TY - JOUR
T1 - Cryo-electron tomography of plunge-frozen whole bacteria and vitreous sections to analyze the recently described bacterial cytoplasmic structure, the Stack
AU - Delgado, Lidia
AU - Martinez, Gema
AU - Lopez-Iglesias, Carmen
AU - Mercade, Elena
PY - 2015/3
Y1 - 2015/3
N2 - Cryo-electron tomography (CET) of plunge-frozen whole bacteria and vitreous sections (CETOVIS) were used to revise and expand the structural knowledge of the "Stack", a recently described cytoplasmic structure in the Antarctic bacterium Pseudomonas deceptionensis M1(T). The advantages of both techniques can be complementarily combined to obtain more reliable insights into cells and their components with three-dimensional imaging at different resolutions. Cryo-electron microscopy (Cryo-EM) and CET of frozen-hydrated P. deceptionensis M1(T) cells confirmed that Stacks are found at different locations within the cell cytoplasm, in variable number, separately or grouped together, very close to the plasma membrane (PM) and oriented at different angles (from 35? to 90?) to the PM, thus establishing that they were not artifacts of the previous sample preparation methods. CET of plunge-frozen whole bacteria and vitreous sections verified that each Stack consisted of a pile of oval disc-like subunits, each disc being surrounded by a lipid bilayer membrane and separated from each other by a constant distance with a mean value of 5.2?1.3nm. FM4-64 staining and confocal microscopy corroborated the lipid nature of the membrane of the Stacked discs. Stacks did not appear to be invaginations of the PM because no continuity between both membranes was visible when whole bacteria were analyzed. We are still far from deciphering the function of these new structures, but a first experimental attempt links the Stacks with a given phase of the cell replication process. The Authors.
AB - Cryo-electron tomography (CET) of plunge-frozen whole bacteria and vitreous sections (CETOVIS) were used to revise and expand the structural knowledge of the "Stack", a recently described cytoplasmic structure in the Antarctic bacterium Pseudomonas deceptionensis M1(T). The advantages of both techniques can be complementarily combined to obtain more reliable insights into cells and their components with three-dimensional imaging at different resolutions. Cryo-electron microscopy (Cryo-EM) and CET of frozen-hydrated P. deceptionensis M1(T) cells confirmed that Stacks are found at different locations within the cell cytoplasm, in variable number, separately or grouped together, very close to the plasma membrane (PM) and oriented at different angles (from 35? to 90?) to the PM, thus establishing that they were not artifacts of the previous sample preparation methods. CET of plunge-frozen whole bacteria and vitreous sections verified that each Stack consisted of a pile of oval disc-like subunits, each disc being surrounded by a lipid bilayer membrane and separated from each other by a constant distance with a mean value of 5.2?1.3nm. FM4-64 staining and confocal microscopy corroborated the lipid nature of the membrane of the Stacked discs. Stacks did not appear to be invaginations of the PM because no continuity between both membranes was visible when whole bacteria were analyzed. We are still far from deciphering the function of these new structures, but a first experimental attempt links the Stacks with a given phase of the cell replication process. The Authors.
KW - Cryo-electron microscopy
KW - Cryo-electron tomography
KW - Vitreous sections
KW - Plunge freezing
KW - High-pressure freezing
KW - Stacks
U2 - 10.1016/j.jsb.2015.01.008
DO - 10.1016/j.jsb.2015.01.008
M3 - Article
C2 - 25617813
SN - 1047-8477
VL - 189
SP - 220
EP - 229
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 3
ER -