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1.
Elife ; 122024 Apr 15.
Article in English | MEDLINE | ID: mdl-38619530

ABSTRACT

Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early endosomes (EE), late endosomes (LE), and recycling endosomes (RE). To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for EE, LE, and RE. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events.


Subject(s)
Endosomes , Trypanosoma , Membranes , Cell Membrane , Transport Vesicles
2.
Cells ; 13(7)2024 Mar 29.
Article in English | MEDLINE | ID: mdl-38607033

ABSTRACT

Research into the neonatal Fc receptor (FcRn) has increased dramatically ever since Simister and Mostov first purified a rat version of the receptor. Over the years, FcRn has been shown to function not only as a receptor that transfers immunity from mother to fetus but also performs an array of different functions that include transport and recycling of immunoglobulins and albumin in the adult. Due to its important cellular roles, several clinical trials have been designed to either inhibit/enhance FcRn function or develop of non-invasive therapeutic delivery system such as fusion of drugs to IgG Fc or albumin to enhance delivery inside the cells. Here, we report the accidental identification of several FcRn alternatively spliced variants in both mouse and human cells. The four new mouse splice variants are capable of binding immunoglobulins' Fc and Fab portions. In addition, we have identified FcRn-specific vesicles in which immunoglobulins and albumin can be stored and that are involved in the endosomal-lysosomal system. The complexity of FcRn functions offers significant potential to design and develop novel and targeted therapeutics.


Subject(s)
Albumins , Immunoglobulin G , Animals , Mice , Rats , Humans , Immunoglobulin G/metabolism , Albumins/metabolism , Endosomes/metabolism , Receptors, Fc/genetics , Receptors, Fc/metabolism
3.
Protein Sci ; 33(5): e4980, 2024 May.
Article in English | MEDLINE | ID: mdl-38607248

ABSTRACT

Endosomal trafficking ensures the proper distribution of lipids and proteins to various cellular compartments, facilitating intracellular communication, nutrient transport, waste disposal, and the maintenance of cell structure. Retromer, a peripheral membrane protein complex, plays an important role in this process by recruiting the associated actin-polymerizing WASH complex to establish distinct sorting domains. The WASH complex is recruited through the interaction of the VPS35 subunit of retromer with the WASH complex subunit FAM21. Here, we report the identification of two separate fragments of FAM21 that interact with VPS35, along with a third fragment that binds to the VPS29 subunit of retromer. The crystal structure of VPS29 bound to a peptide derived from FAM21 shows a distinctive sharp bend that inserts into a conserved hydrophobic pocket with a binding mode similar to that adopted by other VPS29 effectors. Interestingly, despite the network of interactions between FAM21 and retromer occurring near the Parkinson's disease-linked mutation (D620N) in VPS35, this mutation does not significantly impair the direct association with FAM21 in vitro.


Subject(s)
Endosomes , Parkinson Disease , Humans , Mutation , Protein Transport , Vesicular Transport Proteins/genetics
4.
Commun Biol ; 7(1): 439, 2024 Apr 10.
Article in English | MEDLINE | ID: mdl-38600297

ABSTRACT

The phenomenal diversity of neuronal types in the central nervous system is achieved in part by the asymmetric division of neural precursors. In zebrafish neural precursors, asymmetric dispatch of Sara endosomes (with its Notch signaling cargo) functions as fate determinant which mediates asymmetric division. Here, we found two distinct pools of neural precursors based on Sara endosome inheritance and spindle-microtubule enrichment. Symmetric or asymmetric levels of spindle-microtubules drive differently Sara endosomes inheritance and predict neural precursor lineage. We uncover that CAMSAP2a/CAMSAP3a and KIF16Ba govern microtubule asymmetry and endosome motility, unveiling the heterogeneity of neural precursors. Using a plethora of physical and cell biological assays, we determined the physical parameters and molecular mechanisms behind microtubule asymmetries and biased endosome motility. Evolutionarily, the values of those parameters explain why all sensory organ precursor cells are asymmetric in flies while, in zebrafish spinal cord, two populations of neural precursors (symmetric vs asymmetric) are possible.


Subject(s)
Drosophila Proteins , Zebrafish , Animals , Endosomes , Microtubules , Spinal Cord
5.
J Cell Biol ; 223(7)2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38578286

ABSTRACT

The AP-1 adaptor complex is found in all eukaryotes, but it has been implicated in different pathways in different organisms. To look directly at AP-1 function, we generated stably transduced HeLa cells coexpressing tagged AP-1 and various tagged membrane proteins. Live cell imaging showed that AP-1 is recruited onto tubular carriers trafficking from the Golgi apparatus to the plasma membrane, as well as onto transferrin-containing early/recycling endosomes. Analysis of single AP-1 vesicles showed that they are a heterogeneous population, which starts to sequester cargo 30 min after exit from the ER. Vesicle capture showed that AP-1 vesicles contain transmembrane proteins found at the TGN and early/recycling endosomes, as well as lysosomal hydrolases, but very little of the anterograde adaptor GGA2. Together, our results support a model in which AP-1 retrieves proteins from post-Golgi compartments back to the TGN, analogous to COPI's role in the early secretory pathway. We propose that this is the function of AP-1 in all eukaryotes.


Subject(s)
Golgi Apparatus , Membrane Proteins , Protein Transport , Transcription Factor AP-1 , Humans , Adaptor Proteins, Vesicular Transport/metabolism , Cell Membrane/metabolism , Endosomes/genetics , Endosomes/metabolism , Golgi Apparatus/genetics , Golgi Apparatus/metabolism , HeLa Cells , Membrane Proteins/metabolism , trans-Golgi Network/metabolism , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism
6.
J Cell Biol ; 223(6)2024 Jun 03.
Article in English | MEDLINE | ID: mdl-38578646

ABSTRACT

Phosphoinositides are a small family of phospholipids that act as signaling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organization and is commonly done by the overexpression of biosensors. However, this leads to cellular perturbations and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of phosphoinositides in fixed cells and tissue, based on recombinant biosensors with self-labeling SNAP tags. These are highly specific and reliably visualize the subcellular distributions of phosphoinositides across scales, from 2D or 3D cell culture to Drosophila tissue. Further, these probes enable super-resolution approaches, and using STED microscopy, we reveal the nanoscale organization of PI(3)P on endosomes and PI(4)P on the Golgi. Finally, multiplex staining reveals an unexpected presence of PI(3,5)P2-positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualization of multiple phosphoinositide species in an unprecedented manner.


Subject(s)
Biosensing Techniques , Phosphatidylinositols , Endosomes/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphatidylinositols/chemistry , Phosphatidylinositols/metabolism , Biosensing Techniques/methods
7.
Sci Adv ; 10(13): eadl0608, 2024 Mar 29.
Article in English | MEDLINE | ID: mdl-38552021

ABSTRACT

The Golgi-localized golgins golgin-97 and golgin-245 capture transport vesicles arriving from endosomes via the protein TBC1D23. The amino-terminal domain of TBC1D23 binds to the golgins, and the carboxyl-terminal domain of TBC1D23 captures the vesicles, but how it recognizes specific vesicles was unclear. A search for binding partners of the carboxyl-terminal domain unexpectedly revealed direct binding to carboxypeptidase D and syntaxin-16, known cargo proteins of the captured vesicles. Binding is via a threonine-leucine-tyrosine (TLY) sequence present in both proteins next to an acidic cluster. A crystal structure reveals how this acidic TLY motif binds to TBC1D23. An acidic TLY motif is also present in the tails of other endosome-to-Golgi cargo, and these also bind TBC1D23. Structure-guided mutations in the carboxyl-terminal domain that disrupt motif binding in vitro also block vesicle capture in vivo. Thus, TBC1D23 attached to golgin-97 and golgin-245 captures vesicles by a previously undescribed mechanism: the recognition of a motif shared by cargo proteins carried by the vesicle.


Subject(s)
Golgi Apparatus , Membrane Proteins , Golgi Matrix Proteins/metabolism , Membrane Proteins/metabolism , Golgi Apparatus/metabolism , Biological Transport , Endosomes/metabolism , Protein Binding
8.
ACS Nano ; 18(14): 10324-10340, 2024 Apr 09.
Article in English | MEDLINE | ID: mdl-38547369

ABSTRACT

A major challenge in using nanocarriers for intracellular drug delivery is their restricted capacity to escape from endosomes into the cytosol. Here, we significantly enhance the drug delivery efficiency by accurately predicting and regulating the transition pH (pH0) of peptides to modulate their endosomal escape capability. Moreover, by inverting the chirality of the peptide carriers, we could further enhance their ability to deliver nucleic acid drugs as well as antitumor drugs. The resulting peptide carriers exhibit versatility in transfecting various cell types with a high efficiency of up to 90% by using siRNA, pDNA, and mRNA. In vivo antitumor experiments demonstrate a tumor growth inhibition of 83.4% using the peptide. This research offers a potent method for the rapid development of peptide vectors with exceptional transfection efficiencies for diverse pathophysiological indications.


Subject(s)
Drug Delivery Systems , Endosomes , Pharmaceutical Preparations , Endosomes/metabolism , Peptides/metabolism , Hydrogen-Ion Concentration
9.
J Cell Biol ; 223(6)2024 Jun 03.
Article in English | MEDLINE | ID: mdl-38536036

ABSTRACT

Organelles of the endomembrane system contain Rab GTPases as identity markers. Their localization is determined by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). It remains largely unclear how these regulators are specifically targeted to organelles and how their activity is regulated. Here, we focus on the GAP Gyp7, which acts on the Rab7-like Ypt7 protein in yeast, and surprisingly observe the protein exclusively in puncta proximal to the vacuole. Mistargeting of Gyp7 to the vacuole strongly affects vacuole morphology, suggesting that endosomal localization is needed for function. In agreement, efficient endolysosomal transport requires Gyp7. In vitro assays reveal that Gyp7 requires a distinct lipid environment for membrane binding and activity. Overexpression of Gyp7 concentrates Ypt7 in late endosomes and results in resistance to rapamycin, an inhibitor of the target of rapamycin complex 1 (TORC1), suggesting that these late endosomes are signaling endosomes. We postulate that Gyp7 is part of regulatory machinery involved in late endosome function.


Subject(s)
Endosomes , Saccharomyces cerevisiae Proteins , rab GTP-Binding Proteins , ras GTPase-Activating Proteins , Biological Transport , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Signal Transduction , Vacuoles , ras GTPase-Activating Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism
10.
Nat Commun ; 15(1): 2598, 2024 Mar 22.
Article in English | MEDLINE | ID: mdl-38519468

ABSTRACT

Activation of the mechanistic target of rapamycin (mTOR) is a key metabolic checkpoint of pro-inflammatory T-cell development that contributes to the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), however, the underlying mechanisms remain poorly understood. Here, we identify a functional role for Rab4A-directed endosome traffic in CD98 receptor recycling, mTOR activation, and accumulation of mitochondria that connect metabolic pathways with immune cell lineage development and lupus pathogenesis. Based on integrated analyses of gene expression, receptor traffic, and stable isotope tracing of metabolic pathways, constitutively active Rab4AQ72L exerts cell type-specific control over metabolic networks, dominantly impacting CD98-dependent kynurenine production, mTOR activation, mitochondrial electron transport and flux through the tricarboxylic acid cycle and thus expands CD4+ and CD3+CD4-CD8- double-negative T cells over CD8+ T cells, enhancing B cell activation, plasma cell development, antinuclear and antiphospholipid autoantibody production, and glomerulonephritis in lupus-prone mice. Rab4A deletion in T cells and pharmacological mTOR blockade restrain CD98 expression, mitochondrial metabolism and lineage skewing and attenuate glomerulonephritis. This study identifies Rab4A-directed endosome traffic as a multilevel regulator of T cell lineage specification during lupus pathogenesis.


Subject(s)
Glomerulonephritis , Lupus Erythematosus, Systemic , Animals , Mice , CD8-Positive T-Lymphocytes/metabolism , Endosomes/metabolism , Glomerulonephritis/metabolism , Kynurenine/metabolism , Mitochondria/metabolism , Mitophagy , TOR Serine-Threonine Kinases/metabolism , rab4 GTP-Binding Proteins/metabolism
11.
Org Biomol Chem ; 22(14): 2844-2850, 2024 04 03.
Article in English | MEDLINE | ID: mdl-38516851

ABSTRACT

Internal stimuli-responsive controlled release from liposomal vesicles is an innovative approach for site-specific delivery of therapeutic drugs. In this study, to enhance the endosomal pH control of drug release from liposomes, a series of histidine-modified pH-sensitive Cn-His (n = 8, 12, 18) agents were designed and utilized as triggers for liposomal content release. The pH-dependent properties of Cn-His-incorporated liposomes were characterized using dynamic light scattering, ζ-potential, and fluorescence spectroscopy. The liposomes maintained a relatively uniform size across all pH conditions. However, the ζ-potential exhibited positive values at endosomal acidic pH levels and neutral or negative values at physiological pH levels. Furthermore, acidic pH-dependent release of both polar content (carboxyfluorescein) and nonpolar content (Nile red) was observed from the Cn-His-incorporated liposomes. Notably, with C12-His as the pH sensitizer, the pH dependence of liposomal content release was significantly evident. This work establishes endosomal pH-controllable liposome platforms, laying the groundwork for developing clinically applicable triggered release formulations.


Subject(s)
Histidine , Liposomes , Liposomes/chemistry , Hydrogen-Ion Concentration , Polymers/chemistry , Endosomes , Drug Delivery Systems/methods
12.
ACS Appl Mater Interfaces ; 16(13): 15819-15831, 2024 Apr 03.
Article in English | MEDLINE | ID: mdl-38517139

ABSTRACT

Nanoparticles usually enter cells through energy-dependent endocytosis that involves their cytosolic entry via biomembrane-coated endosomes. In contrast, direct translocation of nanoparticles with straight access to cytosol/subcellular components without any membrane coating is limited to very selective conditions/approaches. Here we show that nanoparticles can switch from energy-dependent endocytosis to energy-independent direct membrane penetration once an amphiphile is electrostatically bound to their surface. Compared to endocytotic uptake, this direct cell translocation is faster and nanoparticles are distributed inside the cytosol without any lysosomal trafficking. We found that this direct cell translocation option is sensitive to the charges of both the nanoparticles and the amphiphile. We propose that an electrostatically bound amphiphile induces temporary opening of the cell membrane, which allows direct cell translocation of nanoparticles. This approach can be adapted for efficient subcellular targeting of nanoparticles and nanoparticle-based drug delivery application, bypassing the endosomal trapping and lysosomal degradation.


Subject(s)
Nanoparticles , Cytosol/metabolism , Nanoparticles/metabolism , Endocytosis , Endosomes/metabolism , Drug Delivery Systems
13.
Nat Commun ; 15(1): 2508, 2024 Mar 20.
Article in English | MEDLINE | ID: mdl-38509070

ABSTRACT

In the secretory pathway the destination of trafficking vesicles is determined by specific proteins that, with the notable exception of SNAREs, are recruited from soluble pools. Previously we have shown that microinjected proteoliposomes containing early or late endosomal SNAREs, respectively, are targeted to the corresponding endogenous compartments, with targeting specificity being dependent on the recruitment of tethering factors by some of the SNAREs. Here, we show that targeting of SNARE-containing liposomes is refined upon inclusion of polyphosphoinositides and Rab5. Intriguingly, targeting specificity is dependent on the concentration of PtdIns(3)P, and on the recruitment of PtdIns(3)P binding proteins such as rabenosyn-5 and PIKfyve, with conversion of PtdIns(3)P into PtdIns(3,5)P2 re-routing the liposomes towards late endosomes despite the presence of GTP-Rab5 and early endosomal SNAREs. Our data reveal a complex interplay between permissive and inhibitory targeting signals that sharpen a basic targeting and fusion machinery for conveying selectivity in intracellular membrane traffic.


Subject(s)
SNARE Proteins , rab GTP-Binding Proteins , SNARE Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Phosphatidylinositols/metabolism , Liposomes/metabolism , Endosomes/metabolism , Membrane Fusion
14.
Nanoscale ; 16(14): 6820-6836, 2024 Apr 04.
Article in English | MEDLINE | ID: mdl-38502114

ABSTRACT

The remarkable success of two lipid nanoparticle-mRNA vaccines against coronavirus disease (COVID-19) has placed the therapeutic and prophylactic potential of messenger RNA (mRNA) in the spotlight. It has also drawn attention to the indispensable role of lipid nanoparticles in enabling the effects of this nucleic acid. To date, lipid nanoparticles are the most clinically advanced non-viral platforms for mRNA delivery. This is thanks to their favorable safety profile and efficiency in protecting the nucleic acid from degradation and allowing its cellular uptake and cytoplasmic release upon endosomal escape. Moreover, the development of lipid nanoparticle-mRNA therapeutics was already a very active area of research even before the COVID-19 pandemic, which has likely only begun to bear its fruits. In this Review, we first discuss key aspects of the development of lipid nanoparticles as mRNA carriers. We then highlight promising preclinical and clinical studies involving lipid nanoparticle-mRNA formulations against infectious diseases and cancer, and to enable protein replacement or supplementation and genome editing. Finally, we elaborate on the challenges in advancing lipid nanoparticle-mRNA technology to widespread therapeutic use.


Subject(s)
COVID-19 , Liposomes , Nanoparticles , Humans , COVID-19/therapy , COVID-19 Vaccines , Pandemics , Endosomes , RNA, Messenger/genetics , Nanoparticles/therapeutic use
15.
Commun Biol ; 7(1): 334, 2024 Mar 15.
Article in English | MEDLINE | ID: mdl-38491121

ABSTRACT

VPS37A, an ESCRT-I complex component, is required for recruiting a subset of ESCRT proteins to the phagophore for autophagosome closure. However, the mechanism by which VPS37A is targeted to the phagophore remains obscure. Here, we demonstrate that the VPS37A N-terminal domain exhibits selective interactions with highly curved membranes, mediated by two membrane-interacting motifs within the disordered regions surrounding its Ubiquitin E2 variant-like (UEVL) domain. Site-directed mutations of residues in these motifs disrupt ESCRT-I localization to the phagophore and result in defective phagophore closure and compromised autophagic flux in vivo, highlighting their essential role during autophagy. In conjunction with the UEVL domain, we postulate that these motifs guide a functional assembly of the ESCRT machinery at the highly curved tip of the phagophore for autophagosome closure. These results advance the notion that the distinctive membrane architecture of the cup-shaped phagophore spatially regulates autophagosome biogenesis.


Subject(s)
Autophagosomes , Autophagy , Autophagosomes/metabolism , Autophagy/physiology , Intracellular Membranes/metabolism , Endosomes/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism
16.
PLoS One ; 19(3): e0290672, 2024.
Article in English | MEDLINE | ID: mdl-38483897

ABSTRACT

Viral and cellular particles too large to freely diffuse have two different types of mobility in the eukaryotic cell cytoplasm: directed motion mediated by motor proteins moving along cytoskeletal elements with the particle as its load, and motion in random directions mediated by motor proteins interconnecting cytoskeletal elements. The latter motion is referred to as "active diffusion." Mechanisms of directed motion have been extensively studied compared to mechanisms of active diffusion, despite the observation that active diffusion is more common for many viral and cellular particles. Our previous research showed that active diffusion of vesicular stomatitis virus (VSV) ribonucleoproteins (RNPs) in the cytoplasm consists of hopping between traps and that actin filaments and myosin II motors are components of the hop-trap mechanism. This raises the question whether similar mechanisms mediate random motion of larger particles with different physical and biological properties. Live-cell fluorescence imaging and a variational Bayesian analysis used in pattern recognition and machine learning were used to determine the molecular mechanisms of random motion of VSV inclusion bodies and cellular early endosomes. VSV inclusion bodies are membraneless cellular compartments that are the major sites of viral RNA synthesis, and early endosomes are representative of cellular membrane-bound organelles. Like VSV RNPs, inclusion bodies and early endosomes moved from one trapped state to another, but the distance between states was inconsistent with hopping between traps, indicating that the apparent state-to-state movement is mediated by trap movement. Like VSV RNPs, treatment with the actin filament depolymerizing inhibitor latrunculin A increased VSV inclusion body mobility by increasing the size of the traps. In contrast neither treatment with latrunculin A nor depolymerization of microtubules by nocodazole treatment affected the size of traps that confine early endosome mobility, indicating that intermediate filaments are likely major trap components for these cellular organelles.


Subject(s)
Bridged Bicyclo Compounds, Heterocyclic , Thiazolidines , Vesicular Stomatitis , Animals , Humans , Vesicular Stomatitis/metabolism , Bayes Theorem , Vesicular stomatitis Indiana virus/genetics , Vesiculovirus , Transport Vesicles , Endosomes/metabolism , Inclusion Bodies , Mammals
17.
Sci Adv ; 10(10): eadj6380, 2024 Mar 08.
Article in English | MEDLINE | ID: mdl-38446889

ABSTRACT

Nanomaterials offer unique opportunities to engineer immunomodulatory activity. In this work, we report the Toll-like receptor agonist activity of a nanoscale adjuvant zeolitic imidazolate framework-8 (ZIF-8). The accumulation of ZIF-8 in endosomes and the pH-responsive release of its subunits enable selective engagement with endosomal Toll-like receptors, minimizing the risk of off-target activation. The intrinsic adjuvant properties of ZIF-8, along with the efficient delivery and biomimetic presentation of a severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain trimer, primed rapid humoral and cell-mediated immunity in a dose-sparing manner. Our study offers insights for next-generation adjuvants that can potentially impact future vaccine development.


Subject(s)
COVID-19 , Zeolites , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Adjuvants, Immunologic , Endosomes , Toll-Like Receptors , Zeolites/pharmacology
18.
Mol Biol Cell ; 35(5): ar63, 2024 May 01.
Article in English | MEDLINE | ID: mdl-38446621

ABSTRACT

Regulation of the luminal pH of late endocytic compartments in continuously fed mammalian cells is poorly understood. Using normal rat kidney fibroblasts, we investigated the reversible assembly/disassembly of the proton pumping V-ATPase when endolysosomes are formed by kissing and fusion of late endosomes with lysosomes and during the subsequent reformation of lysosomes. We took advantage of previous work showing that sucrosomes formed by the uptake of sucrose are swollen endolysosomes from which lysosomes are reformed after uptake of invertase. Using confocal microscopy and subcellular fractionation of NRK cells stably expressing fluorescently tagged proteins, we found net recruitment of the V1 subcomplex during sucrosome formation and loss during lysosome reformation, with a similar time course to RAB7a loss. Addition of invertase did not alter mTORC1 signalling, suggesting that the regulation of reversible V-ATPase assembly/disassembly in continuously fed cells differs from that in cells subject to amino acid depletion/refeeding. Using live cell microscopy, we demonstrated recruitment of a fluorescently tagged V1 subunit during endolysosome formation and a dynamic equilibrium and rapid exchange between the cytosolic and membrane bound pools of this subunit. We conclude that reversible V-ATPase assembly/disassembly plays a key role in regulating endolysosomal/lysosomal pH in continuously fed cells.


Subject(s)
Vacuolar Proton-Translocating ATPases , Rats , Animals , Vacuolar Proton-Translocating ATPases/metabolism , beta-Fructofuranosidase/metabolism , Endosomes/metabolism , Signal Transduction , Lysosomes/metabolism , Mammals/metabolism
19.
J Exp Med ; 221(4)2024 Apr 01.
Article in English | MEDLINE | ID: mdl-38442270

ABSTRACT

Genome-wide association studies in systemic lupus erythematosus (SLE) have linked loss-of-function mutations in phagocytic NADPH oxidase complex (NOX2) genes, including NCF1 and NCF2, to disease pathogenesis. The prevailing model holds that reduced NOX2 activity promotes SLE via defective efferocytosis, the immunologically silent clearance of apoptotic cells. Here, we describe a parallel B cell-intrinsic mechanism contributing to breaks in tolerance. In keeping with an important role for B cell Toll-like receptor (TLR) pathways in lupus pathogenesis, NOX2-deficient B cells exhibit enhanced signaling downstream of endosomal TLRs, increased humoral responses to nucleic acid-containing antigens, and the propensity toward humoral autoimmunity. Mechanistically, TLR-dependent NOX2 activation promotes LC3-mediated maturation of TLR-containing endosomes, resulting in signal termination. CRISPR-mediated disruption of NCF1 confirmed a direct role for NOX2 in regulating endosomal TLR signaling in primary human B cells. Together, these data highlight a new B cell-specific mechanism contributing to autoimmune risk in NCF1 and NCF2 variant carriers.


Subject(s)
Lupus Erythematosus, Systemic , NADPH Oxidases , Humans , NADPH Oxidases/genetics , Genome-Wide Association Study , Autoimmunity/genetics , Endosomes , Lupus Erythematosus, Systemic/genetics
20.
Proc Natl Acad Sci U S A ; 121(11): e2307800120, 2024 Mar 12.
Article in English | MEDLINE | ID: mdl-38437552

ABSTRACT

Lipid nanoparticles (LNPs) have recently emerged as a powerful and versatile clinically approved platform for nucleic acid delivery, specifically for mRNA vaccines. A major bottleneck in the field is the release of mRNA-LNPs from the endosomal pathways into the cytosol of cells where they can execute their encoded functions. The data regarding the mechanism of these endosomal escape processes are limited and contradicting. Despite extensive research, there is no consensus regarding the compartment of escape, the cause of the inefficient escape and are currently lacking a robust method to detect the escape. Here, we review the currently known mechanisms of endosomal escape and the available methods to study this process. We critically discuss the limitations and challenges of these methods and the possibilities to overcome these challenges. We propose that the development of currently lacking robust, quantitative high-throughput techniques to study endosomal escape is timely and essential. A better understanding of this process will enable better RNA-LNP designs with improved efficiency to unlock new therapeutic modalities.


Subject(s)
Endosomes , RNA , Consensus , Cytosol , RNA, Messenger/genetics
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