Figure 1: Cryo-electron tomograms of intact cells reveal molecular landscapes, bridging the gap of dimensions between molecular resolution and prominent model systems in cell and developmental biology.
Figure 2: Cryo-focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms (adapted from Mahamid et al. J Struct Biol 2015).
The Mahamid group combines correlative approaches, cryo-focused ion beam milling and electron tomography to study mesoscale assemblies, such as centrosomes and stress granules, in intact cells and model organisms at molecular resolution.
Previous and current research
Our team brings together two fields in biology; namely, the emerging field of phase-separated assemblies in cell biology and state-of-the-art cellular cryo-electron tomography, to advance our understanding on the molecular organisation of the cytoplasm.
Eukaryotes organise their biochemical reactions into functionally distinct compartments. Intriguingly, an increasing number of reports indicate that many cellular compartments are not membrane enclosed. Rather, they assemble dynamically by phase separation, typically triggered upon a specific event. Non membrane-bound assemblies occupy an intermediate length scale between the nanoscale of individual macromolecules and the microscale of cells. They are large, non-deterministic and non-stoichiometric assemblies, the structures and function of which cannot be studied in isolation, outside the context of a living cell. Cryo-electron tomography is currently the only method providing the opportunity for obtaining in situ structural information across various scales – from whole cells to individual macromolecules.
Our recent work on cellular cryo-electron tomography shows that we can now obtain site-specific preparations in suitable eukaryotic systems, which allow direct examination of the structural features of compartments in their native, functional environment; cryo-focused ion beam (FIB) micromachining literally opens ‘electron-transparent windows’ into cells, otherwise too thick to be directly examined by TEM. Now, it is reliably applied to a wide variety of cells grown in culture. We further demonstrated that cryo-sample preparation of voluminous biological samples, encompassing whole model organisms, by cryo-lift-out is feasible and will develop into a routine application. This reduces the need to employ simplified cell culture models for the study of processes inherently unique to metazoans. Our development of cryo-correlative light and electron microscopy in 3D holds promise to attain site-specific targeting of molecular assemblies, which are then rendered accessible in any cellular volume using FIB. Today’s in situ cryo-tomograms are faithful, high-resolution representations of unperturbed molecular landscapes and contain an enormous amount of information at the molecular level. Owing to the unprecedented quality of the data, computational extraction of this information in an objective and quantitative manner is now possible with high fidelity.
Future projects and goals
Our team will continue developing and employing techniques for in situ structural biology to elucidate the structural principles and cytoplasmic environment driving the dynamic assembly of phase-separated compartments: stress granules in HeLa cells, which are RNA bodies that form rapidly in the cytoplasm upon cellular stress, and centrosomes, which are sites of microtubule nucleation, in developing C. elegans embryos. We will combine these studies with a quantitative description of the crowded nature of cytoplasm and of its local variations, to provide a direct readout of the impact of excluded volume on molecular assembly in living cells.