Yoshiyuki Fukuda

A molecular census of 26S proteasomes in intact neurons

A detailed look at proteasomes in situ The 26S proteasome is a protein machine that degrades intracellular proteins in the cytosol. The proteasome is critical for protein quality control and for the regulation of numerous cellular processes in eukaryotic cells. The structure of isolated proteasomes is well established, but how intact proteasomes look within the cell is less clear. Asano et al. used an improved approach to electron cryotomography to look at proteasomes in intact hippocampal neurons. Their analysis suggests that these cells only use about 20% of their proteasomes in an unstressed state, which leaves significant spare capacity to deal with proteotoxic stress. Science, this issue p. 439 Only 20% of proteasomes are being used in unstressed hippocampal neurons. The 26S proteasome is a key player in eukaryotic protein quality control and in the regulation of numerous cellular processes. Here, we describe quantitative in situ structural studies of this highly dynamic molecular machine in intact hippocampal neurons. We used electron cryotomography with the Volta phase plate, which allowed high fidelity and nanometer precision localization of 26S proteasomes. We undertook a molecular census of single- and double-capped proteasomes and assessed the conformational states of individual complexes. Under the conditions of the experiment—that is, in the absence of proteotoxic stress—only 20% of the 26S proteasomes were engaged in substrate processing. The remainder was in the substrate-accepting ground state. These findings suggest that in the absence of stress, the capacity of the proteasome system is not fully used.

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.

Coordinate transformation based cryo-correlative methods for electron tomography and focused ion beam milling.

Correlative microscopy allows imaging of the same feature over multiple length scales, combining light microscopy with high resolution information provided by electron microscopy. We demonstrate two procedures for coordinate transformation based correlative microscopy of vitrified biological samples applicable to different imaging modes. The first procedure aims at navigating cryo-electron tomography to cellular regions identified by fluorescent labels. The second procedure, allowing navigation of focused ion beam milling to fluorescently labeled molecules, is based on the introduction of an intermediate scanning electron microscopy imaging step to overcome the large difference between cryo-light microscopy and focused ion beam imaging modes. These methods make it possible to image fluorescently labeled macromolecular complexes in their natural environments by cryo-electron tomography, while minimizing exposure to the electron beam during the search for features of interest.

Bringing one-dimensional photonic crystals to a new light: an electrophotonic platform for chemical mass transport visualisation and cell monitoring

Photonic sensor technologies represent an important milestone in monitoring complex physical, chemical and biological systems. We present an integrated chemo- and bio-photonic sensing scheme drawing on the integration of one-dimensional (1D) stimuli-responsive photonic crystals (PCs) with an electrophotonic visualisation platform. We demonstrate various modi operandi, including the real-time mapping of spatial concentration distribution of a chemical analyte and the in situ monitoring of adhesive cell cultures, enabled by the modular combination of stimuli-responsive 1D PCs with various light emitters and detectors.

Morphologies of synaptic protein membrane fusion interfaces

Significance Neurotransmitter release occurs upon fusion of synaptic vesicles with the plasma membrane, and it is orchestrated by synaptic proteins, including SNAREs, synaptotagmin, complexin, and other factors. The system is exquisitely fine-tuned to increase the probability of membrane fusion by orders of magnitude upon Ca2+ binding to a Ca2+ sensor, such as synaptotagmin. Although crystal structures are available for some of the key complexes of soluble fragments of synaptic proteins, and the process has been reconstituted, the association of these complexes with the synaptic and plasma membranes remains unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca2+-triggered release.

In situ structural studies of tripeptidyl peptidase II (TPPII) reveal spatial association with proteasomes

Significance Tripeptidyl peptidase II (TPPII) is a key player in protein catabolism. It is common among eukaryotes and believed to act downstream of the 26S proteasome. The giant exopeptidase shows a pronounced polymorphism in vitro; that is, its state of assembly is dependent on protein concentration. In situ structural studies by cryo-electron tomography with the Volta phase plate allow assessment of TPPII supramolecuar organization in a physiological setting. Moreover, cryo-electron tomography allows researchers to probe the spatial association of TPPII with proteasomes. This spatial association is consistent with biochemical data suggesting that TPPII acts as a postproteasomal protease. Our results suggest that the spatial association facilitates sequential protein breakdown by physical proximity. Tripeptidyl peptidase II (TPPII) is a eukaryotic protease acting downstream of the 26S proteasome; it removes tripeptides from the degradation products released by the proteasome. Structural studies in vitro have revealed the basic architecture of TPPII, a two-stranded linear polymer that assembles to form a spindle-shaped complex of ∼6 MDa. Dependent on protein concentration, TPPII has a distinct tendency for polymorphism. Therefore, its structure in vivo has remained unclear. To resolve this issue, we have scrutinized cryo-electron tomograms of rat hippocampal neurons for the occurrence and spatial distribution of TPPII by template matching. The quality of the tomograms recorded with the Volta phase plate enabled a detailed structural analysis of TPPII despite its low abundance. Two different assembly states (36-mers and 32-mers) coexist as well as occasional extended forms with longer strands. A distance analysis of the relative locations of TPPII and 26S proteasomes confirmed the visual impression that these two complexes spatially associate in agreement with TPPII’s role in postproteasomal degradation.

Practical Aspects and Usage Tips for the Volta Phase Plate

Compared to other technical advances in transmission electron microscopy (TEM), such as brighter electron sources, energy filters, better optics and direct detection cameras, phase plates have lagged behind in both development and applications. The main reason for that is the difficulty in solving practical issues, such as beam-induced electrostatic charging, which cannot be reliably predicted or avoided based on theoretical research. Another reason is that phase plates add more complexity to the operation of the microscope and until not so long ago were not usable for automated data acquisition.

Phase-Contrast Cryo-Electron Tomography of Primary Cultured Neuronal Cells

To understand synaptic function, unveiling molecular architecture of synapse is one of the important issues. TEM observation of plastic embedded specimens stained with heavy metals has fundamentally contributed to our knowledge about basic structural features of neuronal synapses. However, structural investigations of synapse at the close-to-physiological state are required for a further understanding of synaptic architecture and function.

Automated Cryo-tomography and Single Particle Analysis with a New Type of Phase Plate

Recent years have shown an increased interest in the development and use of phase plates in cryo-EM. The oldest and the most productive type of phase plate is the carbon film Zernike phase plate [1]. It has been successfully used in cryo-tomography [2] and single particle analysis applications [3]. Despite its good performance the Zernike phase plate has a few pitfalls. One major practical hindrance is its short lifetime [4]. Typically within 10 days after being installed into the microscope its performance deteriorates to the point where it has to be exchanged. Another disadvantage of the Zernike phase plate is that it produces fringes around high-contrast features in the image, such as lipid membranes, support film edges etc [5]. Despite its shortcomings the Zernike phase plate has been the main motivation and experience generator in the last years.