isolate the phagosomal compartment from tissue culture cells.
Assuntos
Fagossomos/metabolismo , Técnicas de Cultura de Tecidos/métodos , Animais , Células Cultivadas , Camundongos , FagocitoseRESUMO
Lysosomes are an important cellular organelle that receive and degrade macromolecules from the secretory, endocytic, autophagic, and phagocytic membrane-trafficking pathways. Defects in lysosome function lead to the development of disease with often-severe consequences to the individual. Since the discovery of lysosomes by Christian de Duve over 50 years ago, research into endocytic and lysosomal biology has allowed for the development of tools to understand further the role of lysosomes in cells. There are now several fluorescent probes that can be used to visualize and assess membrane traffic to the lysosome as well as probes to assess the activity of lysosomal hydrolases in live cells. This chapter describes the current methods used to measure lysosome function in live cells.
Assuntos
Rastreamento de Células/métodos , Corantes Fluorescentes , Proteínas de Membrana Lisossomal/análise , Lisossomos/metabolismo , Animais , Dextranos , Endocitose/fisiologia , Fibroblastos/citologia , Proteínas de Fluorescência Verde/análise , Células HeLa , Humanos , Rim/citologia , Lisossomos/fisiologia , Fagocitose/fisiologia , Transporte Proteico , RatosRESUMO
SNAREs provide energy and specificity to membrane fusion events. Fusogenic trans-SNARE complexes are assembled from glutamine-contributing SNAREs (Q-SNAREs) embedded in one membrane and an arginine-contributing SNARE (R-SNARE) embedded in the other. Regulation of membrane fusion events is crucial for intracellular trafficking. We identify the endosomal protein Varp as an R-SNARE-binding regulator of SNARE complex formation. Varp colocalizes with and binds to VAMP7, an R-SNARE that is involved in both endocytic and secretory pathways. We present the structure of the second ankyrin repeat domain of mammalian Varp in complex with the cytosolic portion of VAMP7. The VAMP7-SNARE motif is trapped between Varp and the VAMP7 longin domain, and hence Varp kinetically inhibits the ability of VAMP7 to form SNARE complexes. This inhibition will be increased when Varp can also bind to other proteins present on the same membrane as VAMP7, such as Rab32-GTP.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Eletroforese em Gel de Poliacrilamida , Endocitose , Humanos , Cinética , Conformação Proteica , Proteínas R-SNARERESUMO
Lysosomes are dynamic organelles that receive and degrade macromolecules from the secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways. Live-cell imaging has shown that fusion with lysosomes occurs by both transient and full fusion events, and yeast genetics and mammalian cell-free systems have identified much of the protein machinery that coordinates these fusion events. Many pathogens that hijack the endocytic pathways to enter cells have evolved mechanisms to avoid being degraded by the lysosome. However, the function of lysosomes is not restricted to protein degradation: they also fuse with the plasma membrane during cell injury, as well as having more specialized secretory functions in some cell types.
Assuntos
Autofagia , Lisossomos/metabolismo , Fusão de Membrana , Fagocitose , Animais , Evolução Biológica , Sistema Livre de Células/metabolismo , Sistema Livre de Células/microbiologia , Sistema Livre de Células/parasitologia , Sistema Livre de Células/ultraestrutura , Sistema Livre de Células/virologia , Humanos , Lisossomos/microbiologia , Lisossomos/parasitologia , Lisossomos/ultraestrutura , Lisossomos/virologia , Proteínas/metabolismoRESUMO
Mucolipin-1 is a membrane protein encoded by the gene MCOLN1, mutations in which result in the lysosomal storage disorder mucolipidosis type IV (MLIV). Efficient lysosomal targeting of mucolipin-1 requires di-leucine motifs in both the N-terminal and the C-terminal cytosolic tails. We have shown that aberrant lactosylceramide trafficking in MLIV cells may be rescued by wild-type mucolipin-1 expression but not by mucolipin-1 mistargeted to the plasma membrane or by lysosome-localized mucolipin-1 mutated in its predicted ion pore-selectivity region. Our data demonstrate that the correct localization of mucolipin-1 and the integrity of its ion pore are essential for its physiological function in the late endocytic pathway.
Assuntos
Antígenos CD/metabolismo , Endocitose/fisiologia , Lactosilceramidas/metabolismo , Lisossomos/metabolismo , Canais de Cátion TRPM/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Colesterol/metabolismo , Humanos , Leucina/metabolismo , Lisossomos/ultraestrutura , Dados de Sequência Molecular , Mucolipidoses/genética , Mucolipidoses/metabolismo , Mutação , Ratos , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Canais de Cátion TRPM/genética , Canais de Potencial de Receptor TransitórioRESUMO
Lysosomes are dynamic organelles receiving membrane traffic input from the biosynthetic, endocytic and autophagic pathways. They may be regarded as storage organelles for acid hydrolases and are capable of fusing with late endosomes to form hybrid organelles where digestion of endocytosed macromolecules occurs. Reformation of lysosomes from the hybrid organelles involves content condensation and probably removal of some membrane proteins by vesicular traffic. Lysosomes can also fuse with the plasma membrane in response to cell surface damage and a rise in cytosolic Ca(2+) concentration. This process is important in plasma membrane repair. The molecular basis of membrane traffic pathways involving lysosomes is increasingly understood, in large part because of the identification of many proteins required for protein traffic to vacuoles in the yeast Saccharomyces cerevisiae. Mammalian orthologues of these proteins have been identified and studied in the processes of vesicular delivery of newly synthesized lysosomal proteins from the trans-Golgi network, fusion of lysosomes with late endosomes and sorting of membrane proteins into lumenal vesicles. Several multi-protein oligomeric complexes required for these processes have been identified. The present review focuses on current understanding of the molecular mechanisms of fusion of lysosomes with both endosomes and the plasma membrane and on the sorting events required for delivery of newly synthesized membrane proteins, endocytosed membrane proteins and other endocytosed macromolecules to lysosomes.
Assuntos
Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Animais , Cálcio/fisiologia , Endocitose/fisiologia , HumanosRESUMO
Both heterotypic and homotypic fusion events are required to deliver endocytosed macromolecules to lysosomes and remodel late endocytic organelles. A trans-SNARE complex consisting of Q-SNAREs syntaxin 7, Vti1b and syntaxin 8 and the R-SNARE VAMP8 has been shown by others to be responsible for homotypic fusion of late endosomes. Using antibody inhibition experiments in rat liver cell-free systems, we confirmed this result, but found that the same Q-SNAREs can combine with an alternative R-SNARE, namely VAMP7, for heterotypic fusion between late endosomes and lysosomes. Co-immunoprecipitation demonstrated separate syntaxin 7 complexes with either VAMP7 or VAMP8 in solubilized rat liver membranes. Additionally, overexpression of the N-terminal domain of VAMP7, in cultured fibroblastic cells, inhibited the mixing of a preloaded lysosomal content marker with a marker delivered to late endosomes. These data show that combinatorial interactions of SNAREs determine whether late endosomes undergo homotypic or heterotypic fusion events.