Electron cryotomography of vitrified cells with a Volta phase plate

Fukuda, Y.; Laugks, Ulrike; Lučić, Vladan; Baumeister, Wolfgang; Danev, Radostin

doi:10.1016/j.jsb.2015.03.004

Cited by 144 publications

(116 citation statements)

References 42 publications

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“…Consequently, alignments become very sensitive to defocus artifacts exaggerating resolution loss in the original image when reconstructed in 3D, more so in small particles due to poor alignment features. To overcome the limitations of conventional cryo-ET, we used phase contrast imaging with the Volta phase plate (VPP), allowing in-focus imaging of the samples with significantly higher contrast (23,24).…”

Section: Significancementioning

confidence: 99%

Morphologies of synaptic protein membrane fusion interfaces

Gipson

Fukuda

Danev

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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 Ca 2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca 2+ -triggered release. T he process of vesicle trafficking is central for transporting materials both inside and outside of cells and is essential for maintaining cellular homeostasis (1, 2). Synaptic vesicle fusion releases neurotransmitter molecules into the synaptic cleft upon an action potential-a fast (<5 ms) and highly regulated process. The neuronal SNARE proteins synaptobrevin-2/VAMP2 and syntaxin-1A are anchored in the synaptic vesicle and plasma membranes, respectively, whereas SNAP-25 is peripherally associated with the plasma membrane. The N-to C-terminal zippering of the trans SNARE complex between synaptic and plasma membranes provides the force for membrane juxtaposition and fusion (3), but SNAREs alone are not Ca 2+ -sensitive. Synaptotagmins are membrane-tethered proteins containing two Ca 2+ -binding C2 domains, and a subset of the 16 isoforms of mammalian synaptotagmins act as Ca 2+ sensors for neurotransmitter release: e.g., synaptotagmin-1 (Syt1) is the Ca 2+ sensor for evoked synchronous neurotransmitter release (4). The system is exquisitely fine-tuned to increase the probability of fusion between synaptic vesicles and the plasma membrane by orders of magnitude upon Ca 2+ binding to Syt1. Synaptotagmins simultaneously interact with anionic phospholipid membranes (5) and the neuronal SNARE complex (6, 7). The SNARE complex also interacts with complexin (Cpx), a small soluble protein that both activates evoked release and regulates spontaneous release (8, 9). Moreover, Cpx forms a tripartite complex with the SNARE complex and Syt1 (10). This tripartite complex is part of the prefusion, "primed" state of the synaptic vesicle fusion machinery. Both Munc13 and Munc18 are important for priming, and, at the molecular level, they facilitate proper SNARE complex assembly (11, 12).Although high-resol...

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Section: Significancementioning

confidence: 99%

Morphologies of synaptic protein membrane fusion interfaces

Gipson

Fukuda

Danev

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Phase plates are typically positioned in the back focal plane of the objective lens of the EM and shift the phase of the scattered electron beam such that low-frequency information is amplified. This is the information that contributes to image contrast, and higher contrast makes it possible to detect structural features within the data and, therefore, allows for the direct observation and analysis of the internal 3D structure of cells (Asano et al, 2015;Fukuda et al, 2015;Engel et al, 2015a,b;Dai et al, 2014Dai et al, , 2013 (Fig. 4).…”

Section: Improved Em Optics Impact On What We See Inside the Cellmentioning

confidence: 99%

Cellular structural biology as revealed by cryo-electron tomography

Irobalieva

Martins

Medalia

2016

Journal of Cell Science

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Understanding the function of cellular machines requires a thorough analysis of the structural elements that underline their function. Electron microscopy (EM) has been pivotal in providing information about cellular ultrastructure, as well as macromolecular organization. Biological materials can be physically fixed by vitrification and imaged with cryo-electron tomography (cryo-ET) in a close-to-native condition. Using this technique, one can acquire three-dimensional (3D) information about the macromolecular architecture of cells, depict unique cellular states and reconstruct molecular networks. Technical advances over the last few years, such as improved sample preparation and electron detection methods, have been instrumental in obtaining data with unprecedented structural details. This presents an exciting opportunity to explore the molecular architecture of both individual cells and multicellular organisms at nanometer to subnanometer resolution. In this Commentary, we focus on the recent developments and in situ applications of cryo-ET to cell and structural biology.

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“…An excellent first application of the Volta phase plate is discussed in [66,67] and figure 12d, where whole cell tomograms were processed and proteasomes as model complex inside the cell were analyzed. This clearly shows the potential of the method and it is certain that we shall see more work done with this kind of phase plate.…”

Section: Phase Information Transfer Bymentioning

confidence: 99%

Advances in electron microscopy: A qualitative view of instrumentation development for macromolecular imaging and tomography

Schröder

2015

Archives of Biochemistry and Biophysics

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Electron cryotomography of vitrified cells with a Volta phase plate

Cited by 144 publications

References 42 publications

Morphologies of synaptic protein membrane fusion interfaces

Morphologies of synaptic protein membrane fusion interfaces

Cellular structural biology as revealed by cryo-electron tomography

Advances in electron microscopy: A qualitative view of instrumentation development for macromolecular imaging and tomography

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