Project description:Dysfunction in lysosomal membrane proteins and their associated complexes impairs the lysosome's essential role as a key signaling hub within the cell. This disruption underlies severe neurological disorders, such as lysosomal storage diseases and cancer. While direct visualization of the proteomic membrane environment of isolated lysosomes is crucial for advancing our understanding of its functions, it remains a significant challenge. To overcome this, we developed a method to enrich intact lysosomes using the essential lysosomal ion channel, transient receptor potential mucolipin 1. This enabled us to employ cryo electron tomography to reveal the heterogeneous molecular landscape of lysosomes. Notably, in concordance with quantitative mass spectrometry we identified protein densities on lysosomal membranes consistent with V-ATPase, Flotillin, clathrin-coated vesicles, mTORC1, HOPS, VPS13C, and dynein-dynactin. These findings demonstrate that our method offers a robust platform for advancing the structural and functional understanding of individual lysosomes, facilitating the visualization and resolution of endogenous protein complexes.
Project description:Viral infections invariably require the dismantling of capsids enclosing viral genomes. Yet, discrete intermediates and host factors supporting nucleic acid uncoating in susceptible cells are largely elusive. Using cryo-electron tomography (cryo-ET) and superresolution microscopy we show that human cells lacking the ubiquitin E3-ligase Mindbomb-1 (Mib1) deadlock adenovirus (AdV) particles at ~150nm radial distance from the nuclear pore complex (NPC). Cryo-ET revealed that the NPC-docked intermediate particles exhibit a central cavity in hexon trimers, the major capsid protein. Quantitative proteomics of isolated nuclei indicated that the intermediate particles bear reduced amounts of fiber and penton-base essential to attach the incoming virus particles to plasma membrane receptors, as well as reduced inner proteins VI and X used for endosomal membrane disruption and charge neutralization of viral DNA, respectively. The intermediate particles colocalized with the host nuclear export factor CRM1/exportin-1, independent of RanGTP hydrolysis required for CRM1 cargo export. They detached from the NPC upon CRM1 inhibition by the small compound leptomycin B, and redocked after inhibitor wash-out, indicating a tunable, direct function of CRM1 in NPC tethering of the intermediate particle poised for DNA uncoating. The results have implications on anti-viral strategies, gene therapy and synthetic biology.