Retrovirus-like Gag Protein Arc1 Binds RNA and Traffics across Synaptic Boutons.

Arc/Arg3.1 is required for synaptic plasticity and cognition, and mutations in this gene are linked to autism and schizophrenia. Arc bears a domain resembling retroviral/retrotransposon Gag-like proteins, which multimerize into a capsid that packages viral RNA. The significance of such a domain in a plasticity molecule is uncertain. Here, we report that the Drosophila Arc1 protein forms capsid-like structures that bind darc1 mRNA in neurons and is loaded into extracellular vesicles that are transferred from motorneurons to muscles. This loading and transfer depends on the darc1-mRNA 3' untranslated region, which contains retrotransposon-like sequences. Disrupting transfer blocks synaptic plasticity, suggesting that transfer of dArc1 complexed with its mRNA is required for this function. Notably, cultured cells also release extracellular vesicles containing the Gag region of the Copia retrotransposon complexed with its own mRNA. Taken together, our results point to a trans-synaptic mRNA transport mechanism involving retrovirus-like capsids and extracellular vesicles.

Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress.

Amino acids are essential building blocks of life. However, increasing evidence suggests that elevated amino acids cause cellular toxicity associated with numerous metabolic disorders. How cells cope with elevated amino acids remains poorly understood. Here, we show that a previously identified cellular structure, the mitochondrial-derived compartment (MDC), functions to protect cells from amino acid stress. In response to amino acid elevation, MDCs are generated from mitochondria, where they selectively sequester and deplete SLC25A nutrient carriers and their associated import receptor Tom70 from the organelle. Generation of MDCs promotes amino acid catabolism, and their formation occurs simultaneously with transporter removal at the plasma membrane via the multivesicular body (MVB) pathway. The combined loss of vacuolar amino acid storage, MVBs, and MDCs renders cells sensitive to high amino acid stress. Thus, we propose that MDCs operate as part of a coordinated cell network that facilitates amino acid homeostasis through post-translational nutrient transporter remodeling.

The endosomal sorting complex ESCRT-II mediates the assembly and architecture of ESCRT-III helices.

The endosomal sorting complexes required for transport (ESCRTs) constitute hetero-oligomeric machines that mediate topologically similar membrane-sculpting processes, including cytokinesis, retroviral egress, and multivesicular body (MVB) biogenesis. Although ESCRT-III drives membrane remodeling that creates MVBs, its structure and the mechanism of vesicle formation are unclear. Using electron microscopy, we visualize an ESCRT-II:ESCRT-III supercomplex and propose how it mediates vesicle formation. We define conformational changes that activate ESCRT-III subunit Snf7 and show that it assembles into spiraling ~9 nm protofilaments on lipid monolayers. A high-content flow cytometry assay further demonstrates that mutations halting ESCRT-III assembly block ESCRT function. Strikingly, the addition of Vps24 and Vps2 transforms flat Snf7 spirals into membrane-sculpting helices. Finally, we show that ESCRT-II and ESCRT-III coassemble into ~65 nm diameter rings indicative of a cargo-sequestering supercomplex. We propose that ESCRT-III has distinct architectural stages that are modulated by ESCRT-II to mediate cargo capture and vesicle formation by ordered assembly.

A chemical inhibitor of IST1-CHMP1B interaction impairs endosomal recycling and induces noncanonical LC3 lipidation.

The endosomal sorting complex required for transport (ESCRT) machinery constitutes multisubunit protein complexes that play an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, including cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair, and autophagy. Given the versatile functionality of ESCRTs, and the intricate organizational structure of the ESCRT machinery, the targeted modulation of distinct ESCRT complexes is considerably challenging. This study presents a pseudonatural product targeting IST1-CHMP1B within the ESCRT-III complexes. The compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting, or biogenesis of extracellular vesicles, it rapidly inhibits transferrin receptor recycling in cells, resulting in the accumulation of transferrin in stalled sorting endosomes. Stalled endosomes become decorated by lipidated LC3, suggesting a link between noncanonical LC3 lipidation and inhibition of the IST1-CHMP1B complex.

IST1 regulates select recycling pathways.

ESCRTs (Endosomal Sorting Complex Required for Transports) are a modular set of protein complexes with membrane remodeling activities that include the formation and release of intraluminal vesicles (ILVs) to generate multivesicular endosomes. While most of the 12 ESCRT-III proteins are known to play roles in ILV formation, IST1 has been associated with a wider range of endosomal remodeling events. Here, we extend previous studies of IST1 function in endosomal trafficking and confirm that IST1, along with its binding partner CHMP1B, contributes to scission of early endosomal carriers. Functionally, depleting IST1 impaired delivery of transferrin receptor from early/sorting endosomes to the endocytic recycling compartment and instead increased its rapid recycling to the plasma membrane via peripheral endosomes enriched in the clathrin adaptor AP-1. IST1 is also important for export of mannose 6-phosphate receptor from early/sorting endosomes. Examination of IST1 binding partners on endosomes revealed that IST1 interacts with the MIT domain-containing sorting nexin SNX15, a protein previously reported to regulate endosomal recycling. Our kinetic and spatial analyses establish that SNX15 and IST1 occupy a clathrin-containing subdomain on the endosomal perimeter distinct from those previously implicated in cargo retrieval or degradation. Using live-cell microscopy, we see that SNX15 and CHMP1B alternately recruit IST1 to this subdomain or the base of endosomal tubules. These findings indicate that IST1 contributes to a subset of recycling pathways from the early/sorting endosome.

Multivesicular body morphogenesis.

Multivesicular bodies (MVBs) are unique organelles in the endocytic pathway that contain vesicles in their lumen. Sorting and incorporation of material into such vesicles is a critical cellular process that has been intensely studied following discovery of the ESCRT (endosomal sorting complex required for transport) machinery just more than a decade ago. In this review, we summarize current understanding of the cellular functions of MVBs and how the ESCRT machinery contributes to MVB morphogenesis. We also highlight the importance of MVBs and ESCRTs in human health. We identify critical areas in which further mechanistic and spatiotemporal studies in living cells will advance this exciting area of research.

Hsp90 Mediates Membrane Deformation and Exosome Release.

Hsp90 is an essential chaperone that guards proteome integrity and amounts to 2% of cellular protein. We now find that Hsp90 also has the ability to directly interact with and deform membranes via an evolutionarily conserved amphipathic helix. Using a new cell-free system and in vivo measurements, we show this amphipathic helix allows exosome release by promoting the fusion of multivesicular bodies (MVBs) with the plasma membrane. We dissect the relationship between Hsp90 conformation and membrane-deforming function and show that mutations and drugs that stabilize the open Hsp90 dimer expose the helix and allow MVB fusion, while these effects are blocked by the closed state. Hence, we structurally separated the Hsp90 membrane-deforming function from its well-characterized chaperone activity, and we show that this previously unrecognized function is required for exosome release.

HSP90ß prevents aging-related cataract formation through regulation of the charged multivesicular body protein (CHMP4B) and p53.

Cataract is a leading ocular disease causing global blindness. The mechanism of cataractogenesis has not been well defined. Here, we demonstrate that the heat shock protein 90ß (HSP90ß) plays a fundamental role in suppressing cataractogenesis. HSP90ß is the most dominant HSP in normal lens, and its constitutive high level of expression is largely derived from regulation by Sp1 family transcription factors. More importantly, HSP90ß is significantly down-regulated in human cataract patients and in aging mouse lenses, whereas HSP90ß silencing in zebrafish causes cataractogenesis, which can only be rescued by itself but not other HSP90 genes. Mechanistically, HSP90ß can directly interact with CHMP4B, a newly-found client protein involved in control of cytokinesis. HSP90ß silencing causes upregulation of CHMP4B and another client protein, the tumor suppressor p53. CHMP4B upregulation or overexpression induces excessive division of lens epithelial cells without proper differentiation. As a result, these cells were triggered to undergo apoptosis due to activation of the p53/Bak-Bim pathway, leading to cataractogenesis and microphthalmia. Silence of both HSP90ß and CHMP4B restored normal phenotype of zebrafish eye. Together, our results reveal that HSP90ß is a critical inhibitor of cataractogenesis through negative regulation of CHMP4B and the p53-Bak/Bim pathway.

LRRK1 functions as a scaffold for PTP1B-mediated EGFR sorting into ILVs at the ER-endosome contact site.

Proper control of epidermal growth factor receptor (EGFR) signaling is important for maintaining cellular homeostasis. Given that EGFR signaling occurs at the plasma membrane and endosomes following internalization, endosomal trafficking of EGFR spatiotemporally regulates EGFR signaling. In this process, leucine-rich repeat kinase 1 (LRRK1) has multiple roles in kinase activity-dependent transport of EGFR-containing endosomes and kinase-independent sorting of EGFR into the intraluminal vesicles (ILVs) of multivesicular bodies. Active, phosphorylated EGFR inactivates the LRRK1 kinase activity by phosphorylating Y944. In this study, we demonstrate that LRRK1 facilitates EGFR dephosphorylation by PTP1B (also known as PTPN1), an endoplasmic reticulum (ER)-localized protein tyrosine phosphatase, at the ER-endosome contact site, after which EGFR is sorted into the ILVs of endosomes. LRRK1 is required for the PTP1B-EGFR interaction in response to EGF stimulation, resulting in the downregulation of EGFR signaling. Furthermore, PTP1B activates LRRK1 by dephosphorylating pY944 on the contact site, which promotes the transport of EGFR-containing endosomes to the perinuclear region. These findings provide evidence that the ER-endosome contact site functions as a hub for LRRK1-dependent signaling that regulates EGFR trafficking.

Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes.

Canonical Wnt signaling requires inhibition of Glycogen Synthase Kinase 3 (GSK3) activity, but the molecular mechanism by which this is achieved remains unclear. Here, we report that Wnt signaling triggers the sequestration of GSK3 from the cytosol into multivesicular bodies (MVBs), so that this enzyme becomes separated from its many cytosolic substrates. Endocytosed Wnt colocalized with GSK3 in acidic vesicles positive for endosomal markers. After Wnt addition, endogenous GSK3 activity decreased in the cytosol, and GSK3 became protected from protease treatment inside membrane-bounded organelles. Cryoimmunoelectron microscopy showed that these corresponded to MVBs. Two proteins essential for MVB formation, HRS/Vps27 and Vps4, were required for Wnt signaling. The sequestration of GSK3 extended the half-life of many other proteins in addition to ß-Catenin, including an artificial Wnt-regulated reporter protein containing GSK3 phosphorylation sites. We conclude that multivesicular endosomes are essential components of the Wnt signal-transduction pathway.

Essential role of Alix in regulating cardiomyocyte exosome biogenesis under physiological and stress conditions.

BACKGROUND: Exosomes released by cardiomyocytes are essential mediators of intercellular communications within the heart, and various exosomal proteins and miRNAs are associated with cardiovascular diseases. However, whether the endosomal sorting complex required for transport (ESCRT) and its key component Alix is required for exosome biogenesis within cardiomyocyte remains poorly understood. METHODS: Super-resolution imaging was performed to investigate the subcellular location of Alix and multivesicular body (MVB) in primary cardiomyocytes. Cardiomyocyte-specific Alix-knockout mice were generated using AAV9/CRISPR/Cas9-mediated in vivo gene editing. A stable Alix-knockdown H9c2 cardiomyocyte line was constructed through lentiviral-mediated delivery of short hairpin RNA. In order to determine the role of Alix in controlling exosome biogenesis, exosomes from cardiomyocyte-specific Alix-knockout mice plasma and Alix-knockdown H9c2 culture medium were isolated and examined by western blot, NTA analysis and transmission electron microscopy. Biochemical and immunofluorescence analysis were performed to determine the role of ESCRT machinery in regulating MVB formation. Lastly, transverse aortic constriction (TAC)-induced cardiac pressure overload model was established to further explore the role of Alix-mediated exosome biogenesis under stress conditions. RESULTS: A significant proportion of Alix localized to the MVB membrane within cardiomyocytes. Genetic deletion of Alix in murine heart resulted in a reduction of plasma exosome content without affecting cardiac structure or contractile function. Consistently, the downregulation of Alix in H9c2 cardiomyocyte line also suppressed the biogenesis of exosomes. We found the defective ESCRT machinery and suppressed MVB formation upon Alix depletion caused compromised exosome biogenesis. Remarkably, TAC-induced cardiac pressure overload led to increased Alix, MVB levels, and elevated plasma exosome content, which could be totally abolished by Alix deletion. CONCLUSION: These results establish Alix as an essential and stress-sensitive regulator of cardiac exosome biogenesis and the findings may yield valuable therapeutic implications.

Vps68 cooperates with ESCRT-III in intraluminal vesicle formation.

The endosomal sorting complex required for transport (ESCRT)-III mediates budding and abscission of intraluminal vesicles (ILVs) into multivesicular endosomes. To further define the role of the yeast ESCRT-III-associated protein Mos10 (also known as Vps60) in ILV formation, we screened for new interaction partners by using stable isotope labeling of amino acids in cell culture (SILAC) and mass spectrometry. Here, we focused on the newly identified interaction partner Vps68. Our data suggest that Vps68 cooperates with ESCRT-III in ILV formation. The deletion of VPS68 caused a sorting defect similar to that of the SNF7 deletion strain when the cargo load was high. The composition of ESCRT-III was altered, the level of core components was higher and the level of associated proteins was lower in the VPS68 deletion strain. Our data further indicate that at some point in the functional cycle of ESCRT-III, Snf7 could be replaced by Mos10. Vps68 has an unusual membrane topology. Two of its potential membrane helices are amphipathic helices that localize to the luminal side of the endosomal membrane. Based on this membrane topology, we propose that Vps68 and ESCRT-III cooperate in the abscission step by weakening the luminal and cytosolic leaflets of the bilayer at the abscission site.

Neutral sphingomyelinase 2 controls exosome secretion by counteracting V-ATPase-mediated endosome acidification.

During endosome maturation, neutral sphingomyelinase 2 (nSMase2, encoded by SMPD3) is involved in budding of intraluminal vesicles (ILVs) into late endosomes or multivesicular bodies (MVBs). Fusion of these with the plasma membrane results in secretion of exosomes or small extracellular vesicles (sEVs). Here, we report that nSMase2 activity controls sEV secretion through modulation of vacuolar H+-ATPase (V-ATPase) activity. Specifically, we show that nSMase2 inhibition induces V-ATPase complex assembly that drives MVB lumen acidification and consequently reduces sEV secretion. Conversely, we further demonstrate that stimulating nSMase2 activity with the inflammatory cytokine TNFα (also known as TNF) decreases acidification and increases sEV secretion. Thus, we find that nSMase2 activity affects MVB membrane lipid composition to counteract V-ATPase-mediated endosome acidification, thereby shifting MVB fate towards sEV secretion. This article has an associated First Person interview with the first author of the paper.

Autophagy and multivesicular body pathways cooperate to protect sulfur assimilation and chloroplast functions.

Autophagy and multivesicular bodies (MVBs) represent 2 closely related lysosomal/vacuolar degradation pathways. In Arabidopsis (Arabidopsis thaliana), autophagy is stress-induced, with deficiency in autophagy causing strong defects in stress responses but limited effects on growth. LYST-INTERACTING PROTEIN 5 (LIP5) is a key regulator of stress-induced MVB biogenesis, and mutation of LIP5 also strongly compromises stress responses with little effect on growth in Arabidopsis. To determine the functional interactions of these 2 pathways in Arabidopsis, we generated mutations in both the LIP5 and AUTOPHAGY-RELATED PROTEIN (ATG) genes. atg5/lip5 and atg7/lip5 double mutants displayed strong synergistic phenotypes in fitness characterized by stunted growth, early senescence, reduced survival, and greatly diminished seed production under normal growth conditions. Transcriptome and metabolite analysis revealed that chloroplast sulfate assimilation was specifically downregulated at early seedling stages in the atg7/lip5 double mutant prior to the onset of visible phenotypes. Overexpression of adenosine 5'-phosphosulfate reductase 1, a key enzyme in sulfate assimilation, substantially improved the growth and fitness of the atg7/lip5 double mutant. Comparative multi-omic analysis further revealed that the atg7/lip5 double mutant was strongly compromised in other chloroplast functions including photosynthesis and primary carbon metabolism. Premature senescence and reduced survival of atg/lip5 double mutants were associated with increased accumulation of reactive oxygen species and overactivation of stress-associated programs. Blocking PHYTOALEXIN DEFICIENT 4 and salicylic acid signaling prevented early senescence and death of the atg7/lip5 double mutant. Thus, stress-responsive autophagy and MVB pathways play an important cooperative role in protecting essential chloroplast functions including sulfur assimilation under normal growth conditions to suppress salicylic-acid-dependent premature cell-death and promote plant growth and fitness.

siRNA screening reveals that SNAP29 contributes to exosome release.

Cells release extracellular vesicles (EVs) of different sizes. Small EVs (< 200 nm) can originate from the fusion of multivesicular bodies with the plasma membrane, i.e. exosomes, and from budding of the plasma membrane, i.e. small ectosomes. To investigate the molecular machinery required for the release of small EVs, we developed a sensitive assay based on incorporation of radioactive cholesterol in EV membranes and used it in a siRNA screening. The screening showed that depletion of several SNARE proteins affected the release of small EVs. We focused on SNAP29, VAMP8, syntaxin 2, syntaxin 3 and syntaxin 18, the depletion of which reduced the release of small EVs. Importantly, this result was verified using gold standard techniques. SNAP29 depletion resulted in the largest effect and was further investigated. Immunoblotting analysis of small EVs showed that the release of several proteins considered to be associated with exosomes like syntenin, CD63 and Tsg101 was reduced, while the level of several proteins that have been shown to be released in ectosomes (annexins) or by secretory autophagy (LC3B and p62) was not affected by SNAP29 depletion. Moreover, these proteins appeared in different fractions when the EV samples were further separated by a density gradient. These results suggest that SNAP29 depletion mainly affects the secretion of exosomes. To investigate how SNAP29 affects exosome release, we used microscopy to study the distribution of MBVs using CD63 labelling and CD63-pHluorin to detect fusion events of MVBs with the plasma membrane. SNAP29 depletion caused a redistribution of CD63-labelled compartments but did not change the number of fusion events. Further experiments are therefore needed to fully understand the function of SNAP29. To conclude, we have developed a novel screening assay that has allowed us to identify several SNAREs involved in the release of small EVs.

Bro1 family proteins harmonize cargo sorting with vesicle formation.

The Endosomal Sorting Complexes Required for Transport (ESCRTs) drive membrane remodeling in a variety of cellular processes that include the formation of endosomal intralumenal vesicles (ILVs) during multivesicular body (MVB) biogenesis. During MVB sorting, ESCRTs recognize ubiquitin (Ub) attached to membrane protein cargo and execute ILV formation by controlling the activities of ESCRT-III polymers regulated by the AAA-ATPase Vps4. Exactly how these events are coordinated to ensure proper cargo loading into ILVs remains unclear. Here we discuss recent work documenting the ability of Bro1, an ESCRT-associated Ub-binding protein, to coordinate ESCRT-III and Vps4-dependent ILV biogenesis with upstream events such as cargo recognition.

UBA6 Inhibition Accelerates Lysosomal TRPML1 Depletion and Exosomal Secretion in Lung Cancer Cells.

Ubiquitin-like modifier-activating enzyme 6 (UBA6) is a member of the E1 enzyme family, which initiates the ubiquitin-proteasome system (UPS). The UPS plays critical roles not only in protein degradation but also in various cellular functions, including neuronal signaling, myocardial remodeling, immune cell differentiation, and cancer development. However, the specific role of UBA6 in cellular functions is not fully elucidated in comparison with the roles of the UPS. It has been known that the E1 enzyme is associated with the motility of cancer cells. In this study, we verified the physiological roles of UBA6 in lung cancer cells through gene-silencing siRNA targeting UBA6 (siUBA6). The siUBA6 treatment attenuated the migration of H1975 cells, along with a decrease in lysosomal Ca2+ release. While autophagosomal proteins remained unchanged, lysosomal proteins, including TRPML1 and TPC2, were decreased in siUBA6-transfected cells. Moreover, siUBA6 induced the production of multivesicular bodies (MVBs), accompanied by an increase in MVB markers in siUBA6-transfected H1975 cells. Additionally, the expression of the exosomal marker CD63 and extracellular vesicles was increased by siUBA6 treatment. Our findings suggest that knock-down of UBA6 induces lysosomal TRPML1 depletion and inhibits endosomal trafficking to lysosome, and subsequently, leads to the accumulation of MVBs and enhanced exosomal secretion in lung cancer cells.

Exosome secretion kinetics are controlled by temperature.

When multivesicular endosomes (MVEs) fuse with the plasma membrane, exosomes are released into the extracellular space where they can affect other cells. The ability of exosomes to regulate cells nearby or further away depends on whether they remain attached to the secreting cell membrane. The regulation and kinetics of exosome secretion are not well characterized, but probes for directly imaging single MVE fusion events have allowed for visualization of the fusion and release process. In particular, the design of an exosome marker with a pH-sensitive dye in the middle of the tetraspanin protein CD63 has facilitated studies of individual MVE fusion events. Using TIRF microscopy, single fusion events were measured in A549 cells held at 23-37°C and events were identified using an automated detection algorithm. Stable docking precedes fusion almost always and a decrease in temperature was accompanied by decrease in the rate of content loss and in the frequency of fusion events. The loss of CD63-pHluorin fluorescence was measured at fusion sites and fit with a single or double exponential decay, with most events requiring two components and a plateau because the loss of fluorescence was typically incomplete. To interpret the kinetics, fusion events were simulated as a localized release of tethered/untethered exosomes coupled with the membrane diffusion of CD63. The experimentally observed decay required three components in the simulation: 1) free exosomes, 2) CD63 membrane diffusion from the endosomal membrane into the plasma membrane, and 3) tethered exosomes. Modeling with slow diffusion of the tethered exosomes (0.0015-0.004 µm2/s) accurately fits the experimental data for all temperatures. However, simulating with immobile tethers or the absence of tethers fails to replicate the data. Our model suggests that exosome release from the fusion site is incomplete due to postfusion, membrane attachment.

Exosome Biogenesis and Lysosome Function Determine Podocyte Exosome Release and Glomerular Inflammatory Response during Hyperhomocysteinemia.

Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation in podocytes is reportedly associated with enhanced release of exosomes containing NLRP3 inflammasome products from these cells during hyperhomocysteinemia (hHcy). This study examined the possible role of increased exosome secretion during podocyte NLRP3 inflammasome activation in the glomerular inflammatory response. Whether exosome biogenesis and lysosome function are involved in the regulation of exosome release from podocytes during hHcy in mice and upon stimulation of homocysteine (Hcy) in podocytes was tested. By nanoparticle tracking analysis, treatments of mice with amitriptyline (acid sphingomyelinase inhibitor), GW4869 (exosome biogenesis inhibitor), and rapamycin (lysosome function enhancer) were found to inhibit elevated urinary exosomes during hHcy. By examining NLRP3 inflammasome activation in glomeruli during hHcy, amitriptyline (but not GW4869 and rapamycin) was shown to have an inhibitory effect. However, all treatments attenuated glomerular inflammation and injury during hHcy. In cell studies, Hcy treatment stimulated exosome release from podocytes, which was prevented by amitriptyline, GW4869, and rapamycin. Structured illumination microscopy revealed that Hcy inhibited lysosome-multivesicular body interactions in podocytes, which was prevented by amitriptyline or rapamycin but not GW4869. Thus, the data from this study shows that activation of exosome biogenesis and dysregulated lysosome function are critically implicated in the enhancement of exosome release from podocytes leading to glomerular inflammation and injury during hHcy.

Recent advances in plant endomembrane research and new microscopical techniques.

The endomembrane system consists of various membrane-bound organelles including the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN), endosomes, and the lysosome/vacuole. Membrane trafficking between distinct compartments is mainly achieved by vesicular transport. As the endomembrane compartments and the machineries regulating the membrane trafficking are largely conserved across all eukaryotes, our current knowledge on organelle biogenesis and endomembrane trafficking in plants has mainly been shaped by corresponding studies in mammals and yeast. However, unique perspectives have emerged from plant cell biology research through the characterization of plant-specific regulators as well as the development and application of the state-of-the-art microscopical techniques. In this review, we summarize our current knowledge on the plant endomembrane system, with a focus on several distinct pathways: ER-to-Golgi transport, protein sorting at the TGN, endosomal sorting on multivesicular bodies, vacuolar trafficking/vacuole biogenesis, and the autophagy pathway. We also give an update on advanced imaging techniques for the plant cell biology research.