Wanda Kukulski joined the NCCR TransCure at the University of Bern as a principal investigator in May 2020. In this article, she provides exciting insights in the newest imaging methods used to visualise the inner structures of cells and describes the challenges to fill the gap between molecular and cellular understanding of membrane proteins.
Why is it important to study cellular membrane architecture?
Like all cellular proteins, membrane transporters and channels perform their function within a cellular environment. Although this is a rather obvious statement, we know surprisingly little about how membrane protein complexes are distributed and arranged within the molecular landscape of native cellular membranes. The membranes that make up eukaryotic cells vary immensely in shape, lipid and protein composition, and in their peripheral interactions with components of the cytosol. For many membrane proteins, the specific architecture and environment of their native membrane links their molecular function to their cellular role. A prominent example is the arrangement of protein complexes of the respiratory chain in cristae, the folds of the inner mitochondrial membrane. The individual complexes are in close physical proximity to each other in order to transfer electrons and generate a proton gradient, which is used by ATP synthases positioned at the ridges of cristae to produce ATP (Kühlbrandt, 2015). A paradigm of functional membrane architecture that our group actively investigates is contact sites between organelles. These cellular ultrastructures are formed by the close apposition of the membranes of two organelles, permitting exchange of small molecules such as lipids or calcium ions. The proteins that perform the molecular exchange thus need to interact with both membranes, either with the lipid bilayers or with other membrane proteins. Therefore, to understand the mechanism of such transfer events, it is important to know the structural arrangement of the proteins and the organelle membranes. This information is complementary to the high-resolution structures of isolated proteins, and it is also complementary to knowledge about the coordination of transfer events occurring at different organelle contact sites. Thus, the molecular architecture of the cellular environment in which membrane-bound processes are embedded, bridges the gap between molecular functions and cellular roles.
New imaging techniques to understand cellular ultrastructure
Why is there a gap between molecular and cellular understanding of membrane proteins at all? A major reason has been the lack of appropriate methods that would allow us to combine the necessary information. Recently, however, we have seen a surge in novel cellular imaging methods. These technological advances generate unprecedented opportunities to visualise as well as understand the interior of cells at high resolution. An approach that is proving particularly useful is correlative light and electron microscopy (CLEM), in which a cellular sample is imaged with one and subsequently with the other microscopy method. CLEM allows us to directly link the presence of fluorescently labelled cellular components to ultrastructural information obtained by electron microscopy. The CLEM technique includes a diverse range of protocols, consisting of various combinations of different light and electron microscopy modalities. Which CLEM method to choose depends very much on the biological question asked. One of the CLEM approaches that we use in our own work is based on precisely locating signals of fluorescent proteins in electron tomograms of resin-embedded cells (Kukulski et al., 2011). This permits us to link knowledge of the presence or absence of membrane-associated proteins with membrane morphology and cellular context, visualised in 3D. Using this approach, we have shown that the different proteins that mediate contact sites between endoplasmic reticulum and plasma membrane localise to different parts of the contact sites (Hoffmann et al., 2019). This finding implies that membrane contact sites have an intricate organisation, although we do not yet understand this organisation. Our CLEM data has however provided hints: we found that certain contact site proteins prefer highly curved membranes, indicating that membrane curvature could be an organising principle within the contact area (Hoffmann et al., 2019).
Electron cryo-tomography: a powerful emerging tool
The above-described CLEM method is robust and allows large data sets to be acquired, but the interpretation of proteinaceous cellular structures in the tomograms is limited. This is because the sample preparation procedure does not preserve cellular structures perfectly. Better preservation is achieved by vitrification of the cellular sample, similar to biochemical samples prepared for structure determination by cryo-EM. In the footsteps of the recent cryo-EM revolution (see article on cryo-EM in newsletter #11), electron cryo-tomography (cryo-ET) is emerging as a powerful method to visualise the interior of cells with molecular resolution and in a near-native state. For abundant proteins that can easily be identified in cellular cryo-tomograms, it is possible to align and average their structures to achieve high resolution, allowing unprecedented interpretation of structures inside cells (Tegunov et al., 2021). An exciting aspect of cellular cryo-ET is that some large membrane proteins can be observed directly in their native membrane environment. We used cryo-ET in our previous work to visualise individual ATP synthases in the inner mitochondrial membranes of apoptotic cells. In healthy cells, the functional organisation of ATP synthases are dimers, with a typical tilted arrangement that matches the curvature of the cristae membrane in which they reside (Kühlbrandt, 2015). We found that in apoptotic mitochondria, cristae membranes flatten locally and that this correlates with disassembly of the dimers into monomers (Ader et al., 2019). This finding did not involve any averaging procedure, as it relied on interpreting ATP synthases individually and in correlation to their localisation.
At the interface between cell and structural biology
Although cryo-ET allows us to directly see proteins associated with membranes, it is often difficult to identify the structure of interest, or to know what stage of a process the imaged cell is in. We therefore use CLEM to combine fluorescent information with cryo-ET. In the example described above, using the fluorescent signal of an apoptotic protein allowed us to ensure that the cells we were imaging were in a defined stage of cell death (Ader et al., 2019). Using CLEM in this way to support cryo-ET poses an additional technical challenge, because it requires the fluorescence microscopy to be performed at cryogenic temperatures. However, there are various recent developments and applications of cryogenic fluorescence microscopy showing that this can be done in an almost routine manner (Bharat et al., 2018, Schorb et al., 2016, Arnold et al., 2016).
Despite recent progress, the optimisation of CLEM, cryo-CLEM and cryo-ET workflows and related method developments are still very actively pursued in the EM community. Particularly needed are improvements in the sensitivity, resolution and precision with which proteins can be identified in cryo-ET. However, it is already clear that these new imaging methods offer novel viewpoints and generate a unique interface between cell and structural biology.
NCCR TransCure PI
ADER, N. R., HOFFMANN, P. C., GANEVA, I., BORGEAUD, A. C., WANG, C., YOULE, R. J. & KUKULSKI, W. 2019. Molecular and topological reorganizations in mitochondrial architecture interplay during Bax-mediated steps of apoptosis. Elife, 8.
ARNOLD, J., MAHAMID, J., LUCIC, V., DE MARCO, A., FERNANDEZ, J. J., LAUGKS, T., MAYER, T., HYMAN, A. A., BAUMEISTER, W. & PLITZKO, J. M. 2016. Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy. Biophys J, 110, 860-9.
BHARAT, T. A. M., HOFFMANN, P. C. & KUKULSKI, W. 2018. Correlative Microscopy of Vitreous Sections Provides Insights into BAR-Domain Organization In Situ. Structure, 26, 879-886 e3.
HOFFMANN, P. C., BHARAT, T. A. M., WOZNY, M. R., BOULANGER, J., MILLER, E. A. & KUKULSKI, W. 2019. Tricalbins Contribute to Cellular Lipid Flux and Form Curved ER-PM Contacts that Are Bridged by Rod-Shaped Structures. Dev Cell, 51, 488-502 e8.
KUHLBRANDT, W. 2015. Structure and function of mitochondrial membrane protein complexes. BMC Biol, 13, 89.
KUKULSKI, W., SCHORB, M., WELSCH, S., PICCO, A., KAKSONEN, M. & BRIGGS, J. A. 2011. Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J Cell Biol, 192, 111-9.
SCHORB, M., GAECHTER, L., AVINOAM, O., SIECKMANN, F., CLARKE, M., BEBEACUA, C., BYKOV, Y. S., SONNEN, A. F., LIHL, R. & BRIGGS, J. A. 2016. New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography. J Struct Biol.
TEGUNOV, D., XUE, L., DIENEMANN, C., CRAMER, P. & MAHAMID, J. 2021. Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.5 A in cells. Nat Methods, 18, 186-193.