RESUMEN
In skeletal muscle, nebulin stabilizes and regulates the length of thin filaments, but the underlying mechanism remains nebulous. In this work, we used cryo-electron tomography and subtomogram averaging to reveal structures of native nebulin bound to thin filaments within intact sarcomeres. This in situ reconstruction provided high-resolution details of the interaction between nebulin and actin, demonstrating the stabilizing role of nebulin. Myosin bound to the thin filaments exhibited different conformations of the neck domain, highlighting its inherent structural variability in muscle. Unexpectedly, nebulin did not interact with myosin or tropomyosin, but it did interact with a troponin T linker through two potential binding motifs on nebulin, explaining its regulatory role. Our structures support the role of nebulin as a thin filament "molecular ruler" and provide a molecular basis for studying nemaline myopathies.
Asunto(s)
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Miofibrillas/ultraestructura , Actinas/química , Actinas/metabolismo , Animales , Tomografía con Microscopio Electrónico , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Proteínas Musculares/genética , Mutación , Miocardio/química , Miocardio/metabolismo , Miocardio/ultraestructura , Miofibrillas/química , Miofibrillas/metabolismo , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/metabolismo , Miosinas/química , Miosinas/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Músculos Psoas/química , Músculos Psoas/metabolismo , Músculos Psoas/ultraestructura , Sarcómeros/química , Sarcómeros/metabolismo , Sarcómeros/ultraestructuraRESUMEN
Electron cryo-tomography (cryoET) is currently the only technique that allows the direct observation of proteins in their native cellular environment. Sub-volume averaging of electron tomograms offers a route to increase the signal-to-noise of repetitive biological structures, such improving the information content and interpretability of tomograms. We discuss the potential for sub-volume averaging in highlighting and investigating specific processes in situ, focusing on microtubule structure and viral infection. We show that (i) in situ sub-volume averaging from single tomograms can guide and complement segmentation of biological features, (ii) the in situ determination of the structure of individual viruses is possible as they infect a cell, and (iii) novel, transient processes can be imaged with high levels of detail.