Cellular Architecture

Current projects

Deciphering (ultra)structural mechanisms of Golgi organization (Delnia Nazari Banyarani, Ph.D. student)

Proper glycosylation of proteins in the endomembrane system is crucial for many biological processes, such as lysosomal sorting, extracellular matrix structuring, and signal transduction. Improper glycosylation, in turn, is associated with neurodegeneration, cancer, and autoimmune diseases. It is, thus, vital for cells to maintain the order of their central glycan modification hub, the Golgi apparatus.

In this context, the Golgi matrix, in concert with other players like the cytoskeleton, guides protein distribution amongst the individual Golgi cisternae, controls their shape, and keeps them stacked. Nevertheless, how the Golgi matrix itself is structured to achieve these functionalities remains largely unknown, as the usability of classical approaches based on fluorescence microscopy, room temperature electron microscopy, and in vitro reconstitution is limited by the small size and high complexity of the system. To overcome this, we will employ state-of-the-art in situ cryo-electron tomography to visualize the Golgi matrix and its interactors. Applying subtomogram averaging to selected players will further allow us to determine their structures at (sub-) nanometer resolution in their intracellular environment.

This will provide an (ultra-) structural framework for the integration of orthogonal data provided by reverse genetics, cell biology, and biochemistry techniques. Together, we will use these approaches to provide unprecedented insights into the structure-function relationship of the Golgi apparatus organization.

Cryo-CLEM labels for minimizing artifacts and background (Deborah Cezar Mendonca, PostDoc)

Cryo-electron tomography (cryo-ET) can provide unique ultrastructural insights into the molecular architecture of natively preserved cells. Combining it with subtomogram averaging (STA) allows for determining subnanometer resolution structures in situ. However, localizing proteins of interest (POIs) within cells during data acquisition and processing remains a bottleneck for this approach. For solving structures of the Arp2/3 complex in its branch junction state, we recently employed its specific accumulation in lamellipodia and its characteristic integration into actin networks to select target sites for tilt-series and individual particle positions, respectively. For POIs, whose localization is less predictable from cell morphology alone, employing fluorescently tagged POIs for cryo-correlative light and electron microscopy (cryo-CLEM) can guide lamella preparation and data acquisition. However, fluorescence microscopy (FM) data will typically not support identifying POIs in cryo-electron tomograms for further processing.
Thus a tagging approach marking proteins in both imaging modalities would be highly desirable. At best, the resulting label should be able to recruit membrane-permeable synthetic fluorophores to allow for reliable detection of POIs at endogenous expression levels in FM, result in a uniquely shaped strong density in cryo-ET, and cause neglectable perturbations for cell physiology and protein function. This project aimes to provide a genetically encoded two-component cryo-CLEM labeling system that can meet those challenging demands.