Apoptosis linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining giant unilamellar vesicle-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.

The hetero-multimeric protein machinery known as Endosomal Sorting Complex Required for Transport (ESCRT) operates across a spectrum of major cellular pathways including endosomal sorting, intralumenal vesicle biogenesis, and repair of endo-lysosomal and plasma membranes. Because of its pivotal roles in membrane remodeling, the ESCRT machinery is also exploited by many viral and bacterial pathogens for replication and intracellular survival. We recently reported that the protozoan pathogen Toxoplasma gondii (Tg) also subverts the ESCRT machinery, at least in one case for the uptake and degradation of host cytosolic proteins to support of its metabolic needs. Although apoptosis-linked gene 2 (ALG-2) is among the various ESCRT-related proteins that Tg recruits to its intracellular niche, the parasitophorous vacuole (PV), no other pathogens are known to exploit ALG-2; hence, the mechanism behind recruitment of ALG-2 to the PV remained unknown. ALG-2, an EF hand calcium-binding ESCRT accessory protein involved in membrane repair and other functions, binds to other proteins bearing certain short linear amino acid motifs. By searching for Tg secretory proteins containing such motifs, we identified TgGRA8 as a candidate with multiple putative ALG-2 binding elements. Reciprocal immunoprecipitation-mass spectrometry experiments suggested an interaction between TgGRA8 and ALG-2 in infected cells, along with other known ALG-2 binding proteins including ALG-2 interacting protein X (ALIX) and TSG101. Accordingly, by knocking out TgGRA8 we found that it is necessary for ALG-2 and ALIX recruitment to the PV. TgGRA8 also contributes to TSG101 recruitment. Biochemical experiments confirmed a direct and unexpectedly Ca2+-independent interaction between ALG-2 and a peptide from TgGRA8 encompassing one of its three conserved ALG-2 binding elements. Also unexpectedly, binding of GRA8 peptide to ALG-2 generated large oligomers suggestive of the peptide's ability to link ALG-2 molecules together. Collectively, our work identifies TgGRA8 as the first microbial effector targeting host ALG-2. Ongoing studies aim to unravel the biochemical mechanisms of TgGRA8 binding to ALG-2 under native cellular conditions along with defining the specific purpose for which Tg recruits ALG-2 to its PV. We acknowledge the funding support from NIH-NIAID (R01 AI120607) for this research work.

Lysosomes are central players in cellular catabolism, signaling, and metabolic regulation. Cellular and environmental stresses that damage lysosomal membranes can compromise their function and release toxic content into the cytoplasm. Here, we examine how cells respond to osmotic stress within lysosomes. Using sensitive assays of lysosomal leakage and rupture, we examine acute effects of the osmotic disruptant glycyl-L-phenylalanine 2-naphthylamide (GPN). Our findings reveal that low concentrations of GPN rupture a small fraction of lysosomes, but surprisingly trigger Ca 2+ release from nearly all. Chelating cytoplasmic Ca 2+ makes lysosomes more sensitive to GPN-induced rupture, suggesting a role for Ca 2+ in lysosomal membrane resilience. GPN-elicited Ca 2+ release causes the Ca 2+ -sensor Apoptosis Linked Gene-2 (ALG-2), along with Endosomal Sorting Complex Required for Transport (ESCRT) proteins it interacts with, to redistribute onto lysosomes. Functionally, ALG-2, but not its ESCRT binding-disabled ΔGF 122 splice variant, increases lysosomal resilience to osmotic stress. Importantly, elevating juxta-lysosomal Ca 2+ without membrane damage by activating TRPML1 also recruits ALG-2 and ESCRTs, protecting lysosomes from subsequent osmotic rupture. These findings reveal that Ca 2+ , through ALG-2, helps bring ESCRTs to lysosomes to enhance their resilience and maintain organelle integrity in the face of osmotic stress.

Many neurodegenerative diseases, including Huntington's disease (HD) and Alzheimer's disease (AD), occur due to an accumulation of aggregation-prone proteins, which results in neuronal death. Studies in animal and cell models show that reducing the levels of these proteins mitigates disease phenotypes. We previously reported a small molecule, NCT-504, which reduces cellular levels of mutant huntingtin (mHTT) in patient fibroblasts as well as mouse striatal and cortical neurons from an HdhQ111 mutant mouse. Here, we show that NCT-504 has a broader potential, and in addition reduces levels of Tau, a protein associated with Alzheimer's disease, as well as other tauopathies. We find that in untreated cells, Tau and mHTT are degraded via autophagy. Notably, treatment with NCT-504 diverts these proteins to multivesicular bodies (MVB) and the ESCRT pathway. Specifically, NCT-504 causes a proliferation of endolysosomal organelles including MVB, and an enhanced association of mHTT and Tau with endosomes and MVB. Importantly, depletion of proteins that act late in the ESCRT pathway blocked NCT-504 dependent degradation of Tau. Moreover, NCT-504-mediated degradation of Tau occurred in cells where Atg7 is depleted, which indicates that this pathway is independent of canonical autophagy. Together, these studies reveal that upregulation of traffic through an ESCRT-dependent MVB pathway may provide a therapeutic approach for neurodegenerative diseases.

The endosomal sorting complexes required for transport (ESCRT) machinery comprises a set of interacting protein complexes first implicated in delivering proteins to the vacuole in yeast and subsequently found to act in forming the intraluminal vesicles (ILVs) that characterize the late endosomal multivesicular body (MVB) and in other cellular processes that typically share a common topology of budding away from the cytoplasm. More than two decades of work has defined the structural organization and function of this versatile machinery on membranes throughout the cell. In this article, we will briefly introduce the ESCRT machinery and then move around the cell to highlight both established and emerging functions.

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.

The cross-talk between angiogenesis and immunity within the tumor microenvironment (TME) is critical for tumor prognosis. While pro-angiogenic and immunosuppressive TME promote tumor growth, anti-angiogenic and immune stimulatory TME inhibit tumor progression. Therefore, there is a great interest in achieving vascular normalization to improve drug delivery and enhance antitumor immunity. However, anti-vascular endothelial growth factor (VEGF) mechanisms to normalize tumor vessels have offered limited therapeutic efficacies for patients with cancer. Here, we report that Myct1, a direct target of ETV2, was nearly exclusively expressed in endothelial cells. In preclinical mouse tumor models, Myct1 deficiency reduced angiogenesis, enhanced high endothelial venule formation, and promoted antitumor immunity, leading to restricted tumor progression. Analysis of The Cancer Genome Atlas (TCGA) datasets revealed a significant (P < 0.05) correlation between MYCT1 expression, angiogenesis, and antitumor immunity in human cancers, as suggested by decreased FOXP3 expression and increased antitumor macrophages in patients with low MYCT1 expression. Mechanistically, MYCT1 interacted with tight junction protein Zona Occludens 1 and regulated Rho GTPase-mediated actin cytoskeleton dynamics, thereby promoting endothelial motility in the angiogenic environment. Myct1-deficient endothelial cells facilitated transendothelial migration of cytotoxic T lymphocytes and polarization of M1 macrophages. Myct1 targeting combined with anti-PD1 treatment significantly (P < 0.05) increased complete tumor regression and long-term survival in anti-PD1-responsive and -refractory tumor models in mice. Our data collectively support a critical role for Myct1 in controlling tumor angiogenesis and reprogramming tumor immunity. Myct1-targeted vascular control, in combination with immunotherapy, may become an exciting therapeutic strategy.

The lysosome is an essential organelle to recycle cellular materials and maintain nutrient homeostasis, but the mechanism to down-regulate its membrane proteins is poorly understood. In this study, we performed a cycloheximide (CHX) chase assay to measure the half-lives of approximately 30 human lysosomal membrane proteins (LMPs) and identified RNF152 and LAPTM4A as short-lived membrane proteins. The degradation of both proteins is ubiquitin dependent. RNF152 is a transmembrane E3 ligase that ubiquitinates itself, whereas LAPTM4A uses its carboxyl-terminal PY motifs to recruit NEDD4-1 for ubiquitination. After ubiquitination, they are internalized into the lysosome lumen by the endosomal sorting complexes required for transport (ESCRT) machinery for degradation. Strikingly, when ectopically expressed in budding yeast, human RNF152 is still degraded by the vacuole (yeast lysosome) in an ESCRT-dependent manner. Thus, our study uncovered a conserved mechanism to down-regulate lysosome membrane proteins.

The ESCRT (endosomal complex required for transport) machinery remodels membranes to bud vesicles away from the cytoplasm. In addition to this classic role, ESCRTs are now understood to repair damage in the plasma membrane, nuclear envelope, and throughout the endolysosomal network. Wounds in endolysosomal membranes are caused by pathogens, particulates, and other chemical or metabolic stresses. Nanoscale damage in these membranes promotes activation and engagement of ESCRT proteins. A full understanding of damage signals, molecular sensing, and the mechanism of membrane repair is yet to be developed. Nevertheless, a triggering role for calcium and ESCRT-I in recruiting ESCRT-III machinery for membrane remodeling is a repeated theme in functional studies of this response. In our current understanding of the continuum of cellular responses to lipid bilayer damage, the ESCRT machinery is fast, sensitive, and deployed independently of other systems.

The lysosome is an essential organelle to recycle cellular materials and maintain nutrient homeostasis, but the mechanism to down-regulate lysosomal membrane proteins is poorly understood. In this study, we developed a cycloheximide chase assay to measure the half-lives of ~30 human lysosomal membrane proteins, and identified RNF152 as a short-lived protein. The degradation of RNF152 depends on ubiquitin and the endosomal sorting complexes required for transport (ESCRT) machinery. Ubiquitinated RNF152 is sorted and internalized by the ESCRT machinery into the lysosomal lumen for degradation. Strikingly, when expressed in budding yeast, human RNF152 is also degraded by the vacuole (yeast lysosome) in an ESCRT-dependent manner. Thus, our study uncovered a conserved mechanism to down-regulate lysosome membrane proteins.

Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for monitoring EV release using the EV-enriched tetraspanin CD63 fused to the small, ATP-independent reporter enzyme, Nanoluciferase. Release of CD63-containing EVs from stably expressing cell lines was monitored by comparing luciferase activity in culture media to that remaining in cells. HEK293, U2OS, U87 and SKMel28 cells released 0.3%-0.6% of total cellular CD63 in the form of EVs over 5 hrs, varying by cell line. To identify cellular machinery important for secretion of CD63-containing EVs, we performed a screen of biologically active chemicals in HEK293 cells. While a majority of compounds did not significantly affect EV release, treating cells with the plecomacrolides bafilomycin or concanamycin, known to inhibit the V-ATPase, dramatically increased EV release. Interestingly, alkalization of the endosomal lumen using weak bases had no effect, suggesting a pH-independent enhancement of EV release by V-ATPase inhibitors. The ability to quantify EVs in small samples will enable future detailed studies of release kinetics as well as further chemical and genetic screening to define pathways involved in EV secretion.

Endolysosomes can be damaged by diverse materials. Terminally damaged compartments are degraded by lysophagy, but pathways that repair salvageable organelles are poorly understood. Here we found that the endosomal sorting complex required for transport (ESCRT) machinery, known to mediate budding and fission on endolysosomes, also plays an essential role in their repair. ESCRTs were rapidly recruited to acutely injured endolysosomes through a pathway requiring calcium and ESCRT-activating factors that was independent of lysophagy. We used live-cell imaging to demonstrate that ESCRTs responded to small perforations in endolysosomal membranes and enabled compartments to recover from limited damage. Silica crystals that disrupted endolysosomes also triggered ESCRT recruitment. ESCRTs thus provide a defense against endolysosomal damage likely to be relevant in physiological and pathological contexts.

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that guide chemical modifications of structural RNAs. Whereas snoRNAs primarily localize in the nucleolus, where their canonical function is to target nascent ribosomal RNAs for 2'-O-methylation, recent studies provide evidence that snoRNAs traffic out of the nucleus. Furthermore, RNA-Seq data indicate that extracellular vesicles released from cells contain snoRNAs. However, it is not known whether snoRNA secretion is regulated or whether secreted snoRNAs are functional. Here, we show that inflammation stimulates secretion of Rpl13a snoRNAs U32a (SNORD32a), U33 (SNORD33), U34 (SNORD34), and U35a (SNORD35a) from cultured macrophages, in mice, and in human subjects. Secreted snoRNAs co-fractionate with extracellular vesicles and are taken up by recipient cells. In a murine parabiosis model, we demonstrate that snoRNAs travel through the circulation to function in distant tissues. These findings support a previously unappreciated link between inflammation and snoRNA secretion in mice and humans and uncover a potential role for secreted snoRNAs in cell-cell communication.

Mycobacterium tuberculosis causes tuberculosis, which kills more people than any other infection. M. tuberculosis grows in macrophages, cells that specialize in engulfing and degrading microorganisms. Like many intracellular pathogens, in order to cause disease, M. tuberculosis damages the membrane-bound compartment (phagosome) in which it is enclosed after macrophage uptake. Recent work showed that when chemicals damage this type of intracellular compartment, cells rapidly detect and repair the damage, using machinery called the endosomal sorting complex required for transport (ESCRT). Therefore, we hypothesized that ESCRT might also respond to pathogen-induced damage. At the same time, our previous work showed that the EsxG-EsxH heterodimer of M. tuberculosis can inhibit ESCRT, raising the possibility that M. tuberculosis impairs this host response. Here, we show that ESCRT is recruited to damaged M. tuberculosis phagosomes and that EsxG-EsxH undermines ESCRT-mediated endomembrane repair. Thus, our studies demonstrate a battle between host and pathogen over endomembrane integrity.

Extracellular vesicles (EVs) are proposed to play important roles in intercellular communication. Two classes of EVs can be distinguished based on their intracellular origin. Exosomes are generated within endosomes and released when these fuse with the plasma membrane, whereas ectosomes bud directly from the plasma membrane. Studies of EV function have been hindered by limited understanding of their biogenesis. Components of the endosomal sorting complex required for transport (ESCRT) machinery play essential roles in topologically equivalent processes at both the endosome and the plasma membrane and are consistently recovered in EVs, but whether they are generally required to produce EVs is still debated. Here, we study the effects of inhibiting the ESCRT-associated AAA+ ATPase VPS4 on EV release from cultured cells using two methods for EV recovery, differential centrifugation and polyethylene glycol precipitation followed by lectin affinity chromatography. We find that inhibiting VPS4 in HEK293 cells decreases release of EV-associated proteins and miRNA as well as the overall number of EV particles. The tetraspanins CD63 and CD9 are among the most frequently monitored EV proteins, but they differ in their subcellular localization, with CD63 primarily in endosomes and CD9 on the plasma membrane. We find that CD63 and CD9 are enriched in separable populations of EVs that are both sensitive to VPS4 inhibition. Serum stimulation increases release of both types of EVs and is also reduced by inhibiting VPS4. Taken together, our data indicate that VPS4 activity is important for generating exosomes and ectosomes, thereby generally implicating the ESCRT machinery in EV biogenesis.

Torsins are developmentally essential AAA+ proteins, and mutation of human torsinA causes the neurological disease DYT1 dystonia. They localize in the ER membranes, but their cellular function remains unclear. We now show that dTorsin is required in Drosophila adipose tissue, where it suppresses triglyceride levels, promotes cell growth, and elevates membrane lipid content. We also see that human torsinA at the inner nuclear membrane is associated with membrane expansion and elevated cellular lipid content. Furthermore, the key lipid metabolizing enzyme, lipin, is mislocalized in dTorsin-KO cells, and dTorsin increases levels of the lipin substrate, phosphatidate, and reduces the product, diacylglycerol. Finally, genetic suppression of dLipin rescues dTorsin-KO defects, including adipose cell size, animal growth, and survival. These findings identify that torsins are essential regulators of cellular lipid metabolism and implicate disturbed lipid biology in childhood-onset DYT1 dystonia.