The overall aim of work being conducted by the Medalia group is to understand the cellular remodeling of biomacromolecules and structure-function relations of large protein machines during cellular processes, using three-dimensional (3D) microscopy approaches (mainly cryo-electron microscopy and tomography). Projects range from the development of tools for structural investigation to structural analysis of the nuclear membrane and cytoskeleton organization, to the retrieval of structures of large macromolecular complexes in situ, using 3D averaging of sub-tomograms from eukaryotic cells.
One of the projects in the Medalia laboratory is designed to solve the mysteries of lamins. Lamins are nuclear intermediate filament proteins found in metazoan cells that assemble into fibrous structures positioned between the inner nuclear membrane and the peripheral chromatin, although a small fraction of lamins is present in the nucleoplasm. Lamins are required for maintaining nuclear structure and, together with many interaction partners, are involved in most nuclear activities. Although mutations in lamins cause a group of over 14 distinct diseases called laminopathies, it is still not clear how lamins are organized in the cell and how these mutations affect lamin functions. Recently, findings from the Medalia group have provided the first glimpse into lamin filaments in somatic and mammalian cells.
Another project in the Medalia lab seeks to resolve the structure of the nuclear pore complex (NPC) at high resolution. In particular, Prof. Medalia and his colleagues are interested in the structural dynamics of the NPC, the sole gateway to the cell nucleus. In earlier work, the Medalia team analyzed the structure of the human NPC in situ, namely inside mammalian cells. Although the structure was viewed at the modest resolution ~6 nm, it indicated that provided a suitable sample, cryo-ET can resolve nuclear structure in situ in intact cells. The team subsequently determined the structure of the frog oocyte NPC up to a resolution of 2 nm by sub-tomogram averaging of native nuclear envelopes. This work also provided the first structural views into the NPC channel, a region thought to be intrinsically disordered but which could be resolved by Medalia’s group using cryo-electron tomography. Despite these advances, an even higher resolution map is needed to understand the process of nuclear transport via the NPC.
These and other efforts rely on the availability of suitable microscopy techniques. For example, the analysis of whole mount biological samples requires chemical fixation and the application of sectioning procedures prior to imaging by cryo-electron tomography. Recently, the Medalia laboratory has developed a simple workflow for vitrifying multicellular tissues and organisms directly on electron microscopy grids, followed by thinning, using the cryo-focused ion beam (CFIB) platform, and the addition of fiducial gold markers on the surface of the CFIB-milled sample under cryogenic conditions. This novel approach allowed the Medalia team to obtain tomograms of vitrified C. elegans embryos and worms under various imaging condition and reconstruct 3D volumes that provided unprecedented views into intracellular organization. The method offers a general solution for acquiring high-resolution tomograms on multicellular samples with cryo-electron tomography and can now be implemented for resolving macromolecular structures within specific tissues and at particular developmental stages in otherwise intact organisms. This approach is under continuous development.