Folliculin (FLCN) is a tumor suppressor that coordinates cellular responses to changes in amino acid availability via regulation of the Rag guanosine triphosphatases. FLCN is recruited to lysosomes during amino acid starvation, where it interacts with RagA/B as a heterodimeric complex with FLCN-interacting proteins (FNIPs). The FLCN-FNIP heterodimer also has GTPase-activating protein (GAP) activity toward RagC/D. These properties raised two important questions. First, how is amino acid availability sensed to regulate lysosomal abundance of FLCN? Second, what is the relationship between FLCN lysosome localization, RagA/B interactions, and RagC/D GAP activity? In this study, we show that RagA/B nucleotide status determines the FLCN-FNIP1 recruitment to lysosomes. Starvation-induced FLCN-FNIP lysosome localization requires GAP activity toward Rags 1 (GATOR1), the GAP that converts RagA/B to the guanosine diphosphate (GDP)-bound state. This places FLCN-FNIP recruitment to lysosomes under the control of amino acid sensors that act upstream of GATOR1. By binding to RagA/BGDP and acting on RagC/D, FLCN-FNIP can coordinate nucleotide status between Rag heterodimer subunits in response to changes in amino acid availability.

Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.

Pancreatic islets are highly vascularized mini-organs, and vascular endothelial growth factor (VEGF)-A is a critical factor in the development of islet vascularization. To investigate the role of VEGF-A and endothelial cells (ECs) in adult islets, we used complementary genetic approaches to temporally inactivate VEGF-A in developing mouse pancreatic and islet progenitor cells or in adult β-cells. Inactivation of VEGF-A early in development dramatically reduced pancreatic and islet vascularization, leading to reduced β-cell proliferation in both developing and adult islets and, ultimately, reduced β-cell mass and impaired glucose clearance. When VEGF-A was inactivated in adult β-cells, islet vascularization was reduced twofold. Surprisingly, even after 3 months of reduced islet vascularization, islet architecture and β-cell gene expression, mass, and function were preserved with only a minimal abnormality in glucose clearance. These data show that normal pancreatic VEGF-A expression is critical for the recruitment of ECs and the subsequent stimulation of endocrine cell proliferation during islet development. In contrast, although VEGF-A is required for maintaining the specialized vasculature observed in normal adult islets, adult β-cells can adapt and survive long-term reductions in islet vascularity. These results indicate that VEGF-A and islet vascularization have a lesser role in adult islet function and β-cell mass.

TDP-43 is an RNA-binding protein that forms cytoplasmic aggregates in multiple neurodegenerative diseases. Although the loss of normal TDP-43 functions likely contributes to disease pathogenesis, the cell biological consequences of human TDP-43 depletion are not well understood. We, therefore, generated human TDP-43 knockout (KO) cells and subjected them to parallel cell biological and transcriptomic analyses. These efforts yielded three important discoveries. First, complete loss of TDP-43 resulted in widespread morphological defects related to multiple organelles, including Golgi, endosomes, lysosomes, mitochondria, and the nuclear envelope. Second, we identified a new role for TDP-43 in controlling mRNA splicing of Nup188 (nuclear pore protein). Third, analysis of multiple amyotrophic lateral sclerosis causing TDP-43 mutations revealed a broad ability to support splicing of TDP-43 target genes. However, as some TDP-43 disease-causing mutants failed to fully support the regulation of specific target transcripts, our results raise the possibility of mutation-specific loss-of-function contributions to disease pathology.

Lysosomes play a pivotal role in coordinating macromolecule degradation and regulating cell growth and metabolism. Despite substantial progress in identifying lysosomal signaling proteins, understanding the pathways that synchronize lysosome functions with changing cellular demands remains incomplete. This study uncovers a role for TANK-binding kinase 1 (TBK1), well known for its role in innate immunity and organelle quality control, in modulating lysosomal responsiveness to nutrients. Specifically, we identify a pool of TBK1 that is recruited to lysosomes in response to elevated amino acid levels. At lysosomes, this TBK1 phosphorylates Rab7 on serine 72. This is critical for alleviating Rab7-mediated inhibition of amino acid-dependent mTORC1 activation. Furthermore, a TBK1 mutant (E696K) associated with amyotrophic lateral sclerosis and frontotemporal dementia constitutively accumulates at lysosomes, resulting in elevated Rab7 phosphorylation and increased mTORC1 activation. This data establishes the lysosome as a site of amino acid regulated TBK1 signaling that is crucial for efficient mTORC1 activation. This lysosomal pool of TBK1 has broader implications for lysosome homeostasis, and its dysregulation could contribute to the pathogenesis of ALS-FTD.

Involution of the mammary gland after lactation is a dramatic example of coordinated cell death. Weaning causes distension of the alveolar structures due to the accumulation of milk, which, in turn, activates STAT3 and initiates a caspase-independent but lysosome-dependent cell death (LDCD) pathway. Although the importance of STAT3 and LDCD in early mammary involution is well established, it has not been entirely clear how milk stasis activates STAT3. In this report, we demonstrate that protein levels of the PMCA2 calcium pump are significantly downregulated within 2-4 h of experimental milk stasis. Reductions in PMCA2 expression correlate with an increase in cytoplasmic calcium in vivo as measured by multiphoton intravital imaging of GCaMP6f fluorescence. These events occur concomitant with the appearance of nuclear pSTAT3 expression but prior to significant activation of LDCD or its previously implicated mediators such as LIF, IL6, and TGFβ3, all of which appear to be upregulated by increased intracellular calcium. We further demonstrate that increased intracellular calcium activates STAT3 by inducing degradation of its negative regulator, SOCS3. We also observed that milk stasis, loss of PMCA2 expression and increased intracellular calcium levels activate TFEB, an important regulator of lysosome biogenesis through a process involving inhibition of CDK4/6 and cell cycle progression. In summary, these data suggest that intracellular calcium serves as an important proximal biochemical signal linking milk stasis to STAT3 activation, increased lysosomal biogenesis, and lysosome-mediated cell death.

Prolonged T-cell receptor (TCR) signaling is required for the proliferation of T lymphocytes. Ligation of the TCR activates signaling, but also causes internalization of the TCR from the cell surface. How TCR signaling is sustained for many hours despite lower surface expression is unknown. Using genetic inhibition of endocytosis, we show here that TCR internalization promotes continued TCR signaling and T-lymphocyte proliferation. T-cell-specific deletion of dynamin 2, an essential component of endocytosis, resulted in reduced TCR signaling strength, impaired homeostatic proliferation, and the inability to undergo clonal expansion in vivo. Blocking endocytosis resulted in a failure to maintain mammalian target of rapamycin (mTOR) activity and to stably induce the transcription factor myelocytomatosis oncogene (c-Myc), which led to metabolic stress and a defect in cell growth. Our results support the concept that the TCR can continue to signal after it is internalized from the cell surface, thereby enabling sustained signaling and cell proliferation.

The large GTPase dynamin plays a key role in endocytosis but is also localized at numerous actin rich sites. We investigated dynamin functions at podosomes/invadosomes, actin-based cellular adhesion structures implicated in tissue invasion. Podosomes/invadosomes are constituted of long F-actin bundles perpendicular to the substratum (actin cores), connected to randomly arranged F-actin fibers parallel to the substratum (actin cloud). We show here that dynamin depletion in v-Src-transformed fibroblasts triggers a massive disorganization of podosomes/invadosomes (isolated or in rosettes), with a corresponding inhibition of their invasive properties. The action of dynamin at podosomes/invadosomes requires a functional full-length protein, suggesting that the effects of dynamin at these sites and in membrane remodelling during endocytosis are mediated by similar mechanisms. In order to determine direct effect of dynamin depletion on invadosome, an optogenetic approach based on the photosensitizer KillerRed was developed. Acute dynamin photo-inactivation leads to a very rapid disorganization of invadosome without affecting focal adhesions. Dynamin therefore is a key regulator of the architecture of actin in podosomes/invadosomes.

Sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) is critical for lymphocyte egress from lymphoid organs. Lymphocytes encounter low S1P concentrations near exit sites before transmigration, yet S1PR1 signaling is rapidly terminated after exposure to S1P. How lymphocytes maintain S1PR1 signaling in a low S1P environment near egress sites is unknown. Here we identify dynamin 2, an essential component of endocytosis, as a novel regulator of T cell egress. Mice with T cell-specific dynamin 2 deficiency had profound lymphopenia and impaired egress from lymphoid organs. Dynamin 2 deficiency caused impaired egress through regulation of S1PR1 signaling, and transgenic S1PR1 overexpression rescued egress in dynamin 2 knockout mice. In low S1P concentrations, dynamin 2 was essential for S1PR1 internalization, which enabled continuous S1PR1 signaling and promoted egress from both thymus and lymph nodes. In contrast, dynamin 2-deficient cells were only capable of a pulse of S1PR1 signaling, which was insufficient for egress. Our results suggest a possible mechanism by which T lymphocytes positioned at exit portals sense low S1P concentrations, promoting their egress into circulatory fluids.

Decreasing glucagon action lowers the blood glucose and may be useful therapeutically for diabetes. However, interrupted glucagon signaling leads to α cell proliferation. To identify postulated hepatic-derived circulating factor(s) responsible for α cell proliferation, we used transcriptomics/proteomics/metabolomics in three models of interrupted glucagon signaling and found that proliferation of mouse, zebrafish, and human α cells was mTOR and FoxP transcription factor dependent. Changes in hepatic amino acid (AA) catabolism gene expression predicted the observed increase in circulating AAs. Mimicking these AA levels stimulated α cell proliferation in a newly developed in vitro assay with L-glutamine being a critical AA. α cell expression of the AA transporter Slc38a5 was markedly increased in mice with interrupted glucagon signaling and played a role in α cell proliferation. These results indicate a hepatic α islet cell axis where glucagon regulates serum AA availability and AAs, especially L-glutamine, regulate α cell proliferation and mass via mTOR-dependent nutrient sensing.

Astrocytes are critical components of the neurovascular unit that support blood-brain barrier (BBB) function. Pathological transformation of astrocytes to reactive states can be protective or harmful to BBB function. Here, using a human induced pluripotent stem cell (iPSC)-derived BBB co-culture model, we show that tumor necrosis factor (TNF) transitions astrocytes to an inflammatory reactive state that causes BBB dysfunction through activation of STAT3 and increased expression of SERPINA3, which encodes alpha 1-antichymotrypsin (α1ACT). To contextualize these findings, we correlated astrocytic STAT3 activation to vascular inflammation in postmortem human tissue. Further, in murine brain organotypic cultures, astrocyte-specific silencing of Serpina3n reduced vascular inflammation after TNF challenge. Last, treatment with recombinant Serpina3n in both ex vivo explant cultures and in vivo was sufficient to induce BBB dysfunction-related molecular changes. Overall, our results define the TNF-STAT3-α1ACT signaling axis as a driver of an inflammatory reactive astrocyte signature that contributes to BBB dysfunction.

Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell- or pancreas-specific recombination, also drive Cre expression in the brain.

Clathrin-mediated endocytosis is a fundamental cellular process conserved from yeast to mammals and is an important endocytic route for the internalization of many specific cargos, including activated growth factor receptors. Here we examined changes in tyrosine phosphorylation, a representative output of growth factor receptor signaling, in cells in which endocytic clathrin-coated pits are frozen at a deeply invaginated state, that is, cells that lack dynamin (fibroblasts from dynamin 1, dynamin 2 double conditional knockout mice). The major change observed in these cells relative to wild-type cells was an increase in the phosphorylation state, and thus activation, of activated Cdc42-associated kinase (Ack), a nonreceptor tyrosine kinase. Ack is concentrated at clathrin-coated pits, and binds clathrin heavy chain via two clathrin boxes. RNA interference-based approaches and pharmacological manipulations further demonstrated that the phosphorylation of Ack requires both clathrin assembly into endocytic clathrin-coated pits and active Cdc42. These findings reveal a link between progression of clathrin-coated pits to endocytic vesicles and an activation-deactivation cycle of Ack.

Macrophages internalize pathogens through phagocytosis, entrapping them into organelles called phagosomes. Phagosomes then fuse with lysosomes to mature into phagolysosomes, acquiring an acidic and hydrolytic lumen that kills the pathogens. During an ongoing infection, macrophages can internalize dozens of bacteria. Thus, we hypothesized that an initial round of phagocytosis might boost lysosome function and bactericidal ability to cope with subsequent rounds of phagocytosis. To test this hypothesis, we employed Fcγ-receptor-mediated phagocytosis and endocytosis, which internalize immunoglobulin G (IgG)-opsonized particles and polyvalent IgG immune complexes, respectively. We report that Fcγ receptor activation in macrophages enhances lysosome-based proteolysis and killing of subsequently phagocytosed E. coli compared to naive macrophages. Importantly, we show that Fcγ receptor activation causes nuclear translocation of TFEB, a transcription factor that boosts expression of lysosome genes. Indeed, Fc receptor activation is accompanied by increased expression of specific lysosomal proteins. Remarkably, TFEB silencing represses the Fcγ-receptor-mediated enhancements in degradation and bacterial killing. In addition, nuclear translocation of TFEB requires phagosome completion and fails to occur in cells silenced for MCOLN1, a lysosomal Ca(2+) channel, suggesting that lysosomal Ca(2+) released during phagosome maturation activates TFEB. Finally, we demonstrate that non-opsonic phagocytosis of E. coli also enhances lysosomal degradation in a TFEB-dependent manner, suggesting that this phenomenon is not limited to Fcγ receptors. Overall, we show that macrophages become better killers after one round of phagocytosis and suggest that phagosomes and lysosomes are capable of bi-directional signaling.

Cell-cell fusion is an evolutionarily conserved process that leads to the formation of multinucleated myofibers, syncytiotrophoblasts and osteoclasts, allowing their respective functions. Although cell-cell fusion requires the presence of fusogenic membrane proteins and actin-dependent cytoskeletal reorganization, the precise machinery allowing cells to fuse is still poorly understood. Using an inducible knockout mouse model to generate dynamin 1- and 2-deficient primary osteoclast precursors and myoblasts, we found that fusion of both cell types requires dynamin. Osteoclast and myoblast cell-cell fusion involves the formation of actin-rich protrusions closely associated with clathrin-mediated endocytosis in the apposed cell. Furthermore, impairing endocytosis independently of dynamin also prevented cell-cell fusion. Since dynamin is involved in both the formation of actin-rich structures and in endocytosis, our results indicate that dynamin function is central to the osteoclast precursors and myoblasts fusion process, and point to an important role of endocytosis in cell-cell fusion.

Progranulin is a lysosomal protein whose haploinsufficiency causes frontotemporal dementia, while homozygous loss of progranulin causes neuronal ceroid lipofuscinosis, a lysosomal storage disease. The sensitivity of cells to progranulin deficiency raises important questions about how cells coordinate intracellular trafficking of progranulin to ensure its efficient delivery to lysosomes. In this study, we discover that progranulin interactions with prosaposin, another lysosomal protein, first occur within the lumen of the endoplasmic reticulum (ER) and are required for the efficient ER exit of progranulin. Mechanistically, we identify an interaction between prosaposin and Surf4, a receptor that promotes loading of lumenal cargos into COPII-coated vesicles, and establish that Surf4 is critical for the efficient export of progranulin and prosaposin from the ER. Collectively, this work demonstrates that a network of interactions occurring early in the secretory pathway promote the ER exit and subsequent lysosomal delivery of newly translated progranulin and prosaposin.

Mutations in VPS13C cause early-onset, autosomal recessive Parkinson's disease (PD). We have established that VPS13C encodes a lipid transfer protein localized to contact sites between the ER and late endosomes/lysosomes. In the current study, we demonstrate that depleting VPS13C in HeLa cells causes an accumulation of lysosomes with an altered lipid profile, including an accumulation of di-22:6-BMP, a biomarker of the PD-associated leucine-rich repeat kinase 2 (LRRK2) G2019S mutation. In addition, the DNA-sensing cGAS-STING pathway, which was recently implicated in PD pathogenesis, is activated in these cells. This activation results from a combination of elevated mitochondrial DNA in the cytosol and a defect in the degradation of activated STING, a lysosome-dependent process. These results suggest a link between ER-lysosome lipid transfer and innate immune activation in a model human cell line and place VPS13C in pathways relevant to PD pathogenesis.

Posttransplantation diabetes mellitus (PTDM) is a common and significant complication related to immunosuppressive agents required to prevent organ or cell transplant rejection. To elucidate the effects of 2 commonly used agents, the calcineurin inhibitor tacrolimus (TAC) and the mTOR inhibitor sirolimus (SIR), on islet function and test whether these effects could be reversed or prevented, we investigated human islets transplanted into immunodeficient mice treated with TAC or SIR at clinically relevant levels. Both TAC and SIR impaired insulin secretion in fasted and/or stimulated conditions. Treatment with TAC or SIR increased amyloid deposition and islet macrophages, disrupted insulin granule formation, and induced broad transcriptional dysregulation related to peptide processing, ion/calcium flux, and the extracellular matrix; however, it did not affect regulation of β cell mass. Interestingly, these β cell abnormalities reversed after withdrawal of drug treatment. Furthermore, cotreatment with a GLP-1 receptor agonist completely prevented TAC-induced β cell dysfunction and partially prevented SIR-induced β cell dysfunction. These results highlight the importance of both calcineurin and mTOR signaling in normal human β cell function in vivo and suggest that modulation of these pathways may prevent or ameliorate PTDM.

Inadequate pancreatic β cell function underlies type 1 and type 2 diabetes mellitus. Strategies to expand functional cells have focused on discovering and controlling mechanisms that limit the proliferation of human β cells. Here, we developed an engraftment strategy to examine age-associated human islet cell replication competence and reveal mechanisms underlying age-dependent decline of β cell proliferation in human islets. We found that exendin-4 (Ex-4), an agonist of the glucagon-like peptide 1 receptor (GLP-1R), stimulates human β cell proliferation in juvenile but not adult islets. This age-dependent responsiveness does not reflect loss of GLP-1R signaling in adult islets, since Ex-4 treatment stimulated insulin secretion by both juvenile and adult human β cells. We show that the mitogenic effect of Ex-4 requires calcineurin/nuclear factor of activated T cells (NFAT) signaling. In juvenile islets, Ex-4 induced expression of calcineurin/NFAT signaling components as well as target genes for proliferation-promoting factors, including NFATC1, FOXM1, and CCNA1. By contrast, expression of these factors in adult islet β cells was not affected by Ex-4 exposure. These studies reveal age-dependent signaling mechanisms regulating human β cell proliferation, and identify elements that could be adapted for therapeutic expansion of human β cells.

Identification of cell-surface markers specific to human pancreatic β cells would allow in vivo analysis and imaging. Here we introduce a biomarker, ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), that is expressed on the cell surface of essentially all adult human β cells, including those from individuals with type 1 or type 2 diabetes. NTPDase3 is expressed dynamically during postnatal human pancreas development, appearing first in acinar cells at birth, but several months later its expression declines in acinar cells while concurrently emerging in islet β cells. Given its specificity and membrane localization, we utilized an NTPDase3 antibody for purification of live human β cells as confirmed by transcriptional profiling, and, in addition, for in vivo imaging of transplanted human β cells. Thus, NTPDase3 is a cell-surface biomarker of adult human β cells, and the antibody directed to this protein should be a useful new reagent for β cell sorting, in vivo imaging, and targeting.