Biomedical subjects
Publications and source records attributed to L A Staehelin.
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The photosynthetic green bacterium Chlorobium limicola 6230 has been examined by freeze-fracture electron microscopy to investigate the size, form, distribution and supramolecular architecture of its chlorosomes (chlorobium vesicles) as well as the chlorosome attachment sites on the cytoplasmic membrane. The oblong chlorosomes that underlie the cytoplasmic membrane show a considerable variation in size from about 40 X 70 nm to 100 X 260 nm and exhibit no particular orientation. The chlorosome core, which appears to be hydrophobic in nature, contains between 10 and 30 rod-shaped elements (approx. 10 nm in diameter) surrounded by an unetchable matrix. The rod elements are closely packed and extend the full length of the chlorosome. Separating the chlorosome core from the cytoplasm is a approx. 3 nm thick lipid-like envelope layer, which exhibits no substructure. A 5-6 nm thick, crystalline baseplate connects the chlorosome to the cytoplasmic membrane. The ridges of the baseplate lattice make an angle of between 40 degrees and 60 degrees with the longitudinal axis of the chlorosome and have a repeating distance of approx. 6 nm. In addition, each ridge exhibits a granular substructure with a periodicity of approx. 3.3 nm. The cytoplasmic membrane regions adjacent to the baseplates are enriched in large (greater than 9 nm) intramembrane particles, most of which belong to approx. 10 nm and approx. 12.5 nm particle size categories. Each chlorosome attachment site contains between 20 and 30 very large (greater than 12.0 nm diameter) intramembrane particles. The following interpretive model of a chlorosome is discussed in terms of biophysical, biochemical and structural information reported by others: it is proposed that the bacteriochlorophyll c (BChl c; chlorobium chlorophyll) is located in the rod elements of the core and that it is complexed with specific proteins. The cytoplasm-associated envelope layer is depicted as consisting of a monolayer of galactosyl diacylglycerol molecules. BChl alpha-protein complexes in a planar lattice configuration most likely make up the crystalline baseplate. The greater than 12-nm particles in the chlorosome attachment sites of the cytoplasmic membrane, finally, may correspond to complexes containing a reaction center and non-crystalline light-harvesting BChl alpha. The crystalline nature of the baseplate is consistent with the notion that it serves two functions: besides transferring excitation energy to the reaction centers it could also function as a distributor of this energy amongst the reaction centers.
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Freeze-fracture replicas of the photosynthetic prokaryote Prochloron sp., collected at Coconut Island, Hawaii, show that the thylakoids are differentiated into stacked and unstacked regions much like the thylakoids of green algae and higher plants. On the exoplasmic (E) fracture face, the particle density is greater in stacked regions ( approximately 3100 particles/mum(2)) than in unstacked regions ( approximately 925 particles/mum(2)). On the complementary protoplasmic (P) fracture face, the particle density is lower in stacked regions ( approximately 2265 particles/mum(2) than in unstacked regions ( approximately 4980 particles/mum(2)). The histogram of the diameters of E face particles in unstacked regions shows a single broad peak centered at 80 A. In stacked regions, four distinct peaks, at 75, 105, 130, and 160 A, are observed. These size classes are virtually identical to those found on E faces of thylakoids of the green alga Chlamydomonas and of greening pea chloroplasts. In the latter systems, the different size classes of E face particles are believed to represent photosystem II units surrounded by variable amounts of chlorophyll a/b light-harvesting complex. We propose that the same interpretation applies to the thylakoids of Prochloron, which contain a similar chlorophyll a/b complex. Our results add to the evidence supporting the view of the chlorophyll a/b complex as both a light-harvesting complex and a membrane adhesion factor. The similarity of the architecture of the thylakoids of Prochloron to that of green algal and plant chloroplasts also provides additional evidence of an evolutionary relationship between Prochloron and the chloroplasts of green plants.
A highly purified chlorophyll a/b light-harvesting complex (chl a/b LHC; chl a/b ratio 1.2) was obtained from Triton-solubilized chloroplast membranes of pea and barley according to the method of Burke et al. (1978, Arch. Biochem. Biophys. 187: 252--263). Gel electrophoresis of the cation-precipitated chl a/b LHC from peas reveals the presence of four polypeptides in the 23- to 28-kdalton size range. Three of these peptides appear to be identical to those derived from re-electrophoresed CPII and CPII* bands. In freeze-fracture replicas, the cation-precipitated chl a/b LHC appears as a semicrystalline aggregate of membranous sheets containing closely spaced granules. Upon removal of the cations by dialysis, the aggregates break up into their constituent membranous sheets without changing their granular substructure. These membranous sheets can be resolubilized in 1.5% Triton X-100, and the chl a/b LHC particles then reconstituted into soybean lecithin liposomes. Freeze-fracture micrographs of the reconstituted chl a/b LHC vesicles suspended in a low salt medium reveal randomly dispersed approximately 80-A particles on both concave and convex fracture faces as well as some crystalline particle arrays, presumably resulting from incompletely solubilized fragments of the membranous sheets. Based on the approximately 80-A diameter of the particles, and on the assumption that one freeze-fracture particle represents the structural unit of one chl a/b LHC aggregate, a theoretical mol wt of approximately 200 kdalton has been calculated for the chl a/b LHC. Deep-etching and negative-staining techniques reveal that the chl a/b LHC particles are also exposed on the surface of the bilayer membranes. Addition of greater than or equal to 2 mM MgCl2 or greater than or equal to 60 mM NaCl to the reconstituted vesicles leads to their aggregation and, with divalent cations, to the formation of extensive membrane stacks. At the same time, the chl a/b LHC particles become clustered into the adhering membrane regions. Under these conditions the particles in adjacent membranes usually become precisely aligned. Evidence is presented to aupport the hypothesis that adhesion between the chl a/b LHC particles is mediated by hydrophobic interactions, and that the cations are needed to neutralize surface charges on the particles.
Highly ordered arrays of intramembrane particles are observed in freeze-fractured plasma membranes of the green alga Micrasterias denticulata during the synthesis of the secondary cell wall. The observable architecture of the complex consists primarily of a precise hexagonal array of from 3 to 175 rosettes, consisting of 6 particles each, which fracture with the P-face. The complexes are observed at the ends of impressions of cellulose fibrils. The distance between rows of rosettes is equal to the center-to-center distance between parallel cellulose fibrils of the secondary wall. Correlation of the structure of the complex with the pattern of deposition indicates that the size of a given fibril is proportional to the number of rosettes engaged in its formation. Vesicles containing hexagonal arrays of rosettes are found in the cytoplasm and can be observed in the process of fusing with the plasma membrane, suggesting that the complexes are first assembled in the cytoplasm and then incorporated into the plasma membrane, where they become active in fibril formation. Single rosettes appear to be responsible for the synthesis of microfibrils during primary wall growth. Similar rosettes have now been detected in a green alga, in fern protonemata, and in higher plant cells. This structure, therefore, probably represents a significant component of the cellulose synthesizing mechanism in a large variety of plant cells.
Freeze-fracture micrographs of cells of the green alga Micrasterias denticulata stabilized by ultrarapid freezing reveal imprints of polysomes on the rough endoplasmic reticulum membranes. The imprints appear as broad, spiral ridges on the P faces and as corresponding wide grooves on the E faces of the membranes. Distinct 110-A particles with a spacing of 270 +/- 45 A are associated with the P-face ridges. Where imprints of individual ribosomes can be discerned, it is seen that there is a 1:1 relationship between the ribosomes and the 110-A particles, and that the 110-A particles are located in a peripheral position with respect to the polysome spirals. We propose that the 110-A particles could be structural equivalents of ribosome-binding sites, consisting of a molecule each of ribophorins I and II and a nascent polypeptide chain. These observations suggest that the spiral form of polysomes could result from the forces generated by the extrusion of the growing polypeptide chains to one side of the polysome.
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The thylakoids of vegetative cells of the filamentous cyanobacterium, Anabaena cylindrica, are capable of oxygen-evolving photosynthesis and contain both Photosystems I and II (PSI and PSII). The heterocysts, cells specialized for nitrogen fixation, do not produce oxygen and lack Photosystem II activity, the major accessory pigments, and perhaps the chlorophyll a associated with PSII. Freeze-fracture replicas of vegetative cells and of heterocysts reveal differences in the structure of the thylakoids. A histogram of particle sizes on the exoplasmic fracture face (E-face, EF) of vegetative cell thylakoids has two major peaks, at 75 and 100 A. The corresponding histogram for heterocyst thylakoids lacks the 100 A size class, but has a very large peak at about 55 A with a shoulder at 75 A. Histograms of protoplasmic fracture face (P-face, PF) particle diameters show single broad peaks, the mean diameter being 71 A for vegetative cells and 64 A for heterocysts. The thylakoids of both cell types have about 5600 particles/micrometers2 on the P-face. On the E-face, the density drops from 939 particles/micrometers2 on vegetative cell thylakoids to 715 particles/micrometers2 on heterocyst thylakoids. The data suggest that the 100 A E-face particle of vegetative cell thylakoids is a PSII complex. The 55 A EF particle of heterocysts may be part of the nitrogenase complex or a remnant of the PSII complex. The role of the 75 A EF particle is unknown. Other functions localized on cyanobacterial thylakoids, such as respiration and hydrogenase activity, must be considered when interpreting the structure of these complex thylakoids.
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Alterations in membrane structure as a result of lipid phase transitions have been studied in Anacystis nidulans, a blue-green alga. Cells grown at 38 degrees C were subjected to temperature transitions of 38 degrees C leads to 21 degrees C and 38 degrees C leads to 10 degrees C, previously shown to produce substantial changes in photosynthetic activities, and examined by freeze-fracture electron microscopy. As a result of these treatments, large particle-free regions appeared on the fracture faces of both the plasma and thylakoid membranes. Particle density measurements suggest that the displacement of the integral membrane protein complexes occurs in both lateral and vertical directions. Returning the cells to 38 degrees C resulted in the restoration of normal membrane morphology, indicating that the proteins were not lost from the membrane. Such displacement of the integral membrane protein complexes could contribute significantly to the temperature-dependent alterations in the functional activity of membrane-bound enzymatic complexes.
The apical cytoplasm of epithelial cells of the small and large intestines has been examined by freeze-etch techniques as well as conventional and high voltage electron microscopy of sectioned material to gain a better understanding of the fine structural organization of the terminal web region. In the small intestine the terminal web exhibits a distinct stratification caused by the association of different sets of filaments with the three members of the junctional complex. Individual filaments of this network are closely associated with the sealing elements of the tight junctions, the surface of the core microfilament bundles, and the intermicrovillar plasma membrane. This region of the terminal web is the apical zone. The adherens zone appears as a band of interwoven filaments of two different diameters extending across the cytoplasm at the level of the intermediate junction. Within this region of the terminal web, individual 60-70 A actin-like filaments separate from the bundles of core microfilaments to interact with one another and with filaments of similar diameter from the zonula adherens. 100 A tonofilaments also contribute to the adherens zone, presumably stabilizing the orientation of the actin-like filaments. The basal zone which underlies the adherens zone consists of closely interwoven bundles of tonofilaments that are anchored to and interconnect the spot desmosomes. Within the large intestine the cytoplasmic microfilaments form a looser and less clearly stratified network which nevertheless retains the same basic organization found in the small intestine. Transmembrane linkers appear to originate within the cytoplasmic plaques of the spot desmosomes, pass through the plasma membranes, and meet in a staggered configuration in the intercellular space; these linkers may thus mediate the actual mechanical coupling between the cytoskeletal networks of tonofilament bundles of adjacent cells. This integrated system of cytoplasmic filaments and intercellular junctions endows the apical cytoplasm with both the flexibility and the stability necessary for the normal functioning of the epithelium.
We have studied fluid secretion by the contractile vacuole apparatuss of the trypanosomatid flagellate Leptomonas collosoma with thin sections and freeze-fracture replicas of cells stabilized by ultrarapid freezing without prior fixation or cryoprotection. The ultrarapid freezing has revealed membrane specializations related to fluid segregation and transport as well as membrane rearrangements which may accompany water expulsion at systole. This osmoregulatory apparatu consists of the spongiome, the contractile vacuole, and the fluid discharge site. The coated tubules of the spongiome converge on the contractile vacuole from all directions. These 60- to 70-nm tubules contain characteristic double rows of 11-nm intramembrane particles in a helical configuration which fracture predominantly with the E face. Short double rows of similar particles are also frequently found on both faces of the contractile vacuole itself, in addition to many smaller particles on the P face. The spongiome tubules fuse with the vacuole during the filling stage of each cycle and then detach before secretion. The contractile vacuole membrane is permanently attached to the plasma membrane of the flagellar pocket by a dense adhesion plaque. In some ultrarapidly frozen cells, 20- to 40-nm perforations can be visualized within the plaque and the adjacent membranes during the presumptive time of discharge. The formation of the plaque perforations and the membrane channels occurs without fusion of the vacuole and the plasma membrane and does not require extracellular calcium. On the basis of our results, we have developed a model for water secretion which suggests that the adhesion plaque may induce pore formation in the adjoining lipid bilayers, thereby allowing bulk expulsion of the fluid.
Sheep red blood cell (SRBC) ghosts were incubated with preparations of anti-SRBC IgG, antigen-binding fragments of IgG (Fab') or IgG coupled to horseradish peroxidase (HRPO). Frozen samples of the labelled ghosts were deep-etched and replicated with platinum-carbon to visualize their surface features in the transmission electron microscope. The surfaces of control ghosts contain a very low number of 'background' particles (42 +/- 8 particles/micron 2) that vary in size from 4.5 to 25 nm. After labelling with whole IgG the density of surface particles (average diameter 12.3 nm) increases dramatically to 480 +/- 54 per micron 2. Fab'-labelled ghosts exhibited both significantly fewer (87 +/- 14 particles/micron 2) and smaller (average diameter 9.8 nm) surface particles. Ghosts labelled with IgG-HRPO conjugates possessed 590 +/- 45 particles/micron 2 with an average diameter of 15.3 nm. When these ghosts were incubated with diaminobenzidine and hydrogen peroxide the average size but not the density of the particles increased. Based on these and other observations we conclude that an organic surface marker for freeze-etched membranes has to have a diameter of greater than 15 nm if it is to be consistently seen over extended areas and against the background granularity of the surface of a red blood cell ghost. Somewhat better resolution may be expected if markers consisting of inorganic crystals with a distinct shape and coupled to Fab' fragments can be made.
Sheep red blood cell (SRBC) ghosts were incubated with preparations of anti-SRBC IgG, antigen-binding fragments (Fab') or IgG coupled to horseradish peroxidase (HRPO). Frozen samples of the labeled ghosts were deep-etched and replicated with platinum-carbon to visualize their surface features in the transmission electron microscope. An analysis of both size and density of surface "particles" observed on labeled ghosts was performed to evaluate which markers could be practically used to label these membranes. It was concluded that in this system a marker must exhibit a diameter of greater than 150 A (the apparent size of IgG-HRPO conjugates) to be consistently seen over extensive surface areas and to be distinguished from the background granularity of the SRBC ghost surface. This rules out the use of Fab' or IgG as markers in this system.
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