Negative Staining and Cryoelectron Microscopy

Harris, J. Robin

doi:10.1017/s1551929500060016

Cited by 91 publications

(112 citation statements)

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“…To confirm the findings we conducted parallel experiments to label the PilQ protein using immunonegative staining (Chirica et al, 2002). This technique allows labelling of surface-localized epitopes only (Beveridge et al, 1994;Harris, 1997). With the K010 antibody, these experiments failed to visualize the PilQ protein on the meningococcal surface (data not shown).…”

Section: Discussionmentioning

confidence: 74%

Topology of the outer-membrane secretin PilQ from Neisseria meningitidis

et al. 2006

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Neisseria meningitidis is the causative agent of epidemic meningococcal meningitis and septicaemia. Type IV pili are surface organelles that mediate a variety of functions, including adhesion, twitching motility, and competence for DNA binding and uptake in transformation. The secretin PilQ is required for type IV pilus expression at the cell surface, and forms a dodecameric cage-like macromolecular complex in the meningococcal outer membrane. PilQ-null mutants are devoid of surface pili, and prevailing evidence suggests that the PilQ complex facilitates extrusion and retraction of type IV pili across the outer membrane. Defining the orientation of the meningococcal PilQ complex in the membrane is a prerequisite for understanding the structure-function relationships of this important protein in pilus biology. In order to begin to define the topology of the PilQ complex in the outer membrane, polyhistidine insertions in N-and C-terminal regions of PilQ were constructed, and their subcellular locations examined. Notably, the insertion epitopes at residues 205 and 678 were located within the periplasm, whereas residue 656 was exposed at the outer surface of the outer membrane. Using electron microscopy with Ni-NTA gold labelling, it was demonstrated that the insertion at residue 205 within the N-terminus mapped to a site on the arm-like features of the 3D structure of the PilQ multimer. Interestingly, mutation of the same region gave rise to an increase in vancomycin permeability through the PilQ complex. The results yield novel information on the PilQ N-terminal location and function in the periplasm, and reveal a complex organization of the membrane-spanning secretin in vivo.

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Section: Discussionmentioning

confidence: 74%

Topology of the outer-membrane secretin PilQ from Neisseria meningitidis

et al. 2006

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“…2G) showed spectacular contrast enhancement. This technique, which is still in its early stages, combines the benefits of vitrification with the increased contrast characterizing negatively stained images (17). However, it was not possible to use either cryo-TEM or cryo-TEM with uranyl acetate or phosphotungstate staining to visualize the RPR120535͞DNA complexes from zones A and B, because when the charge ratio was negative, the number of complexes present was limited, and when the charge ratio was neutral, large RPR120535͞DNA complexes increased the thickness of the film of the vitreous solution, which made optimal imaging of the structure more difficult.…”

Section: Resultsmentioning

confidence: 99%

Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer

Pitard¹,

Aguerre²,

Airiau³

et al. 1997

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

145

125

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Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine͞plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine͞plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine͞plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 Å, irrespective of the lipid͞DNA ratio. The most active lipopolyamine͞DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains.

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“…For negative staining analysis, 4 μl of the sample was placed on copper grids covered with formvar and carbon and counterstained with 2% uranyl acetate, using the droplet technique [26]. Specimens were examined in a Zeus 902 transmission electron microscope operated at an acceleration voltage of 80 kV.…”

Section: Structural Characterization Of Aβ Aggregatesmentioning

confidence: 99%

Oligomeric and fibrillar species of β-amyloid (Aβ42) both impair mitochondrial function in P301L tau transgenic mice

et al. 2008

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We recently provided evidence for a mitochondrial dysfunction in P301L tau transgenic mice, a strain modeling the tau pathology of Alzheimer's disease (AD) and frontotemporal dementia (FTD). In addition to tau aggregates, the AD brain is further characterized by Aβ peptide-containing plaques. When we addressed the role of Aβ, this indicated a synergistic action of tau and Aβ pathology on the mitochondria. In the present study, we compared the toxicity of different Aβ42 conformations in light of recent studies suggesting that oligomeric rather than fibrillar Aβ might be the actual toxic species. Interestingly, both oligomeric and fibrillar, but not disaggregated (mainly monomeric) Aβ42 caused a decreased mitochondrial membrane potential in cortical brain cells obtained from FTD P301L tau transgenic mice. This was not observed with cerebellar preparations indicating selective vulnerability of cortical neurons. Furthermore, we found reductions in state 3 respiration, the respiratory control ratio, and uncoupled respiration when incubating P301L tau mitochondria either with oligomeric or fibrillar preparations of Aβ42. Finally, we found that aging specifically increased the sensitivity of mitochondria to oligomeric Aβ42 damage indicating that oligomeric and fibrillar Aβ42 are both toxic, but exert different degrees of toxicity.

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Negative Staining and Cryoelectron Microscopy

Cited by 91 publications

References 0 publications

Topology of the outer-membrane secretin PilQ from Neisseria meningitidis

Topology of the outer-membrane secretin PilQ from Neisseria meningitidis

Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer

Oligomeric and fibrillar species of β-amyloid (Aβ42) both impair mitochondrial function in P301L tau transgenic mice

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