The Structure and Subunit Organization of the Bacterial Flagellar Motor by Scanning Transmission Electron Microscopy and Cryoelectron Microscopy

1990 ◽  
Vol 48 (1) ◽  
pp. 278-279
Author(s):  
Gina E. Sosinsky ◽  
Noreen R. Francis ◽  
Charles D. DeRosier ◽  
David J. DeRosier ◽  
James Hainfeld ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Basal Body ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Cryoelectron Microscopy ◽  
Data Set ◽  
Bacterial Flagellum ◽  
The Individual ◽  
The Masses ◽  
Subunit Organization

The bacterial flagellum is unique in having a rotary motor. In Salmonella typhimurium, the basal body, a component of the motor, consists of four rings (denoted M, S, L, and P) threaded on a coaxial rod. The M, L, and P rings are each composed of a different protein: FliF=61 kD, FlgH=22 kD, and FlgI=36 kD, respectively. The rod contains at least four different proteins: FlgB=15 kD, FlgC=14 kD, FlgF=26 kD, and FlgG=28 kD. Using quantitative gel analysis, Jones et al. estimated that there are about 26 copies of FlgG, FlgH, Flgl and FliF, and 6 copies of FlgB, FlgC and FlgF per basal body. The total mass of these 7 proteins per basal body is ∽4200 kD. There appear to be additional proteins in the basal body, but their locations and amounts are not known. Our aim is to produce subcomplexes of the basal body and determine their structures and masses using electron microscopy. This approach is complementary to that of Jones et al. and can reveal the presence and amounts of as yet unidentified components. We find, in pH3- or pH4-treated preparations of basal bodies, four subcomplexes of the hook basal body complex (HBB): the HLPRS (hook, L and P rings on the distal rod, proximal rod, S ring); the HLPR (lacks the M and S rings), the HLP (lacks the M, S, and proximal rod); and the LP complex (Figs. 1 and 2). We have been able to visualize the three-dimensional structure and the subunit organization using the combined techniques of cryoelectron microscopy and image analysis. These studies suggest that the S ring is a separate component from the rod or M ring and that the rod consists of two sections. Because the different sub-complexes are distinguishable in a field of particles, we measured the molecular masses of the individual subcomplexes using the Brookhaven STEM even though these preparations are not homogeneous (Fig. 3). All the structures analyzed so far had hooks attached. We measured the length and mass/length from STEM images and then subtracted the mass of the hook. Preliminary results show that the molecular mass of the hookless basal body is 4400−500 kD (n=165), that of the LP-rod (proximal and distal) is 3500±300 kD (n=52), and that of the LP-distal rod is 2300±450 kD (n=76) (Fig. 4). The difference between these three molecular weights gives estimates of the mass of the M and S rings (4400 - 3500 = 900 kD) and proximal rod, 3500 − 2300 = 1200 kD. The mass of the M and S rings may be underestimated due to the undetected presence of HLPRS subcomplexes in the basal body data set. We are presently measuring and re-evaluating masses for the subcomplexes in order to get more accurate estimates of the masses and numbers of subunits.

The three-dimensional structure of frozen-hydrated left- and right-handed forms of bacterial flagellar filaments from an identical serotype

1986 ◽  
Vol 44 ◽  
pp. 298-299
Author(s):  
S. Trachtenberg ◽  
D. J. DeRosier
Keyword(s):  
Ionic Strength ◽  
Basal Body ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Protein Species ◽  
Liquid Environment ◽  
Bacterial Flagellum ◽  
Left And Right ◽  
Rotary Motor ◽  
Helical Parameters

The bacterial cell is propelled through the liquid environment by means of one or more rotating flagella. The bacterial flagellum is composed of a basal body (rotary motor), hook (universal coupler), and filament (propellor). The filament is a rigid helical assembly of only one protein species — flagellin. The filament can adopt different morphologies and change, reversibly, its helical parameters (pitch and hand) as a function of mechanical stress and chemical changes (pH, ionic strength) in the environment.

Comparison of IVEM and quick-freeze, deep-etch, rotary shadow images of the cell cytoplasm

1993 ◽  
Vol 51 ◽  
pp. 66-67
Author(s):  
Carolyn A. Larabell
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Dimensional Structure ◽  
Whole Mount ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Excellent Method ◽  
Fine Structures ◽  
The Individual

The techniques of freeze-fracture and quick-freeze, deep-etch, rotary-shadow electron microscopy have provided information about cellular structures that cannot be obtained from thin section electron microscopy. Freeze-fracture replicas provide extensive views of membranes and the arrangement of integral membrane proteins, but filamentous structures such as the cytoskeleton and extracellular matrix are not visualized because they are embedded in ice. In order to visualize such fine structures, it is necessary to deeply etch (or freeze-dry) cells that have been rapidly frozen, thus revealing about 0.1 to 0.3 μm of three dimensional structure. Replicas of cells prepared in this fashion provide excellent high magnification views of the individual filaments and their interactions with the plasma membrane as well as with membrane-bounded organelles. Another excellent method for visualization of cytoskeletal structures is whole mount electron microscopy. In this paper, I describe views of the egg cytoskeleton obtained from visualization of the isolated egg cortex prepared as a whole mount at 400 kV and compare these results with those obtained from replicas of cells that have been rapidly frozen men fractured and deeply etched.

Electron Microscopy of Cytoplasmic Contractile Proteins

1978 ◽  
Vol 36 (3) ◽  
pp. 606-614 ◽  
Author(s):  
T.D. Pollard ◽  
P. Maupin
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Uranyl Acetate ◽  
Contractile Proteins ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
Cytoplasmic Matrix ◽  
Computer Image Processing ◽  
Nonmuscle Cells

In this paper we review some of the contributions that electron microscopy has made to the analysis of actin and myosin from nonmuscle cells. We place particular emphasis upon the limitations of the ultrastructural techniques used to study these cytoplasmic contractile proteins, because it is not widely recognized how difficult it is to preserve these elements of the cytoplasmic matrix for electron microscopy. The structure of actin filaments is well preserved for electron microscope observation by negative staining with uranyl acetate (Figure 1). In fact, to a resolution of about 3nm the three-dimensional structure of actin filaments determined by computer image processing of electron micrographs of negatively stained specimens (Moore et al., 1970) is indistinguishable from the structure revealed by X-ray diffraction of living muscle.

High-voltage electron microscopy of maxillary gland of spirochete-infected brine shrimp: lesion of efferent tubule

1988 ◽  
Vol 46 ◽  
pp. 362-363
Author(s):  
G. E. Tyson ◽  
M. J. Song
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Brine Shrimp ◽  
Natural Populations ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Infected Adult

Natural populations of the brine shrimp, Artemia, may possess spirochete- infected animals in low numbers. The ultrastructure of Artemia's spirochete has been described by conventional transmission electron microscopy. In infected shrimp, spirochetal cells were abundant in the blood and also occurred intra- and extracellularly in the three organs examined, i.e. the maxillary gland (segmental excretory organ), the integument, and certain muscles The efferent-tubule region of the maxillary gland possessed a distinctive lesion comprised of a group of spirochetes, together with numerous small vesicles, situated in a cave-like indentation of the base of the tubule epithelium. in some instances the basal lamina at a lesion site was clearly discontinuous. High-voltage electron microscopy has now been used to study lesions of the efferent tubule, with the aim of understanding better their three-dimensional structure.Tissue from one maxillary gland of an infected, adult, female brine shrimp was used for HVEM study.

Confocal laser microscopy of impregnated central nervous system tissue

1988 ◽  
Vol 46 ◽  
pp. 94-95
Author(s):  
J.N. Turner ◽  
M. Siemens ◽  
D. Szarowski ◽  
D.N. Collins
Keyword(s):  
Electron Microscopy ◽  
Central Nervous System ◽  
Nervous System ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Confocal Laser ◽  
High Contrast ◽  
Central Nervous System Tissue ◽  
Tissue Samples ◽  
Reflected Light

A classic preparation of central nervous system tissue (CNS) is the Golgi procedure popularized by Cajal. The method is partially specific as only a few cells are impregnated with silver chromate usualy after osmium post fixation. Samples are observable by light (LM) or electron microscopy (EM). However, the impregnation is often so dense that structures are masked in EM, and the osmium background may be undesirable in LM. Gold toning is used for a subtle but high contrast EM preparation, and osmium can be omitted for LM. We are investigating these preparations as part of a study to develop correlative LM and EM (particularly HVEM) methodologies in neurobiology. Confocal light microscopy is particularly useful as the impregnated cells have extensive three-dimensional structure in tissue samples from one to several hundred micrometers thick. Boyde has observed similar preparations in the tandem scanning reflected light microscope (TSRLM).

scholarly journals Capturing Enveloped Viruses on Affinity Grids for Downstream Cryo-Electron Microscopy Applications

2013 ◽  
Vol 20 (1) ◽  
pp. 164-174 ◽  
Author(s):  
Gabriella Kiss ◽  
Xuemin Chen ◽  
Melinda A. Brindley ◽  
Patricia Campbell ◽  
Claudio L. Afonso ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Measles Virus ◽  
Influenza A ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Nitrilotriacetic Acid ◽  
Dimensional Structure ◽  
Enveloped Viruses ◽  
Cryo Electron Microscopy ◽  
Human Immunodeficiency Virus Virus

AbstractElectron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.

scholarly journals Site Specific Three-dimensional Structural Analysis in Tissues and Cells Using Automated DualBeam Slice &View

2007 ◽  
Vol 15 (2) ◽  
pp. 26-31 ◽  
Author(s):  
Ben Lich
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Surface Region ◽  
Dimensional Structure ◽  
Near Surface ◽  
Imaging Capability ◽  
Fluorescent Markers ◽  
Confocal And Electron Microscopy

DualBeam instruments that combine the imaging capability of scanning electron microscopy (SEM) with the cutting and deposition capability of a focused ion beam (FIB) provide biologists with a powerful tool for investigating three-dimensional structure with nanoscale (1 nm-100 nm) resolution. Ever since Van Leeuwenhoek used the first microscope to describe bacteria more than 300 years ago, microscopy has played a central role in scientists' efforts to understand biological systems. Light microscopy is generally limited to a useful resolution of about a micrometer. More recently the use of confocal and electron microscopy has enabled investigations at higher resolution. Used with fluorescent markers, confocal microscopy can detect and localize molecular scale features, but its imaging resolution is still limited. SEM is capable of nanometer resolution, but is limited to the near surface region of the sample.

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