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Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid–liquid phase separation (LLPS) 1 , 2 . Biomolecular interactions—particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions—are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems 3 – 6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS 7 – 9 , and has been suggested as a mechanism for intracellular concentration buffering 2 , 7 , 8 , 10 . However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates—including the nucleolus, Cajal bodies, stress granules and P-bodies—implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates. Heterotypic multicomponent interactions are shown to dominate the liquid–liquid phase separation that enables the formation of intracellular condensates.

Listeria -based live-attenuated cancer vaccines represent a promising therapy in many different pre-clinical tumor models and in clinical trials. Enhancing its anti-cancer immunity and increasing its safety profile will advance the clinical applications of Listeria vaccines. By manipulating Listeria monocytogenes flavin metabolism, we engineered a quadruple attenuated intracellular Listeria (QUAIL) vaccine candidate strain that has limited toxicity associated with extracellular growth in major extracellular niches in vivo, including blood and implanted catheter ports. Furthermore, we showed that QUAIL can be effectively programmed to engage innate-like T cells known as mucosal-associated invariant T cells, which could be harnessed for future cancer immunotherapies. The results presented here lay the foundation for further analysis of QUAIL as a safer, yet immunopotent L. monocytogenes vaccine or therapeutic vector.

Rab44 is an atypical Rab GTPase that contains some additional domains such as the EF-hand and coiled-coil domains as well as Rab-GTPase domain. Although Rab44 genes have been found in mammalian genomes, no studies concerning Rab44 have been reported yet. Here, we identified Rab44 as an upregulated protein during osteoclast differentiation. Knockdown of Rab44 by small interfering RNA promotes RANKL-induced osteoclast differentiation of the murine monocytic cell line, RAW-D or of bone marrow-derived macrophages (BMMs). In contrast, overexpression of Rab44 prevents osteoclast differentiation. Rab44 was localized in the Golgi complex and lysosomes, and Rab44 overexpression caused an enlargement of early endosomes. A series of deletion mutant studies of Rab44 showed that the coiled-coil domain and lipidation sites of Rab44 is important for regulation of osteoclast differentiation. Mechanistically, Rab44 affects nuclear factor of activated T-cells c1 (NFATc1) signaling in RANKL-stimulated macrophages. Moreover, Rab44 depletion caused an elevation in intracellular Ca 2+ transients upon RANKL stimulation, and particularly regulated lysosomal Ca 2+ influx. Taken together, these results suggest that Rab44 negatively regulates osteoclast differentiation by modulating intracellular Ca 2+ levels followed by NFATc1 activation.

The use of exosomes in clinical settings is progressively becoming a reality, as clinical trials testing exosomes for diagnostic and therapeutic applications are generating remarkable interest from the scientific community and investors. Exosomes are small extracellular vesicles secreted by all cell types playing intercellular communication roles in health and disease by transferring cellular cargoes such as functional proteins, metabolites and nucleic acids to recipient cells. An in-depth understanding of exosome biology is therefore essential to ensure clinical development of exosome based investigational therapeutic products. Here we summarise the most up-to-date knowkedge about the complex biological journey of exosomes from biogenesis and secretion, transport and uptake to their intracellular signalling. We delineate the major pathways and molecular players that influence each step of exosome physiology, highlighting the routes of interest, which will be of benefit to exosome manipulation and engineering. We highlight the main controversies in the field of exosome research: their adequate definition, characterisation and biogenesis at plasma membrane. We also delineate the most common identified pitfalls affecting exosome research and development. Unravelling exosome physiology is key to their ultimate progression towards clinical applications. 6VAGYNgLFyqkhnacPGvJhW Video Abstract

Lysosomes must maintain the integrity of their limiting membrane to ensure efficient fusion with incoming organelles and degradation of substrates within their lumen. Pancreatic cancer cells upregulate lysosomal biogenesis to enhance nutrient recycling and stress resistance, but it is unknown whether dedicated programmes for maintaining the integrity of the lysosome membrane facilitate pancreatic cancer growth. Using proteomic-based organelle profiling, we identify the Ferlin family plasma membrane repair factor Myoferlin as selectively and highly enriched on the membrane of pancreatic cancer lysosomes. Mechanistically, lysosomal localization of Myoferlin is necessary and sufficient for the maintenance of lysosome health and provides an early acting protective system against membrane damage that is independent of the endosomal sorting complex required for transport (ESCRT)-mediated repair network. Myoferlin is upregulated in human pancreatic cancer, predicts poor survival and its ablation severely impairs lysosome function and tumour growth in vivo. Thus, retargeting of plasma membrane repair factors enhances the pro-oncogenic activities of the lysosome. Gupta et al. show that the membrane repair factor Myoferlin protects against membrane damage of pancreatic cancer lysosomes to sustain enhanced lysosomal function and promote tumour growth.

Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy) 1 . Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons 2 . Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss 3 , interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1 -deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance. The membrane-shaping protein ARL6IP1 is involved in the selective degradation of the endoplasmic reticulum, and this process depends on its ubiquitination and interaction with other membrane-shaping proteins such as FAM134B.

Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses 1 – 3 . Here we show that a previously uncharacterized protein encoded by CXorf21— a gene that is associated with systemic lupus erythematosus 4 , 5 —interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease 4 , 6 – 9 . Loss of this type-I-interferon-inducible protein, which we refer to as ‘TLR adaptor interacting with SLC15A4 on the lysosome’ (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF 10 , 11 . The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus 12 – 14 . The interaction between TASL and SLC15A4 links endolysosomal Toll-like receptors to the transcription factor IRF5, providing a mechanistic explanation for the involvement of the complex in systemic lupus erythematosus.

Haematopoietic expression of the adaptor protein Trib1 is shown to be required for the presence of adipose-tissue-resident macrophages with an M2-like phenotype; Trib1 deficiency leads to aberrant expression of C/EBPα and impaired adipose tissue function. Trib1 protein role in macrophage function Macrophages are classified loosely into two types: M1 cells are immune cells active against microbial infection, and M2 cells have a broad spectrum of activities involving tissue repair, helminth infection, tumour progression and various metabolic disorders. This paper demonstrates that Tribbles homolog 1 (Trib1), an adaptor protein involved in protein degradation through interaction with COP1 ubiquitin ligase, is essential for the development of adipose-tissue-resident macrophages with an M2-like phenotype. Trib1 deficiency leads to aberrant expression of the transcription factor C/EBPα and impaired adipose tissue function. TRIB1 mutations have been implicated in metabolic disorders including atherosclerosis and hyperlipidaemia, and this work points to possible explanation of the relations between TRIB1 and metabolic disorders in humans. Macrophages consist of at least two subgroups, M1 and M2 (refs 1 , 2 , 3 ). Whereas M1 macrophages are proinflammatory and have a central role in host defence against bacterial and viral infections 4 , 5 , M2 macrophages are associated with responses to anti-inflammatory reactions, helminth infection, tissue remodelling, fibrosis and tumour progression 6 . Trib1 is an adaptor protein involved in protein degradation by interacting with COP1 ubiquitin ligase 7 . Genome-wide association studies in humans have implicated TRIB1 in lipid metabolism 8 , 9 , 10 . Here we show that Trib1 is critical for the differentiation of F4/80 + MR + tissue-resident macrophages—that share characteristics with M2 macrophages (which we term M2-like macrophages)—and eosinophils but not for the differentiation of M1 myeloid cells. Trib1 deficiency results in a severe reduction of M2-like macrophages in various organs, including bone marrow, spleen, lung and adipose tissues. Aberrant expression of C/EBPα in Trib1-deficient bone marrow cells is responsible for the defects in macrophage differentiation. Unexpectedly, mice lacking Trib1 in haematopoietic cells show diminished adipose tissue mass accompanied by evidence of increased lipolysis, even when fed a normal diet. Supplementation of M2-like macrophages rescues the pathophysiology, indicating that a lack of these macrophages is the cause of lipolysis. In response to a high-fat diet, mice lacking Trib1 in haematopoietic cells develop hypertriglyceridaemia and insulin resistance, together with increased proinflammatory cytokine gene induction. Collectively, these results demonstrate that Trib1 is critical for adipose tissue maintenance and suppression of metabolic disorders by controlling the differentiation of tissue-resident M2-like macrophages.

Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1β) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1β and GSDMD processing, but abrogates pore formation, thereby preventing IL-1β release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases. Disulfiram is an FDA-approved drug for treating alcoholism. Wu and colleagues show that disulfiram can be repurposed to efficiently inhibit pyroptosis by specifically blocking gasdermin-mediated pore formation.

Compartmentalization of cellular material in droplet-like structures is a hallmark of liquid–liquid phase separation 1 , 2 , but the mechanisms of droplet removal are poorly understood. Evidence suggests that droplets can be degraded by autophagy 3 , 4 , a highly conserved degradation system in which membrane sheets bend to isolate portions of the cytoplasm within double-membrane autophagosomes 5 – 7 . Here we examine how autophagosomes sequester droplets that contain the protein p62 (also known as SQSTM1) in living cells, and demonstrate that double-membrane, autophagosome-like vesicles form at the surface of protein-free droplets in vitro through partial wetting. A minimal physical model shows that droplet surface tension supports the formation of membrane sheets. The model also predicts that bending sheets either divide droplets for piecemeal sequestration or sequester entire droplets. We find that autophagosomal sequestration is robust to variations in the droplet-sheet adhesion strength. However, the two sides of partially wetted sheets are exposed to different environments, which can determine the bending direction of autophagosomal sheets. Our discovery of this interplay between the material properties of droplets and membrane sheets enables us to elucidate the mechanisms that underpin droplet autophagy, or ‘fluidophagy’. Furthermore, we uncover a switching mechanism that allows droplets to act as liquid assembly platforms for cytosol-degrading autophagosomes 8 or as specific autophagy substrates 9 – 11 . We propose that droplet-mediated autophagy represents a previously undescribed class of processes that are driven by elastocapillarity, highlighting the importance of wetting in cytosolic organization. A theoretical model, in vitro reconstitution and in vivo experimentation show that competition between droplet surface tension and membrane sheet instability dictates the form and function of autophagosomal membranes.