RESUMEN
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
Caveolae are plasma membrane invaginations involved in transport, signalling and mechanical membrane sensing in metazoans. Their formation depends upon multiple interactions between membrane-embedded caveolins, lipids and cytosolic cavin proteins. Of the four cavin family members, only cavin1 is strictly required for caveola formation. Here, we demonstrate that an eleven residue (undecad) repeat sequence (UC1) exclusive to cavin1 is essential for caveolar localization and promotes membrane remodelling through binding to phosphatidylserine. In the notochord of mechanically stimulated zebrafish embryos, the UC1 domain is required for caveolar stability and resistance to membrane stress. The number of undecad repeats in the cavin1 UC1 domain varies throughout evolution, and we find that an increased number also correlates with increased caveolar stability. Lastly, we show that the cavin1 UC1 domain induces dramatic remodelling of the plasma membrane when grafted into cavin2 suggesting an important role in membrane sculpting. Overall, our work defines a novel conserved cavin1 modular domain that controls caveolar assembly and stability.
Asunto(s)
Caveolas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Análisis Mutacional de ADN , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células MCF-7 , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Notocorda/metabolismo , Células PC-3 , Proteínas de Unión a Fosfato , Proteínas de Unión al ARN/química , Estrés Mecánico , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
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
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
The cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems, mouse and zebrafish. In both models, Cavin4 localized to T-tubules, and loss of Cavin4 resulted in aberrant T-tubule maturation. In zebrafish, which possess duplicated cavin4 paralogs, Cavin4b was shown to directly interact with the T-tubule-associated BAR domain protein Bin1. Loss of both Cavin4a and Cavin4b caused aberrant accumulation of interconnected caveolae within the T-tubules, a fragmented T-tubule network enriched in Caveolin-3, and an impaired Ca2+ response upon mechanical stimulation. We propose a role for Cavin4 in remodeling the T-tubule membrane early in development by recycling caveolar components from the T-tubule to the sarcolemma. This generates a stable T-tubule domain lacking caveolae that is essential for T-tubule function.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sarcolema/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Caveolas/metabolismo , Línea Celular , Embrión no Mamífero/metabolismo , Imagenología Tridimensional , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/ultraestructura , Unión Proteica , Sarcolema/ultraestructura , Pez Cebra/embriologíaRESUMEN
The zebrafish is a vertebrate model suited to the exploration of cell biology within a whole organism. Hypotheses in cell mechanics can be tested by using the zebrafish notochord as a manipulable experimental system. Here, the methodologies to prepare, label, and simultaneously induce and image mechanical loading on live zebrafish notochord cells via electrical stimulation are described. This approach investigates membrane mechanics in a live, physiological setting and is thus suited for caveola research where observations within the tissues of an intact organism are increasingly relevant. This chapter also aims to introduce fundamental methodologies for the use of zebrafish in "in vivo cell biology."
Asunto(s)
Membrana Celular/metabolismo , Estimulación Eléctrica/métodos , Microscopía Confocal/métodos , Notocorda/citología , Notocorda/metabolismo , Pez Cebra/metabolismo , Animales , Caveolas/metabolismo , Estimulación Eléctrica/instrumentación , Microscopía Confocal/instrumentación , Estrés Mecánico , Pez Cebra/embriologíaRESUMEN
The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction. However, in the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. Here, we use the optically transparent and genetically tractable zebrafish system to probe T-tubule development in vivo. By combining live imaging of transgenic markers with three-dimensional electron microscopy, we derive a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formation in vivo, we develop a quantitative screen for proteins that associate with and modulate early T-tubule formation, including an overexpression screen of the entire zebrafish Rab protein family. We propose an endocytic capture model involving firstly, formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma, secondly, stabilization by myofibrils/sarcoplasmic reticulum and finally, delivery of membrane from the recycling endosome and Golgi complex.
Asunto(s)
Contracción Muscular/fisiología , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestructura , Sarcolema/fisiología , Sarcolema/ultraestructura , Animales , Canales de Calcio/metabolismo , Canales de Calcio/ultraestructura , Canales de Calcio Tipo L/metabolismo , Proteínas Portadoras/metabolismo , Biología Evolutiva , Aparato de Golgi/metabolismo , Masculino , Microscopía Electrónica , Proteínas Musculares/química , Músculo Esquelético/química , Miofibrillas/metabolismo , Sarcolema/química , Retículo Sarcoplasmático/metabolismo , Pez CebraRESUMEN
The embryonic notochord is a flexible structure present during development that serves as scaffold for formation of the vertebrate spine. This rod-like organ is thought to have evolved in non-vertebrate chordates to facilitate locomotion by providing a rigid but flexible midline structure against which the axial muscles can contract. This hydrostatic "skeleton" is exposed to a variety of mechanical forces during oscillation of the body. There is evidence that caveolae, submicroscopic cup-shaped plasma membrane pits, can buffer tension in cells that undergo high levels of mechanical stress. Indeed, caveolae are particularly abundant in the embryonic notochord. In this study, we used the CRISPR/Cas9 system to generate a mutant zebrafish line lacking Cavin1b, a coat protein required for caveola formation. Our cavin1b-/- zebrafish line exhibits reduced locomotor capacity and prominent notochord lesions characterized by necrotic, damaged, and membrane-permeable cells. Notochord diameter and body length are reduced, but remarkably, the mutants recover and are homozygous viable. By manipulating mechanical stress using a number of different assays, we show that progression of lesion severity in the mutant notochord is directly dependent on locomotion. We also demonstrate changes in caveola morphology in vivo in response to mechanical stress. Finally, induction of a catastrophic collapse of live cavin1b-/- mutant notochord cells provides the first real-time observation of caveolae mediating cellular mechanoprotection.