RESUMO
Endoplasmic reticulum exit sites (ERESs) are tubular outgrowths of endoplasmic reticulum that serve as the earliest station for protein sorting and export into the secretory pathway. How these structures respond to different cellular conditions remains unclear. Here, we report that ERESs undergo lysosome-dependent microautophagy when Ca2+ is released by lysosomes in response to nutrient stressors such as mTOR inhibition or amino acid starvation in mammalian cells. Targeting and uptake of ERESs into lysosomes were observed by super-resolution live-cell imaging and focus ion beam scanning electron microscopy (FIB-SEM). The mechanism was ESCRT dependent and required ubiquitinated SEC31, ALG2, and ALIX, with a knockout of ALG2 or function-blocking mutations of ALIX preventing engulfment of ERESs by lysosomes. In vitro, reconstitution of the pathway was possible using lysosomal lipid-mimicking giant unilamellar vesicles and purified recombinant components. Together, these findings demonstrate a pathway of lysosome-dependent ERES microautophagy mediated by COPII, ALG2, and ESCRTS induced by nutrient stress.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório , Proteínas de Ligação ao Cálcio , Retículo Endoplasmático , Complexos Endossomais de Distribuição Requeridos para Transporte , Lisossomos , Microautofagia , Proteínas de Transporte Vesicular , Lisossomos/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Transporte Proteico , Células HeLa , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Autofagia/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Cálcio/metabolismoRESUMO
To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites3,4. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle5,6. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation7,8, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.
Assuntos
Retículo Endoplasmático , Mitocôndrias , Membranas Mitocondriais , Movimento , Proteínas de Transporte Vesicular , Humanos , Esclerose Lateral Amiotrófica/genética , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Mitocôndrias/química , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Transdução de Sinais , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/ultraestrutura , Microscopia Eletrônica , Imageamento Tridimensional , Sítios de Ligação , Difusão , Fatores de Tempo , Mutação , HomeostaseRESUMO
Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted 'Janelia Fluor' (JF) dyes useful for biological imaging experiments in cells and in vivo.
Assuntos
Corantes Fluorescentes/química , Rodaminas/química , Linhagem Celular Tumoral , Humanos , Raios Infravermelhos , Microscopia de Fluorescência/métodos , Estrutura MolecularRESUMO
Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.