RESUMEN
Epithelial homeostasis can be critically influenced by how cells respond to mechanical forces, both local changes in force balance between cells and altered tissue-level forces.1 Coupling of specialized cell-cell adhesions to their cytoskeletons provides epithelia with diverse strategies to respond to mechanical stresses.2,3,4 Desmosomes confer tissue resilience when their associated intermediate filaments (IFs)2,3 stiffen in response to strain,5,6,7,8,9,10,11 while mechanotransduction associated with the E-cadherin apparatus12,13 at adherens junctions (AJs) actively modulates actomyosin by RhoA signaling. Although desmosomes and AJs make complementary contributions to mechanical homeostasis in epithelia,6,8 there is increasing evidence to suggest that these cytoskeletal-adhesion systems can interact functionally and biochemically.8,14,15,16,17,18,19,20 We now report that the desmosome-IF system integrated by desmoplakin (DP) facilitates active tension sensing at AJs for epithelial homeostasis. DP function is necessary for mechanosensitive RhoA signaling at AJs to be activated when tension was applied to epithelial monolayers. This effect required DP to anchor IFs to desmosomes and recruit the dystonin (DST) cytolinker to apical junctions. DP RNAi reduced the mechanical load that was applied to the cadherin complex by increased monolayer tension. Consistent with reduced mechanical signal strength, DP RNAi compromised assembly of the Myosin VI-E-cadherin mechanosensor that activates RhoA. The integrated DP-IF system therefore supports AJ mechanotransduction by enhancing the mechanical load of tissue tension that is transmitted to E-cadherin. This crosstalk was necessary for efficient elimination of apoptotic epithelial cells by apical extrusion, demonstrating its contribution to epithelial homeostasis.
RESUMEN
The actin cytoskeleton is composed of both branched and unbranched actin filaments. In mammals, the unbranched actin filaments are primarily copolymers of actin and tropomyosin. Biochemical and imaging studies indicate that different tropomyosin isoforms are segregated to different actin filament populations in cells and tissues, providing isoform-specific functionality to the actin filament. Intrinsic to this model is the prediction that single-molecule imaging of tropomyosin isoforms would confirm homopolymer formation along the length of single actin filaments, a knowledge gap that remains unaddressed in the cellular environment. We combined chemical labeling of genetically engineered tropomyosin isoforms with electron tomography to locate individual tropomyosin molecules in fibroblasts. We find that the organization of two non-muscle tropomyosins, Tpm3.1 with Tpm4.2, can be distinguished from each other using light and electron microscopy. Visualization of single tropomyosin molecules associated with actin filaments supports the hypothesis that tropomyosins form continuous homopolymers, instead of heteropolymers, in the presence of all physiologically native actin-binding proteins. This is true for both isoforms tested. Furthermore, the data suggest that the tropomyosin molecules on one side of an actin filament may not be in register with those on the opposite side, indicating that each tropomyosin polymer may assembly independently.
Asunto(s)
Caveolas , Dinaminas , Endocitosis , Endocitosis/fisiología , Caveolas/metabolismo , Dinaminas/metabolismo , Animales , HumanosRESUMEN
Lipids are the defining features of cellular membranes. They act collectively to form a variety of different structures, and understanding their complex behavior represents an early example of systems biology. A multidisciplinary approach is needed to analyse the functions of lipids in biological systems, and new work is providing fascinating insights into their roles in membrane biology, metabolism, signaling, subcellular dynamics and various disease processes.
Asunto(s)
Membrana Celular , Metabolismo de los Lípidos , Membrana Celular/metabolismo , Humanos , Transducción de Señal , Animales , Lípidos/química , Biología de Sistemas , Lípidos de la Membrana/metabolismoRESUMEN
Macrophages regulate metabolic homeostasis in health and disease. Macrophage colony-stimulating factor (CSF1)-dependent macrophages contribute to homeostatic control of the size of the liver. This study aimed to determine the systemic metabolic consequences of elevating circulating CSF1. Acute administration of a CSF1-Fc fusion protein to mice led to monocytosis, increased resident tissue macrophages in the liver and all major organs, and liver growth. These effects were associated with increased hepatic glucose uptake and extensive mobilization of body fat. The impacts of CSF1 on macrophage abundance, liver size, and body composition were rapidly reversed to restore homeostasis. The effects of CSF1 on metabolism were independent of several known endocrine regulators and did not impact the physiological fasting response. Analysis using implantable telemetry in metabolic cages revealed progressively reduced body temperature and physical activity with no change in diurnal food intake. These results demonstrate the existence of a dynamic equilibrium between CSF1, the mononuclear phagocyte system, and control of liver-to-body weight ratio, which in turn controls systemic metabolic homeostasis. This novel macrophage regulatory axis has the potential to promote fat mobilization, without changes in appetence, which may have novel implications for managing metabolic syndrome.NEW & NOTEWORTHY CSF1 administration expands tissue macrophages, which transforms systemic metabolism. CSF1 drives fat mobilization and glucose uptake to support liver growth. The effects of CSF1 are independent of normal hormonal metabolic regulation. The effects of CSF1 are rapidly reversible, restoring homeostatic body composition. CSF1-dependent macrophages and liver size are coupled in a dynamic equilibrium.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos , Macrófagos , Animales , Ratones , Factor Estimulante de Colonias de Macrófagos/farmacología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Metabolismo de los Hidratos de Carbono , Glucosa/metabolismo , LípidosRESUMEN
As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nanoscale topography. Here, we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nanoscale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by I-BAR proteins, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physiological scenarios.
Asunto(s)
Actinas , Actinas/metabolismo , Membrana Celular/metabolismo , HomeostasisRESUMEN
Plasma membrane rupture can result in catastrophic cell death. The skeletal muscle fiber plasma membrane, the sarcolemma, provides an extreme example of a membrane subject to mechanical stress since these cells specifically evolved to generate contraction and movement. A quantitative model correlating ultrastructural remodeling of surface architecture with tissue changes in vivo is required to understand how membrane domains contribute to the shape changes associated with tissue deformation in whole animals. We and others have shown that loss of caveolae, small invaginations of the plasma membrane particularly prevalent in the muscle sarcolemma, renders the plasma membrane more susceptible to rupture during stretch.1,2,3 While it is thought that caveolae are able to flatten and be absorbed into the bulk membrane to buffer local membrane expansion, a direct demonstration of this model in vivo has been unachievable since it would require measurement of caveolae at the nanoscale combined with detailed whole-animal morphometrics under conditions of perturbation. Here, we describe the development and application of the "active trapping model" where embryonic zebrafish are immobilized in a curved state that mimics natural body axis curvature during an escape response. The model is amenable to multiscale, multimodal imaging including high-resolution whole-animal three-dimensional quantitative electron microscopy. Using the active trapping model, we demonstrate the essential role of caveolae in maintaining sarcolemmal integrity and quantify the specific contribution of caveolar-derived membrane to surface expansion. We show that caveolae directly contribute to an increase in plasma membrane surface area under physiologically relevant membrane deformation conditions.
Asunto(s)
Caveolas , Pez Cebra , Animales , Membrana Celular , Caveolas/metabolismo , Fibras Musculares Esqueléticas , Microscopía ElectrónicaRESUMEN
As physical barriers, epithelia must preserve their integrity when challenged by mechanical stresses. Cell-cell junctions linked to the cortical cytoskeleton play key roles in this process, often with mechanotransduction mechanisms that reinforce tissues. Caveolae are mechanosensitive organelles that buffer tension via disassembly. Loss of caveolae, through caveolin-1 or cavin1 depletion, causes activation of PtdIns(4, 5)P2 signaling, recruitment of FMNL2 formin, and enhanced-cortical actin assembly. How this equates to physiological responses in epithelial cells containing endogenous caveolae is unknown. Here we examined the effect of mechanically inducing acute disassembly of caveolae in epithelia. We show that perturbation of caveolae, through direct mechanical stress, reinforces the actin cortex at adherens junctions. Increasing interactions with membrane lipids by introducing multiple phosphatidylserine-binding undecad cavin1 (UC1) repeat domains into cavin1 rendered caveolae more stable to mechanical stimuli. This molecular stabilization blocked cortical reinforcement in response to mechanical stress. Cortical reinforcement elicited by the mechanically induced disassembly of caveolae increased epithelial resilience against tensile stresses. These findings identify the actin cortex as a target of caveola mechanotransduction that contributes to epithelial integrity.
Asunto(s)
Actinas , Caveolas , Caveolas/metabolismo , Mecanotransducción Celular , Caveolina 1/metabolismo , Citoesqueleto/metabolismo , Células Epiteliales/metabolismoRESUMEN
Caveolin-1 (CAV1) and CAV3 are membrane-sculpting proteins driving the formation of the plasma membrane (PM) caveolae. Within the PM mosaic environment, caveola assembly is unique as it requires progressive oligomerization of newly synthesized caveolins while trafficking through the biosynthetic-secretory pathway. Here, we have investigated these early events by combining structural, biochemical, and microscopy studies. We uncover striking trafficking differences between caveolins, with CAV1 rapidly exported to the Golgi and PM while CAV3 is initially retained in the endoplasmic reticulum and laterally moves into lipid droplets. The levels of caveolins in the endoplasmic reticulum are controlled by proteasomal degradation, and only monomeric/low oligomeric caveolins are exported into the cis-Golgi with higher-order oligomers assembling beyond this compartment. When any of those early proteostatic mechanisms are compromised, chemically or genetically, caveolins tend to accumulate along the secretory pathway forming non-functional aggregates, causing organelle damage and triggering cellular stress. Accordingly, we propose a model in which disrupted proteostasis of newly synthesized caveolins contributes to pathogenesis.
Asunto(s)
Caveolinas , Proteostasis , Caveolinas/metabolismo , Caveolina 1/metabolismo , Proteínas de la Membrana/metabolismo , Caveolas/metabolismo , Membrana Celular/metabolismo , Aparato de Golgi/metabolismoRESUMEN
Caveolae are plasma membrane invaginations with a distinct lipid composition. Membrane lipids cooperate with the structural components of caveolae to generate a metastable surface domain. Recent studies have provided insights into the structure of essential caveolar components and how lipids are crucial for the formation, dynamics, and disassembly of caveolae. They also suggest new models for how caveolins, major structural components of caveolae, insert into membranes and interact with lipids.
Asunto(s)
Caveolas , Lípidos de la Membrana , Caveolas/química , Caveolas/metabolismo , Lípidos de la Membrana/análisis , Lípidos de la Membrana/metabolismo , Caveolinas/análisis , Caveolinas/metabolismo , EndocitosisRESUMEN
Oxidative stress is a feature of many disease conditions. Oxidative stress can activate a number of cellular pathways leading to cell death, including a distinct iron-dependent pathway involving lipid peroxidation, termed ferroptosis, but cells have evolved complex mechanisms to respond to these stresses. Here, we briefly summarise current evidence linking caveolae to the cellular oxidative stress response. We discuss recent studies in cultured cells and in an in vivo model suggesting that lipid peroxidation driven by oxidative stress causes disassembly of caveolae to release caveola proteins into the cell where they regulate the master transcriptional redox controller, nuclear factor erythroid 2-related factor 2. These studies suggest that caveolae maintain cellular susceptibility to oxidative stress-induced cell death and suggest a crucial role in cellular homeostasis and the response to wounding.
Asunto(s)
Caveolas , Estrés Oxidativo , Caveolas/metabolismo , Células Cultivadas , Oxidación-Reducción , Muerte Celular , Factor 2 Relacionado con NF-E2/metabolismoRESUMEN
The spatial sorting of RNA transcripts is fundamental for the refinement of gene expression to distinct subcellular regions. Although, in non-mammalian early embryogenesis, differential RNA localisation presages cell fate determination, in mammals it remains unclear. Here, we uncover apical-to-basal RNA asymmetries in outer blastomeres of 16-cell stage mouse preimplantation embryos. Basally directed RNA transport is facilitated in a microtubule- and lysosome-mediated manner. Yet, despite an increased accumulation of RNA transcripts in basal regions, higher translation activity occurs at the more dispersed apical RNA foci, demonstrated by spatial heterogeneities in RNA subtypes, RNA-organelle interactions and translation events. During the transition to the 32-cell stage, the biased inheritance of RNA transcripts, coupled with differential translation capacity, regulates cell fate allocation of trophectoderm and cells destined to form the pluripotent inner cell mass. Our study identifies a paradigm for the spatiotemporal regulation of post-transcriptional gene expression governing mammalian preimplantation embryogenesis and cell fate.
Asunto(s)
Embrión de Mamíferos , ARN , Ratones , Animales , ARN/metabolismo , Embrión de Mamíferos/metabolismo , Diferenciación Celular/genética , Blastocisto/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Mamíferos/genéticaRESUMEN
To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.
Asunto(s)
Mitofagia , Fagocitosis , Autofagia/fisiología , ADN Mitocondrial/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mitofagia/fisiología , Humanos , Animales , RatonesRESUMEN
The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live-cell super-resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor-binding-deficient mutants in cultured hippocampal neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG-synaptotagmin-1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1-SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A- and BoNT/E-induced neurointoxication as quantified by SNAP-25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.
Asunto(s)
Toxinas Botulínicas Tipo A , Toxinas Botulínicas Tipo A/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , Animales , RatasRESUMEN
Macrophages are key cellular contributors to the pathogenesis of COVID-19, the disease caused by the virus SARS-CoV-2. The SARS-CoV-2 entry receptor ACE2 is present only on a subset of macrophages at sites of SARS-CoV-2 infection in humans. Here, we investigated whether SARS-CoV-2 can enter macrophages, replicate, and release new viral progeny; whether macrophages need to sense a replicating virus to drive cytokine release; and, if so, whether ACE2 is involved in these mechanisms. We found that SARS-CoV-2 could enter, but did not replicate within, ACE2-deficient human primary macrophages and did not induce proinflammatory cytokine expression. By contrast, ACE2 overexpression in human THP-1-derived macrophages permitted SARS-CoV-2 entry, processing and replication, and virion release. ACE2-overexpressing THP-1 macrophages sensed active viral replication and triggered proinflammatory, antiviral programs mediated by the kinase TBK-1 that limited prolonged viral replication and release. These findings help elucidate the role of ACE2 and its absence in macrophage responses to SARS-CoV-2 infection.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/fisiología , Enzima Convertidora de Angiotensina 2/genética , Citocinas , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Macrófagos/metabolismo , Virión/metabolismoRESUMEN
Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20-40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli, Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes. Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.
Asunto(s)
Sepsis , Infecciones Estafilocócicas , Humanos , Antibacterianos/uso terapéutico , Proteómica , Sepsis/microbiología , Bacterias , Escherichia coli , Klebsiella , Pruebas de Sensibilidad MicrobianaRESUMEN
Caveolae have been linked to many biological functions, but their precise roles are unclear. Using quantitative whole-cell proteomics of genome-edited cells, we show that the oxidative stress response is the major pathway dysregulated in cells lacking the key caveola structural protein, CAVIN1. CAVIN1 deletion compromised sensitivity to oxidative stress in cultured cells and in animals. Wound-induced accumulation of reactive oxygen species and apoptosis were suppressed in Cavin1-null zebrafish, negatively affecting regeneration. Oxidative stress triggered lipid peroxidation and induced caveolar disassembly. The resulting release of CAVIN1 from caveolae allowed direct interaction between CAVIN1 and NRF2, a key regulator of the antioxidant response, facilitating NRF2 degradation. CAVIN1-null cells with impaired negative regulation of NRF2 showed resistance to lipid-peroxidation-induced ferroptosis. Thus, caveolae, via lipid peroxidation and CAVIN1 release, maintain cellular susceptibility to oxidative-stress-induced cell death, demonstrating a crucial role for this organelle in cellular homeostasis and wound response.
Asunto(s)
Caveolas , Factor 2 Relacionado con NF-E2 , Animales , Caveolas/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Pez Cebra/metabolismo , Peroxidación de Lípido , Proteínas de Unión al ARN/metabolismo , Estrés OxidativoRESUMEN
Microbes have developed many strategies to subvert host organisms, which, in turn, evolved several innate immune responses. As major lipid storage organelles of eukaryotes, lipid droplets (LDs) are an attractive source of nutrients for invaders. Intracellular viruses, bacteria, and protozoan parasites induce and physically interact with LDs, and the current view is that they "hijack" LDs to draw on substrates for host colonization. This dogma has been challenged by the recent demonstration that LDs are endowed with a protein-mediated antibiotic activity, which is upregulated in response to danger signals and sepsis. Dependence on host nutrients could be a generic "Achilles' heel" of intracellular pathogens and LDs a suitable chokepoint harnessed by innate immunity to organize a front-line defense. Here, we will provide a brief overview of the state of the conflict and discuss potential mechanisms driving the formation of the 'defensive-LDs' functioning as hubs of innate immunity.
Asunto(s)
Antiinfecciosos , Gotas Lipídicas , Humanos , Gotas Lipídicas/metabolismo , Orgánulos , Bacterias , Inmunidad Innata , Antiinfecciosos/metabolismo , Metabolismo de los LípidosRESUMEN
Caveolae are small membrane invaginations that generally are stably attached to the plasma membrane. Their release is believed to depend on the GTPase dynamin 2 (Dyn2), in analogy with its role in fission of clathrin-coated vesicles. The mechanistic understanding of caveola fission is, however, sparse. Here, we used microscopy-based tracking of individual caveolae in living cells to determine the role of Dyn2 in caveola dynamics. We report that Dyn2 stably associated with the bulb of a subset of caveolae, but was not required for formation or fission of caveolae. Dyn2-positive caveolae displayed longer plasma membrane duration times, whereas depletion of Dyn2 resulted in shorter duration times and increased caveola fission. The stabilizing role of Dyn2 was independent of its GTPase activity and the caveola stabilizing protein EHD2. Thus, we propose that, in contrast to the current view, Dyn2 is not a core component of the caveolae machinery, but rather functions as an accessory protein that restrains caveola internalization.
Asunto(s)
Caveolas , Dinamina II , Caveolas/metabolismo , Dinamina II/genética , Dinamina II/metabolismo , Endocitosis , GTP Fosfohidrolasas/metabolismoRESUMEN
Osteoclasts are giant bone-digesting cells that harbor specialized lysosome-related organelles termed secretory lysosomes (SLs). SLs store cathepsin K and serve as a membrane precursor to the ruffled border, the osteoclast's 'resorptive apparatus'. Yet, the molecular composition and spatiotemporal organization of SLs remains incompletely understood. Here, using organelle-resolution proteomics, we identify member a2 of the solute carrier 37 family (Slc37a2) as a SL sugar transporter. We demonstrate in mice that Slc37a2 localizes to the SL limiting membrane and that these organelles adopt a hitherto unnoticed but dynamic tubular network in living osteoclasts that is required for bone digestion. Accordingly, mice lacking Slc37a2 accrue high bone mass owing to uncoupled bone metabolism and disturbances in SL export of monosaccharide sugars, a prerequisite for SL delivery to the bone-lining osteoclast plasma membrane. Thus, Slc37a2 is a physiological component of the osteoclast's unique secretory organelle and a potential therapeutic target for metabolic bone diseases.