A new view into prokaryotic cell biology from electron cryotomography

Oikonomou, Catherine M.; Jensen, Grant J.

doi:10.1038/nrmicro.2016.7

Cited by 97 publications

(61 citation statements)

References 187 publications

(204 reference statements)

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“…Though the secretory machinery of these cells has been extensively studied by both LM and conventional EM (Rubi et al, 2005, Giordano et al, 2008), we sought to advance those earlier efforts by imaging these cells in a near-native, frozen-hydrated state in 3-D via electron cryo-tomography (ECT) (Oikonomou and Jensen, 2016). We were particularly interested in dense core secretory granules (DCSGs), intracellular compartments which are central to the efficient secretion of hormones including insulin (Kim et al, 2001).…”

Section: Resultsmentioning

confidence: 99%

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

Carter

Mageswaran

Farino

et al. 2018

Journal of Structural Biology

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In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80 K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules. Here we describe a method to distinguish fluorescent protein tags from these autofluorescent sources based on the narrower emission spectrum of the former. The method is first tested on mitochondria and then applied to examine the ultrastructural variability of secretory granules within insulin-secreting pancreatic beta-cell-derived INS-1E cells.

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Section: Resultsmentioning

confidence: 99%

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

Carter

Mageswaran

Farino

et al. 2018

Journal of Structural Biology

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“…Unfortunately, due to the large size of the complex and its position spanning the cell envelope, flagellar motors lose components when purified. Recently, the advent of electron cryo‐tomography (ECT) (Gan & Jensen, ; Oikonomou & Jensen, ) allowed our group and others to reveal the in situ structures of various bacterial flagellar motors within intact cells at macromolecular (~4 nm) resolution. These studies showed the diversity of flagellar motors in different species adapted to unique external environments (Chen et al , ; Zhao et al , ; Minamino & Imada, ; Terashima et al , ; Zhu et al , ; Kaplan et al , ).…”

Section: Introductionmentioning

confidence: 99%

In situ imaging of the bacterial flagellar motor disassembly and assembly processes

et al. 2019

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The self‐assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio‐temporal synchronization of gene expression with proper protein localization and association of dozens of protein components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with the addition of each new component stabilizing the previous one. However, very little is known about flagellar disassembly. Here, using electron cryo‐tomography and sub‐tomogram averaging of intact Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis cells, we study flagellar motor disassembly and assembly in situ. We first show that motor disassembly results in stable outer membrane‐embedded sub‐complexes. These sub‐complexes consist of the periplasmic embellished P‐ and L‐rings, and bend the membrane inward while it remains apparently sealed. Additionally, we also observe various intermediates of the assembly process including an inner‐membrane sub‐complex consisting of the C‐ring, MS‐ring, and export apparatus. Finally, we show that the L‐ring is responsible for reshaping the outer membrane, a crucial step in the flagellar assembly process.

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“…Electron cryotomography (ECT) can image intact cells in a frozen, fully hydrated state, providing macromolecular resolution (~4-6 nm) details about native cellular structures [13]. Here, we used ECT to visualize the structure of the archaellar basal body in vivo in Thermococcus kodakaraensis cells.…”

Section: Introductionmentioning

confidence: 99%

Morphology of the archaellar motor and associated cytoplasmic cone in Thermococcus kodakaraensis

et al. 2017

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Data information: s, S-layer; m, membrane; c, conical structure; a, archaella; r, ring. Scale bars, 100 nm; scale bar in (A) applies to (1-3); scale bar in (B) applies to (B-E). EMBO reportsª 2017 The Authors EMBO reportsStructure of an archaellar motor and cone Ariane Briegel et al EV1 Figure EV2. Double-cone structure observed in Thermococcus kodakaraensis.A tomographic slice through a side view shows two associated conical structures (c1 and c2), both associated with archaella (a). s, S-layer; m, membrane. Scale bar, 100 nm. A, B Tomographic slices through two cells, highlighting the association between the cone and the archaella. C, D 3D segmentations of the cells in (A) and (B), respectively, with cones in blue and archaella in red, embedded in tomographic slices. E Tomographic slices of individual archaella show the varying orientations of archaella with respect to the cell envelope, as well as apparent connections to the cone.Data information: Scale bars, 100 nm in (A) and (B), 50 nm in (E) (applies to all panels in E); segmentations not to scale. Figure EV4. Individual particles from the subtomogram average show heterogeneity in the L3 density and angle of cone density. EMBO reportsª 2017 The Authors EMBO reports Structure of an archaellar motor and cone Ariane Briegel et al EV3The L3 density appears as either two dots of similar (first two panels) or different intensity (third panel), a single dot (fourth panel), or a dot and an extended line (fifth panel). m, membrane; c, conical structure. Scale bar, 10 nm (applies to all panels).ª 2017 The Authors EMBO reportsAriane Briegel et al Structure of an archaellar motor and cone EMBO reports EV4

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A new view into prokaryotic cell biology from electron cryotomography

Cited by 97 publications

References 187 publications

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

In situ imaging of the bacterial flagellar motor disassembly and assembly processes

Morphology of the archaellar motor and associated cytoplasmic cone in Thermococcus kodakaraensis

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