Vladan Lucic

Snapshots of nuclear pore complexes in action captured by cryo-electron tomography

Nuclear pore complexes reside in the nuclear envelope of eukaryotic cells and mediate the nucleocytoplasmic exchange of macromolecules. Traffic is regulated by mobile transport receptors that target their cargo to the central translocation channel, where phenylalanine-glycine-rich repeats serve as binding sites. The structural analysis of the nuclear pore is a formidable challenge given its size, its location in a membranous environment and its dynamic nature. Here we have used cryo-electron tomography to study the structure of nuclear pore complexes in their functional environment, that is, in intact nuclei of Dictyostelium discoideum. A new image-processing strategy compensating for deviations of the asymmetric units (protomers) from a perfect eight-fold symmetry enabled us to refine the structure and to identify new features. Furthermore, the superposition of a large number of tomograms taken in the presence of cargo, which was rendered visible by gold nanoparticles, has yielded a map outlining the trajectories of import cargo. Finally, we have performed single-molecule Monte Carlo simulations of nuclear import to interpret the experimentally observed cargo distribution in the light of existing models for nuclear import.

Cryo-electron tomography: The challenge of doing structural biology in situ

Electron microscopy played a key role in establishing cell biology as a discipline, by producing fundamental insights into cellular organization and ultrastructure. Many seminal discoveries were made possible by the development of new sample preparation methods and imaging modalities. Recent technical advances include sample vitrification that faithfully preserves molecular structures, three-dimensional imaging by electron tomography, and improved image-processing methods. These new techniques have enabled the extraction of high fidelity structural information and are beginning to reveal the macromolecular organization of unperturbed cellular environments.

Quantitative analysis of the native presynaptic cytomatrix by cryoelectron tomography

The filamentous structures that tether exocytic vesicles to the plasma membrane in the active zone are rearranged in response to synaptic stimulation.

Cryo-electron tomography of cells: connecting structure and function.

Cryo-electron tomography (cryo-ET) allows the visualization of cellular structures under close-to-life conditions and at molecular resolution. While it is inherently a static approach, yielding structural information about supramolecular organization at a certain time point, it can nevertheless provide insights into function of the structures imaged, in particular, when supplemented by other approaches. Here, we review the use of experimental methods that supplement cryo-ET imaging of whole cells. These include genetic and pharmacological manipulations, as well as correlative light microscopy and cryo-ET. While these methods have mostly been used to detect and identify structures visualized in cryo-ET or to assist the search for a feature of interest, we expect that in the future they will play a more important role in the functional interpretation of cryo-tomograms.

Cryo–electron tomography reveals a critical role of RIM1α in synaptic vesicle tethering

RIM1α-deficient synapses show structural defects in presynaptic vesicle distribution and tethering to the active zone that can be reversed by proteasome inhibition.

Electron cryotomography of vitrified cells with a Volta phase plate

Electron cryotomography provides a means of studying the three dimensional structure of pleomorphic objects, such as organelles or cells, with a resolution of 1-3nm. A limitation in the study of radiation sensitive biological samples is the low signal-to-noise ratio of the tomograms which may obscure fine details. To overcome this limitation, the recently developed Volta phase plate (VPP) was applied in electron cryotomographic studies of a wide range of cellular structures, from magnetotactic bacteria to primary cultured neurons. The results show that the VPP improves contrast significantly and consequently the signal-to-noise ratio of the tomograms, moreover it avoids disturbing fringing artifacts typical for Zernike phase plates. The contrast improvement provided by the VPP was also confirmed in projection images of relatively thick (∼400nm) samples. In order to investigate the respective contributions of the VPP and the energy filter, images acquired with different combinations of the two were compared. Zero-loss energy filtering reduced the background noise in thicker areas of the sample and improved the contrast of features such as poly-β-hydroxybutyrate granules in magnetotactic bacteria, whereas the VPP provided an overall contrast improvement for all sample areas. After 3D reconstruction, tomograms acquired with the combination of a VPP and an energy filter showed structural features in neuronal processes with outstanding clarity. We also show that the VPP can be combined with focused ion beam milling to examine structures embedded deeply inside cells. Thus, we expect that VPP will become a standard element of the electron cryotomography workflow.

Cryo-electron tomography: methodology, developments and biological applications

Cryo‐electron tomography allows three‐dimensional visualization of frozen‐hydrated, vitrified biological material at molecular resolution. Here, we summarize the most important sample preparation methods and technical aspects relevant for cryo‐electron tomography, as well as its recent biological applications from isolated macromolecular complexes to entire cells and tissues.

Structural modeling of protein interactions by analogy: application to PSD-95.

We describe comparative patch analysis for modeling the structures of multidomain proteins and protein complexes, and apply it to the PSD-95 protein. Comparative patch analysis is a hybrid of comparative modeling based on a template complex and protein docking, with a greater applicability than comparative modeling and a higher accuracy than docking. It relies on structurally defined interactions of each of the complex components, or their homologs, with any other protein, irrespective of its fold. For each component, its known binding modes with other proteins of any fold are collected and expanded by the known binding modes of its homologs. These modes are then used to restrain conventional molecular docking, resulting in a set of binary domain complexes that are subsequently ranked by geometric complementarity and a statistical potential. The method is evaluated by predicting 20 binary complexes of known structure. It is able to correctly identify the binding mode in 70% of the benchmark complexes compared with 30% for protein docking. We applied comparative patch analysis to model the complex of the third PSD-95, DLG, and ZO-1 (PDZ) domain and the SH3-GK domains in the PSD-95 protein, whose structure is unknown. In the first predicted configuration of the domains, PDZ interacts with SH3, leaving both the GMP-binding site of guanylate kinase (GK) and the C-terminus binding cleft of PDZ accessible, while in the second configuration PDZ interacts with GK, burying both binding sites. We suggest that the two alternate configurations correspond to the different functional forms of PSD-95 and provide a possible structural description for the experimentally observed cooperative folding transitions in PSD-95 and its homologs. More generally, we expect that comparative patch analysis will provide useful spatial restraints for the structural characterization of an increasing number of binary and higher-order protein complexes.

Detailed state model of CaMKII activation and autophosphorylation

By combining biochemical experiments with computer modelling of biochemical reactions we elucidated some of the currently unresolved aspects of calcium–calmodulin-dependent protein kinase II (CaMKII) activation and autophosphorylation that might be relevant for its physiological function and provided a model that incorporates in detail the mechanism of CaMKII activation and autophosphorylation at T286 that is based on experimentally determined binding constants and phosphorylation rates. To this end, we developed a detailed state model of CaMKII activation and autophosphorylation based on the currently available literature, and constrained it with data from CaMKII autophosphorylation essays. Our model takes exact phosphorylation patterns of CaMKII holoenzymes into account, and is valid at physiologically relevant conditions where the concentrations of calcium and calmodulin are not saturating. Our results strongly suggest that even when bound to less than fully calcium-bound calmodulin, CaMKII is in the active state, and indicate that the autophosphorylation of T286 by an active non-phosphorylated CaMKII subunit is significantly faster than by an autophosphorylated CaMKII subunit. These results imply that CaMKII can be efficiently activated at significantly lower calcium concentrations than previously thought, which may explain how CaMKII gets activated at calcium concentrations existing at synapses in vivo. We also investigated the significance of CaMKII holoenzyme structure on CaMKII autophosphorylation and obtained estimates of previously unknown binding constants.

Insights into the molecular organization of the neuron by cryo-electron tomography

Despite great progress in the identification and characterization of the key molecular players in neuronal function, remarkably little is known about their supramolecular organization. Cryo-electron tomography (cryo-ET), providing three-dimensional views of the molecular components of the cell in their native, fully hydrated environment, is uniquely positioned to elucidate the native architecture of the molecular machinery of the neuron. In our laboratory, we employ cryo-ET to study neuronal morphology in a variety of experimental systems and develop methods to extract quantitative and functional information from tomographic data. This approach has allowed us to shed light onto the intricate organization of the molecules of the synaptic cleft and the presynaptic cytomatrix, providing evidence for their functional roles. Also, cryo-ET of cultured neurons is beginning to open new perspectives on neuronal ultrastructure and the architecture of synaptic complexes in situ. Here, we will review these findings and discuss future directions towards the elucidation of the molecular landscape of the neuron.