Autophagy, a process of degradation that occurs via the lysosomal pathway, has an essential role in multiple aspects of immunity, including immune system development, regulation of innate and ...adaptive immune and inflammatory responses, selective degradation of intracellular microorganisms, and host protection against infectious diseases
. Autophagy is known to be induced by stimuli such as nutrient deprivation and suppression of mTOR, but little is known about how autophagosomal biogenesis is initiated in mammalian cells in response to viral infection. Here, using genome-wide short interfering RNA screens, we find that the endosomal protein sorting nexin 5 (SNX5)
is essential for virus-induced, but not for basal, stress- or endosome-induced, autophagy. We show that SNX5 deletion increases cellular susceptibility to viral infection in vitro, and that Snx5 knockout in mice enhances lethality after infection with several human viruses. Mechanistically, SNX5 interacts with beclin 1 and ATG14-containing class III phosphatidylinositol-3-kinase (PI3KC3) complex 1 (PI3KC3-C1), increases the lipid kinase activity of purified PI3KC3-C1, and is required for endosomal generation of phosphatidylinositol-3-phosphate (PtdIns(3)P) and recruitment of the PtdIns(3)P-binding protein WIPI2 to virion-containing endosomes. These findings identify a context- and organelle-specific mechanism-SNX5-dependent PI3KC3-C1 activation at endosomes-for initiation of autophagy during viral infection.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
The endopeptidase furin and the trans-Golgi network protein TGN38 are membrane proteins that recycle between the TGN and plasma membrane. TGN38 is transported by a retromer-dependent pathway from ...early endosomes to the TGN, whereas the intracellular transport of furin is poorly defined. Here we have identified the itinerary and transport requirements of furin. Using internalisation assays, we show that furin transits the early and late endosomes en route to the TGN. The GTPase Rab9 and the TGN golgin GCC185, components of the late endosome-to-TGN pathway, were required for efficient TGN retrieval of furin. By contrast, TGN38 trafficking was independent of Rab9 and GCC185. To identify the sorting signals for the early endosome-to-TGN pathway, the trafficking of furin-TGN38 chimeras was investigated. The diversion of furin from the Rab9-dependent late-endosome-to-TGN pathway to the retromer-dependent early-endosome-to-TGN pathway required both the transmembrane domain and cytoplasmic tail of TGN38. We present evidence to suggest that the length of the transmembrane domain is a contributing factor in endosomal sorting. Overall, these data show that furin uses the Rab9-dependent pathway from late endosomes and that retrograde transport directly from early endosomes is dependent on both the transmembrane domain and the cytoplasmic tail.
The intracellular trafficking of β‐site amyloid precursor protein (APP) cleaving enzyme (BACE1) and APP regulates amyloid‐β production. Our previous work demonstrated that newly synthesized BACE1 and ...APP are segregated into distinct trafficking pathways from the trans‐Golgi network (TGN), and that alterations in their trafficking lead to an increase in Aβ production in non‐neuronal and neuronal cells. However, it is not known whether BACE1 and APP are transported through the Golgi stacks together and sorted at the TGN or segregated prior to arrival at the TGN. To address this question, we have used high‐resolution Airyscan technology followed by Huygens deconvolution to quantify the overlap of BACE1 and APP in Golgi subcompartments in HeLa cells and primary neurons. Here, we show that APP and BACE1 are segregated, on exit from the endoplasmic reticulum and in the cis‐Golgi and throughout the Golgi stack. In contrast, the transferrin receptor, which exits the TGN in AP‐1 mediated transport carriers as for BACE1, colocalizes with BACE1, but not APP, throughout the Golgi stack. The segregation of APP and BACE1 is independent of the Golgi ribbon structure and the cytoplasmic domain of the cargo. Overall, our findings reveal the segregation of different membrane cargoes early in the secretory pathway, a finding relevant to the regulation of APP processing events.
Amyloid precursor protein (APP) and the β‐secretase, BACE1, are sorted into different post‐Golgi transport pathways. Here, we investigated the partitioning of APP and BACE1 along the secretory pathway. Using super‐resolution microscopy, we show that APP and BACE1 are segregated in the cis‐Golgi and throughout the Golgi stack in HeLa cells and primary neurons, whereas, the transferrin receptor, colocalizes with BACE1 but not APP. These findings reveal segregation of membrane cargoes early in the secretory pathway, which we propose limits APP processing.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
The neonatal Fc receptor (FcRn) is responsible for the recycling of endocytosed albumin and IgG, and contributes to their long plasma half-life. We recently identified an FcRn-dependent recycling ...pathway from macropinosomes in macrophages; however, little is known about the dynamics of intracellular FcRn-ligand interactions to promote recycling. Here we demonstrate a multiplexed biophysical fluorescent microscopy approach to resolve the spatiotemporal dynamics of albumin-FcRn interactions in living bone marrow-derived macrophages (BMDMs). We used the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) to detect the interaction of a FcRn-mCherry fusion protein with endocytosed Alexa Fluor 488-labeled human serum albumin (HSA-AF488) in BMDMs, and raster image correlation spectroscopy (RICS) analysis of single fluorescent-labeled albumin molecules to monitor the diffusion kinetics of internalized albumin. Our data identified a major fraction of immobile HSA-AF488 molecules in endosomal structures of human FcRn-positive mouse macrophages and an increase in FLIM-FRET following endocytosis, including detection of FRET in tubular-like structures. A nonbinding mutant of albumin showed minimum FLIM-FRET and high mobility. These data reveal the kinetics of FcRn-ligand binding within endosomal structures for recruitment into transport carriers for recycling. These approaches have wide applicability for analyses of intracellular ligand-receptor interactions.
The small G protein Arl5b is localised on the trans‐Golgi network (TGN) and regulates endosomes‐to‐TGN transport. Here, we combined in vivo and in vitro techniques to map the interactive partners and ...near neighbours of Arl5b at the TGN, using constitutively active, membrane‐bound Arl5b(Q70L)‐GFP in stably expressing HeLa cells, and the proximity labelling techniques BioID and APEX2 in parallel with GFP‐Trap pull down. From MS analysis, 22 Golgi proteins were identified; 50% were TGN‐localised Rabs, Arfs and Arls. The scaffold/tethering factors ACBD3 (GCP60) and PIST (GOPC) were also identified, and we show that Arl5b is required for TGN recruitment of ACBD3. Overall, the combination of in vivo labelling and direct pull downs indicates a highly organised complex of small G proteins on TGN membranes.
Identifying interactomes of membrane‐associated small G proteins is challenging. Here, we applied three proteomic approaches to identify the interactive neighbourhood of constitutively active Arl5b, a small G protein of the trans‐Golgi network (TGN). 50% of the hits were TGN/Golgi‐localised small G proteins, indicating an organised complex of small G proteins. Also identified was the tethering molecule ACDB3, and its TGN recruitment was shown to be Arl5b‐dependent.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a ...major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are trans-Golgi-localized GTPases known for roles in maintaining Golgi structure and Golgi-associated trafficking. We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6-GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The regulation of macropinocytosis, a specialised endocytosis pathway, is important for immune cell function. However, it is not known whether the biogenesis of macropinosomes involves one or more ...distinct pathways. We previously identified sorting nexin 5 (SNX5) as a regulator of macropinocytosis in macrophages. Here, we show that bone-marrow-derived macrophages from SNX5-knockout mice had a 60-70% reduction in macropinocytic uptake of dextran or ovalbumin, whereas phagocytosis and retrograde transport from the plasma membrane to the Golgi was unaffected. In contrast, deficiency of SNX5 had no effect on macropinocytosis or antigen presentation by dendritic cells. Activation of macrophages with CSF-1 resulted in a localisation of SNX5 to actin-rich ruffles in a manner dependent on receptor tyrosine kinases. SNX5-deficient macrophages showed a dramatic reduction in ruffling on the dorsal surface following CSF-1 receptor activation, whereas peripheral ruffling and cell migration were unaffected. We demonstrate that SNX5 is acting upstream of actin polymerisation following CSF-1 receptor activation. Overall, our findings reveal the important contribution of dorsal ruffing to receptor-activated macropinocytosis in primary macrophages and show that SNX5 selectively regulates macropinosomes derived from the dorsal ruffles.
The neonatal Fc receptor (FcRn) has a pivotal role in albumin and IgG homeostasis. Internalized IgG captured by FcRn under acidic endosomal conditions is recycled to the cell surface where exocytosis ...and a shift to neutral pH promote extracellular IgG release. Although a similar mechanism is proposed for FcRn-mediated albumin intracellular trafficking and recycling, this pathway is less well defined but is relevant to the development of therapeutics exploiting FcRn to extend the half-life of short-lived plasma proteins. Recently, a long-acting recombinant coagulation factor IX–albumin fusion protein (rIX-FP) has been approved for the management of hemophilia B. Fusion to albumin potentially enables internalized proteins to engage FcRn and escape lysosomal degradation. In this study, we present for the first time a detailed investigation of the FcRn-mediated recycling of albumin and the albumin fusion protein rIX-FP. We demonstrate that following internalization via FcRn at low pH, rIX-FP, like albumin, is detectable within the early endosome and rapidly (within 10–15 min) traffics into the Rab11+ recycling endosomes, from where it is exported from the cell. Similarly, rIX-FP and albumin taken up by fluid-phase endocytosis at physiological pH traffics into the Rab11+ recycling compartment in FcRn-positive cells but into the lysosomal compartment in FcRn-negative cells. As expected, recombinant factor IX (without albumin fusion) and an FcRn interaction–defective albumin variant localized to the lysosomal compartments of both FcRn-expressing and nonexpressing cells. These results indicate that FcRn-mediated recycling via the albumin moiety is a mechanism for the half-life extension of rIX-FP observed in clinical studies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The retrograde transport pathways from early/recycling endosomes are critical for recycling a range of endogenous cargo, as well as internalisation of bacterial and plant toxins. We have previously ...shown that the retrograde transport of the two model cargos, TGN38 and Shiga toxin, differs in the requirement for TGN golgins; transport of TGN38 requires the TGN golgin GCC88 whereas that of Shiga toxin requires GCC185. Here we have further defined the retrograde transport requirements of these two cargos. Tracking the transport of these cargos demonstrated that the bulk of Shiga toxin is transported from early endosomes to recycling endosomes en route to the TGN whereas the bulk of TGN38 is transported from early endosomes to the TGN with only low levels detected in recycling endosomes. In cells depleted of the TGN t-SNARE syntaxin 16, TGN38 accumulated predominantly in early endosomes whereas Shiga toxin accumulated in Rab11-positive recycling endosomes, suggesting distinct routes for each cargo. Retrograde transport of Shiga toxin and TGN38 requires retromer, however, whereas sorting nexin 1 (SNX1) is specifically required for transport of Shiga toxin, sorting nexin 2 (SNX2) is required for the transport of TGN38. Overall, our data have identified different itineraries for the retrograde transport of Shiga toxin and TGN38 and distinct retromer components that regulate the transport of these cargos.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Abstract
In response to longstanding healthcare inequities unmasked by the Coronavirus Disease 2019 pandemic, the infectious diseases (ID) section at the Yale School of Medicine designed and ...implemented a pilot curriculum integrating Infectious Disease Diversity, Equity, and Antiracism (ID2EA) into ID educational training and measured program outcomes. We herein describe a mixed-methods assessment of section members on whether the ID2EA curriculum affected their beliefs and behaviors regarding racism and healthcare inequities. Participants rated the curriculum as useful (92% averaging across sessions) and effective in achieving stated learning objectives (89% averaging across sessions), including fostering understanding of how inequities and racism are linked to health disparities and identifying strategies to effectively deal with racism and inequities. Despite limitations in response rates and assessment of longer-term behavioral change, this work demonstrates that training in diversity, equity, and antiracism can be successfully integrated into ID physicians’ educational activities and affect physicians’ perspectives on these topics.
To complement existing infectious diseases training on bias and microaggressions, we designed a curriculum that examined historical and geographical factors that led to healthcare inequities. Notably, our program integrated community members, so their voices were part of the educational experience.
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