Cellular Architecture
Cellular Architecture
Our team is interested in how cells establish, maintain, and alter the shapes and orientations of their large-scale components, like organelles or the cytoskeleton. We are convinced that a holistic understanding of this cellular architecture can only be achieved by visualizing the machinery that organizes it. To do this under the most native conditions and at (sub-) nanometer resolutions, we rely on cryo-electron tomography and subtomogram averaging performed on cellular specimen. In situ structural and ultrastructural insights obtained by this workflow can then serve as framework for the integration of results we gather from reverse genetics, light microscopy, biochemistry, and in vitro structural approaches.
Current Team Members:
PI:
Florian Faessler
Engineer:
Chantal Weber
PostDoc:
Deborah Cezar Mendonca
Ph.D. Student:
Delnia Nazari Banyarani
Former Team Members:
Master Student:
Amina Sabar
Intern:
Paula Guía
Current projects
>> Open PostDoc position: Revealing the native structures underlying large scale intracellular transport <<
Project:
The project will focus on large-scale intracellular transport machinery previously undescribed by in-situ structural biology. It is built from actin filaments, microtubules, and membranous compartments, providing ample opportunity for ultrastructural analysis and in-situ structure determination using cryo-electron tomography and subtomogram averaging. There will also be the option of developing new processing pipelines for both of these approaches. For more details on the project, reach out to florian.faessler@igbmc.fr
Application:
Send the following documents (in English) compiled as a single PDF to florian.faessler@igbmc.fr latest on the 27th of September 2024:
-A motivation letter covering your education/scientific experiences and how they have prepared/motivated you to work on this project (max. 1 page)
-Your CV
-A copy of your PhD degree/a certification that the degree will be issued
-A description of the biggest technical challenge of your PhD study, including how you overcame it and what you learned by doing so (max. 1 page)
-The names and contacts of 2 referees
Microtubule organizing center (MTOC) fingerprint
Microtubules (MTs) are crucial for cell division and directional cell migration and, thus, tumor growth and metastasis. MT organizing centers (MTOCs) transmit distinct characteristics to the associated MTs. So, exhibit MTs organized by the centrosome more repair sites and slower motor protein-mediated transport than MTs associated with the Golgi apparatus, which is the other major MTOC in many mammalian cell types. The mechanisms by which MTOCs leave such functional “fingerprints” on MTs are still unknown but likely depend on the recruitment of MT-associated proteins (MAPs).
We will describe these fingerprints in more detail and aim to determine what principal types of MAPs might confer these characteristics from the MTOCs to the MTs and how they are distributed along MT filaments to do so. For this, we will employ genetic and pharmacological perturbations and characterize MTs (ultra-) structurally employing focused ion beam milling enabled in-situ cryo-electron tomography combined with a dedicated subtomogram averaging pipeline.
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.
Funding and partners
Recent publications
B. Zens, F. Fäßler, J. M. Hansen, R. Hauschild, J. Datler, V.-V. Hodirnau, V. Zheden, J. Alanko, M. Sixt, F. K. M Schur (2023):
Lift-out cryo-FIBSEM and cryo-ET reveal the ultrastructural landscape of extracellular matrix.
Journal of Cell Biology. 223, 6
F. Fäßler, M. G. Javoor, F. K. M. Schur (2023):
Deciphering the molecular mechanisms of actin cytoskeleton regulation in cell migration using cryo-EM.
Biochemical Society Transactions. 51, 87–99
F. Fäßler, M. G. Javoor, J. Datler, H. Döring, F. W. Hofer, G. Dimchev, V.-V. Hodirnau, J. Faix, K., Rottner, F. K. M. Schur (2023):
ArpC5 isoforms regulate Arp2/3 complex–dependent protrusion through differential Ena/VASP positioning.
Science Advances. 9, eadd6495
W. J. Nicolas, F. Fäßler, P. Dutka, F. K. M. Schur, G. Jensen, E. Meyerowitz (2022):
Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose fibers and reticulated homogalacturonan networks.
Current Biology. 32, 2375-2389.e6
G. Dimchev, B. Amiri, F. Fäßler, M. Falcke, F. K. M. Schur (2021):
Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data.
Journal of Structural Biology. 213, 107808
F. Fäßler, G. Dimchev, V.-V. Hodirnau, W. Wan, F. K. M. Schur (2020):
Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction.
Nat Commun. 11, 6437
F. Fäßler, B. Zens, R. Hauschild, F. K. M. Schur (2020):
3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy.
Journal of Structural Biology. 212, 107633