Biomedical subjects

G E Sosinsky

Publications and source records attributed to G E Sosinsky.

9 recordsLinked to original sources

PubMed1995

Preparation, characterization, and structure of half gap junctional layers split with urea and EGTA.

Gap junctions, collections of membrane channels responsible for intercellular communication, contain two paired hemichannels (also called connexons). We have investigated conditions for splitting the membrane pair using urea. We have developed a protocol which consistently splits the gap junction samples with 60-90% efficiency. Our results indicate that hydrophobic forces are important in holding the two connexons together but that Ca2+ ions are also important in the assembly of the membrane pair. Greater yields and better structural integrity of split junctions were obtained with a starting preparation of gap junctions which had been detergent treated. Image analysis of edge views of single connexon layers reveal an asymmetry in the appearance of the cytoplasmic and extracellular surface. Cryo-electron microscopy and image analysis of split junctions show that the packing and structural detail of membranes containing arrays of single connexons are the same as for intact junctions, and that the urea treatment causes no gross structural changes in the connexon assembly.

Amino Acid Sequence↗

PubMed1992

Mass determination and estimation of subunit stoichiometry of the bacterial hook-basal body flagellar complex of Salmonella typhimurium by scanning transmission electron microscopy.

The basal body, a part of the rotary motor of the bacterial flagellum, is a multiprotein assembly that consists of four rings (denoted M, S, P, and L) and an axial rod (denoted R). From analysis of scanning transmission electron microscopy images of hook-basal body preparations isolated from Salmonella typhimurium, we have determined the masses of the basal body and three of its subcomplexes. The mass of the basal body (i.e., the four rings and rod) is 4400 +/- 490 kDa (mean +/- SD; n = 54). The mass of the LPR subcomplex (i.e., L and P rings and the whole rod) is 2600 +/- 380 kDa (n = 55), that of the L and P rings and the distal part of the rod is 2100 +/- 320 kDa (n = 25), and the mass of the L and P ring subcomplex is 1700 +/- 260 kDa (n = 514). These results, together with the masses of the component proteins, indicate that the rings contain approximately 26 subunits each and that the mass of the rod is consistent with a composition of approximately 6 copies each of three of the rod proteins FlgB, FlgC, and FlgF and approximately 26 copies of FlgG as determined by Jones et al. [Jones, C. J., Macnab, R. M., Okino, H. & Aizawa, S.-I. (1990) J. Mol. Biol. 212, 377-387] using quantitative gel electrophoresis. The results of Jones et al., together with ours, account for all proteins in the basal body to within approximately 5% (or 200 kDa).

Bacterial Proteins↗

PubMed1992

Substructure of the flagellar basal body of Salmonella typhimurium.

The Salmonella typhimurium basal body, a part of the flagellar rotary motor, consists of four rings (denoted M, S, P and L) and a coaxial rod. Using low-dose electron microscopy and image averaging methods on negatively stained and frozen-hydrated preparations, we examined whole basal body complexes and subcomplexes obtained by dissociation in acid. Dissociation occurs in steps, allowing us to obtain images of substructures lacking the M ring, lacking the M and S rings, and lacking the M and S rings and the proximal portion of the rod. We obtained images of the L and P ring subcomplex. The existence of a subcomplex missing only the M ring suggests either that the S and M rings derive from two different proteins, or that the M ring is a labile domain of a single protein, which makes up both rings. At the 25 to 30 A resolution of our averaged images, the L, P and S rings appear cylindrically symmetric. Images of the M ring show variability that may be due to differences in angular orientation of the grid, but equally could be due to structural variations. Three-dimensional reconstructions of these structures from the averaged images reveal the internal structure and spatial organization of these components.

Flagella↗

PubMed1992

Image analysis of gap junction structures.

Isolated gap junction plaques contain hexagonal crystalline arrays of membrane channels called connexons which are a suitable specimen for electron crystallography. Image analysis of gap junction lattices has shown that while there is sufficient lattice order for structural analysis to approximately 25 A, there is enough disorder in both the lattice and the connexon to create a family of related images. This review is focused on how these images can be interpreted in terms of what is known about both the connexon and its constituent protein, connexin.

Animals↗

PubMed1991

Structural studies of tropomyosin by cryoelectron microscopy and x-ray diffraction.

A comparison has been made between cryoelectron microscope images and the x-ray structure of one projection of the Bailey tropomyosin crystal. The computed transforms of the electron micrographs extend to a resolution of approximately 18 A compared with the reflections from x-ray crystallography which extend to 15 A. After correction of the images for lattice distortions and the contrast transfer function, the structure factors were constrained to the plane group (pmg) symmetry of this projection. Amplitude and phase data for five images were compared with the corresponding view from the three-dimensional x-ray diffraction data (Phillips, G.N., Jr., J.P. Fillers, and C. Cohen. 1986. J. Mol. Biol. 192: 111-131). The average R factor between the electron microscopy and x-ray amplitudes was 15%, with an amplitude-weighted mean phase difference of 4.8 degrees. The density maps derived from cryoelectron microscopy contain structural features similar to those from x-ray diffraction: these include the width and run of the filaments and their woven appearance at the crossover regions. Preliminary images obtained from frozen-hydrated tropomyosin/troponin cocrystals suggest that this approach may provide structural details not readily obtainable from x-ray diffraction studies.

Animals↗

PubMed1990

Correlation analysis of gap junction lattice images.

Fourier averages of connexon images computed from low-irradiation electron micrographs of isolated negatively stained gap junction domains exhibited differences in stain distribution and connexon orientation. To analyze these polymorphic structures, correlation averaging methods were applied to images from negatively stained and frozen-hydrated specimens. For the negatively stained specimens, separate averages over two subsets of connexons with differing degrees of stain accumulation in the axial channel were obtained. Two populations of connexons with opposite skew orientations were distinguishable within a single junctional domain of a frozen-hydrated specimen. Correlation maps calculated using the left- and right-skewed references showed that the selected connexons tend to locally cluster. Using correlation methods to analyze packing disorder in a typical connexon lattice, we estimated the root-mean-square variation in the nearest neighbor pair separation to be approximately 11% of the lattice constant. Displacements of the connexons relative to each other increased with increasing pair separation in the lattice, rather like a liquid, although long-range orientation order was conserved as in a crystal. These results support the hypothesis that the hexagonal ordering of the connexons results from short-range repulsive forces.

Biophysical Phenomena↗

PubMed1989

Image reconstruction of the flagellar basal body of Caulobacter crescentus.

The bacterium Caulobacter crescentus has a single polar flagellum, which is present for only a portion of its cell cycle. The flagellum is ejected from the swarmer cell and then synthesized de novo later in the cell cycle. The flagellum is composed of a transmembrane basal body, a hook and a filament. Single-particle averaging and image reconstruction methods were applied to the electron micrographs of negatively stained basal bodies from C. crescentus. These basal bodies have five rings threaded on a rod. The L and P rings are connected by a bridge of material at their outer radii. The E ring is a thin, flat disk. The S ring has a triangular cross section, the sides of the triangle abutting the E ring, the rod and the M ring. The M ring, which is at the inner membrane of the cell, has a different structure depending on the method of preparation. With one method, the M ring makes a snug contact with the S ring and is often capped by an axial button, a new component apparently distinct from the M ring. With the other method, the M ring is similar to that of S. typhimurium; that is, it contacts the S ring only at an outer radius and lacks the button. Averages of the rod-hook-filament subassembly ejected by swarmer cells reveal that the rod consists of two parts with the E ring marking the approximate position of the break. The structures of basal bodies from two mutants defective in the hook assembly were found to be indistinguishable from wild-type basal bodies, suggesting that the assembly of the basal body is independent of the hook or filament assembly.

Bacteria↗

PubMed1988

Gap junction structures. VIII. Membrane cross-sections.

Profiles of negatively stained gap junctions have been measured by grid sectioning. After normal levels of electron irradiation, the membrane thickness shrinks to about half that of unirradiated controls, but no shrinkage occurs in the hexagonal lattice plane. Even under low irradiation conditions, there is significant thinning of the membranes. Edge views, in which rows of connexons are aligned parallel to the beam, were obtained from grid sections, folds in normal negatively stained specimens, and sections of a positively stained specimen. Averaging these micrographs with the translational and mirror symmetry of the projected lattice image displays conserved and variable features in the stain distribution of different specimens. Variations in the relative amount of negative stain in the gap at the surfaces and in the channel are uncorrelated with the irradiation but appear to depend on the local staining conditions and the integrity of the connexons. The dimensions measured from previously unirradiated grid sections, folds, and positively stained sections are in accord with x-ray diffraction measurements. Radiation-induced shrinkage can be accounted for by mass loss principally from the membrane bilayer. Disordering of the surface structure appears to be correlated with the radiation sensitivity of the bilayer; in contrast, the gap structure is well preserved under a variety of conditions.

Animals↗

PubMed1985

Gap junction structures. VII. Analysis of connexon images obtained with cationic and anionic negative stains.

Micrographs of isolated gap junction specimens, negatively stained with one molybdate, three tungstate and three uranyl stains, were recorded at low and high irradiation. Fourier-averaged images of the negatively stained gap junctions have been self-consistently scaled to identify conserved and variable features. Intrinsic features in the hexagonally averaged images have been distinguished from residual noise by statistical comparisons among similarly prepared specimens. The cationic uranyl stains can penetrate the axial connexon channel, whereas the anionic stains are largely excluded; these observations indicate that the channel is negatively charged. Variability in the extent of the axial stain penetration, and enhancement of this staining by radiation damage and heating may be accounted for by a leaky, labile channel gate. The peripheral stain concentrations marking the perimeter of the skewed, six-lobed connexon image and the stain-excluding region at the 3-fold axis of the lattice, which are seen only under conditions of low irradiation with both anionic and cationic stains, are identified as intrinsic features of the isolated gap junction structure. The stain concentrations located approximately 30 A from the connexon center appear to be symmetrically related on opposite sides of the junction by non-crystallographic 2-fold axes oriented approximately 8 degrees to the lattice axes at the plane of the gap. The radiation-sensitive hexagonal features seen in the negatively stained images may correspond to substructure on the cytoplasmic surfaces of the paired gap junction membranes.

Animals↗