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J E Heuser

Publications and source records attributed to J E Heuser.

At least 55 records · Page 3Linked to original sources
PubMed1987

High-pressure liquid chromatography fractionation of Chlamydomonas dynein extracts and characterization of inner-arm dynein subunits.

A rapid procedure for fractionating salt-stable dynein subunits from high-salt extracts of Chlamydomonas axonemes has been developed using a high-pressure liquid chromatography system with an anion exchange column and gradient salt elution. Five distinct fractions are shown to be highly enriched for five distinct subunits or subunit complexes by SDS/polyacrylamide gel electrophoresis. ATPase activity and electron microscopy. Peaks 1 and 4 contain, respectively, the single-headed gamma-subunit and the two-headed alpha/beta-heteropolymer that form the outer arm in situ and are dissociated by salt exposure; both peaks are absent from the outer arm-less mutant pf-28. Peaks 2, 3 and 5 contain, respectively, two distinct single-headed species and a double-headed species that derive from inner arms; all three peaks are missing from the inner arm-less mutant pf-23. Sucrose-gradient sedimentation analysis confirms these assignments and provides additional information on the intermediate-chain and light-chain composition of the inner-arm species. Electron microscopy of the purified inner-arm species visualized by the quick-freeze deep-etch technique complements a previous analysis of outer-arm species. Each protein is shown to have a unique morphology, and both the inner- and outer-arm proteins clearly belong to a common family whose structural divergence presumably reflects functional specialization.

Adenosine Triphosphatases↗
PubMed1987

Crossbridges in insect flight muscles of the blowfly (Sarcophaga bullata).

Crossbridges in quick-frozen deep-etched blowfly flight muscles (from Sarcophaga bullata) were compared with those observed in the traditional waterbug preparation (Lethocerus) and found to be indistinguishable. Hence, blowfly was chosen as a fresher more accessible tissue for determining the effect of various fixatives and nucleotides on crossbridge structure. In rigor control, crossbridges were most regular in muscles that were stabilized before freezing by prefixation in glutaraldehyde followed by 'hardening' with neutralized tannic acid, so all nucleotide treatments were terminated by such fixation. MgATP (5 mM) converted the rigor pattern of crossbridges into a random array of disconnected thick filament projections. Lower levels of ATP (0.1 mM) caused a variable but generally lesser degree of crossbridge disconnection, as did 5 mM ADP (probably because it slowly converted to ATP inside the muscle fibres). Vanadate (1-2 mM) potentiated muscle relaxation in the latter two nucleotide treatments (i.e. produced a greater degree of crossbridge disconnection). Thus, differences in overall crossbridge abundance were readily apparent in chemically fixed muscles. Structural details within individual crossbridges were less well preserved, however. Chemical prefixation tended to collapse the muscle lattice, add a surface film to the filaments and thus obscure crossbridge details. Rigorous control of fixative pH largely prevented these problems and permitted recognition of the fact that in Sarcophaga flight muscle, as in Lethocerus muscle in rigor, the S1 'heads' of crossbridges attach to the thin filaments in the expected 'arrowhead' configuration.

Actin Cytoskeleton↗
PubMed1987

Deep-etch visualization of 27S clathrin: a tetrahedral tetramer.

It has recently been reported that 8S clathrin trimers or "triskelions" form larger 27S oligomers upon dialysis into low ionic strength buffers (Prasad, K., R. E. Lippoldt, H. Edelhoch, and M. S. Lewis, 1986, Biochemistry, 25:5214-5219). Here, deep-etch electron microscopy of the 27S species reveals that they are closed tetrahedra composed of four clathrin triskelions. This was determined by two approaches. First, standard quick-freezing and freeze-etching of unfixed 27S species suspended in 2 mM 2-(N-morpholino)ethane sulfonic acid (MES) buffer, pH 5.9, yielded unambiguous images of tetrahedra that measured 33 nm on each edge. Second, the technique of freeze-drying molecules on mica (Heuser, J. E., 1983, J. Mol. Biol., 169:155-195) was modified to overcome the low affinity of mica in 2 mM MES, by pretreating the mica with polylysine. Thereafter, 27S species adsorbed avidly to it and collapsed into characteristic configurations containing four globular domains, each linked to the others by three approximately 33-nm struts. The globular domains look like vertices of deep-etched clathrin triskelions and the links, numbering 12 in all, look like four sets of triskelion legs. New light scattering and equilibrium centrifugation data confirm that 27S polymer is four times as massive as one clathrin triskelion. We conclude that in conditions that do not favor the formation of standard clathrin cages, low affinity interactions lead to closed, symmetrical assemblies of four triskelions, each of which assumes a unique puckered, straight-legged configuration to create the edges of a tetrahedron. Tetrahedra are similar in construction to the cubic octomers of clathrin recently found in ammonium sulfate solutions (Sorger, P. K., R. A. Crowther, J. T. Finch, and B. M. F. Pearse, 1986, J. Cell Biol., 103:1213-1219) but are still smaller, involving only half as many clathrin triskelions.

Animals↗
PubMed1987

Nucleated assembly of Chlamydomonas and Volvox cell walls.

The Chlamydomonas reinhardtii cell wall is made up of hydroxyproline-rich glycoproteins, arranged in five distinct layers. The W6 (crystalline) layer contains three major glycoproteins (GP1, GP2, GP3), selectively extractable with chaotropic agents, that self-assemble into crystals in vitro. A system to study W6 assembly in a quantitative fashion was developed that employs perchlorate-extracted Chlamydomonas cells as nucleating agents. Wall reconstitution by biotinylated W6 monomers was monitored by FITC-streptavidin fluorescence and quick-freeze/deep-etch electron microscopy. Optimal reconstitution was obtained at monomer concentrations (0.2-0.3 mg/ml) well below those required for nonnucleated assembly. Assembly occurred from multiple nucleation sites, and faithfully reflected the structure of the intact W6 layer. Specificity of nucleated assembly was demonstrated using two cell-wall mutants (cw-2 and cw-15); neither served as a substrate for assembly of wild-type monomers. In addition, W6 sublayers were assembled from purified components: GP2 and GP3 coassembled to form the inner (W6A) sublayer; this then served as a substrate for self-assembly of GP1 into the outer (W6B) sublayer. Finally, evolutionary relationships between C. reinhardtii and two additional members of the Volvocales (Chlamydomonas eugametos and Volvox carteri) were explored by performing interspecific reconstitutions. Hybrid walls were obtained between C. reinhardtii and Volvox but not with C. eugametos, confirming taxonomic assignments based on structural criteria.

Cell Nucleus↗
PubMed1987

Dissociation and reassociation of trichocyst proteins: biochemical and ultrastructural studies.

Trichocysts, the crystalline exocytotic organelles in Paramecium tetraurelia, are composed of small, acidic proteins existing primarily as disulphide-linked dimers. We have disaggregated trichocyst proteins with heat, simultaneously observing the changes in morphology and protein composition. The tip matrix was most heat-labile; its subunits progressively broke away from the distal end. During this process, breakdown of the cylindrical shaft began. Shafts first became flattened and torn lengthwise, yielding smaller, interconnected pieces still having the crystalline arrangement of their 5 nm thick fibres. Ultimately this pattern became disordered, and discrete fibrils of the same thickness disengaged from the meshwork. In freeze-etched preparations these fibrils were composed of thinner filaments in side-by-side association. Disaggregation of the tip sheath began from the distal end before shaft dissociation was complete. Trichocysts broke down to thin fibrils, but probably not to monomeric subunits. At least three proteins were preferentially released in the initial phase of dissociation. Disulphide-reducing agent present during heating increased the rate of dissociation without altering the sequence of morphological changes or the order of release of individual proteins. The rate and extent of heat-induced dissociation were strongly dependent on pH and cation concentration. The stabilizing effects of low pH and of cations were additive. A cooled suspension of fully dissociated trichocysts reassociated into sedimentable aggregates with discernible filamentous order, but without the crystalline structure of intact trichocysts. Reassociation was dependent upon time, temperature and protein concentration. All but one of the trichocyst proteins re-entered the sedimentable aggregate during reassociation. Reassociation was faster and more complete at pH 6 than at pH 8 and was stimulated by Ca2+, Mg2+ and La3+. Trichocyst proteins dissociated in the presence of dithiothreitol did not reassociate, even after removal of the reducing agent. Trichocysts from mutants defective to varying degrees in trichocyst formation were subjected to similar experimental protocols. Heat-dissociated trichocysts of the mutants scc6 and ptA1 reassociated at rates similar to those of wild-type; ftA3 showed slower reassociation, and tam38 showed little or no reassociation. Reassociation of wild-type trichocyst proteins was blocked by the addition of an equal amount of tam38 trichocyst proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Crystals of the Chlamydomonas reinhardtii cell wall: polymerization, depolymerization, and purification of glycoprotein monomers.

Two of the three major outer layers of the Chlamydomonas reinhardtii cell wall (W6 and W4) can be solubilized from living cells with sodium perchlorate or other chaotropes and will repolymerize in vitro to form milligram amounts of wall crystals. Conditions for optimal crystalization are presented, and conditions that fail to induce polymerization are exploited to maintain monomers in aqueous solution for ion-exchange chromatography. The four major glycoproteins of the complex (GP1, 1.5, 2, and 3) have in this way been purified to apparent homogeneity and have been characterized morphologically by transmission electron microscopy using the quick-freeze, deep-etch technique and by amino acid composition. Three of the four are hydroxyproline-rich species that copolymerize to form the W6 layer. The fourth (GP1.5) is a glycine-rich species that binds to the interior of the in vitro crystal; it is apparently equivalent to the granules within the W4 layer in situ.

Cell Wall↗
PubMed1986

Different structural states of a microtubule cross-linking molecule, captured by quick-freezing motile axostyles in protozoa.

Freeze-etch preparation of the laminated bundles of microtubules in motile axostyles demonstrates that the cross-bridges populating individual layers or laminae are structurally similar to the dynein arms of cilia and flagellae. Also, like dynein, they are extracted by high salt and undergo a change in tilt upon removal of endogenous ATP (while the axostyle as a whole straightens and becomes stiff). On the other hand, the bridges running between adjacent microtubule laminae in the axostyle turn out to be much more delicate and wispy in appearance, and display no similarity to dynein arms. Thus we propose that the internal or "intra-laminar" cross-bridges are the active force-generating ATPases in this system, and that they generate overall bends or changes in the helical pitch of the axostyle by altering the longitudinal and lateral register of microtubules in each lamina individually; e.g., by "warping" each lamina and creating longitudinal shear forces within it. The cross-links between adjacent laminae, on the other hand, would then simply be force-transmitting elements that serve to translate the shearing forces generated within individual laminae into overall helical shape changes. (This hypothesis differs from the views of earlier workers who considered a more active role for the later cross-links, postulating that they cause an active sliding between adjacent layers that somehow leads to axostyle movement.) Also described here are physical connections between adjacent intra-laminar cross-bridges, structurally analogous to the overlapping components of the outer dynein arms of cilia and flagella. As with dynein, these may represent a mechanism for propagating local changes from cross-bridge to cross-bridge down the axostyle, as occurs during the passage of bends down the length of the organelle.

Adenosine Triphosphatases↗
PubMed1985

Substructure of inner dynein arms, radial spokes, and the central pair/projection complex of cilia and flagella.

The substructure of the components of the axoneme interior--the inner dynein arms, the radial spokes, and the central pair/projection complex--was analyzed for Chlamydomonas. Tetrahymena, Strongelocentrotus, and Mnemiopsis using the quick-freeze, deep-etch technique. The inner arms are shown to resemble the outer arms in overall molecular organization, but they are disposed differently on the microtubule and have two distinct morphologies--dyads with two heads and triads with three. The dyads associate with spokes S3 and S2; the triads associate with S1. The spokes form a three-start right-handed helix with a 288-nm rise; the central pair makes a shallow left-handed twist. The spoke heads are shown to be made up of four major subunits; two bind to the spoke shaft and two bind to a pair of central-sheath projections.

Animals↗
PubMed1985

Structure of the Chlamydomonas agglutinin and related flagellar surface proteins in vitro and in situ.

Using the quick-freeze, deep-etch technique, we compare the structure of the cane-shaped plus and minus sexual agglutinin molecules purified from gametes of Chlamydomonas reinhardi. We also describe the structure of three additional gamete-specific fibrillar molecules, called short canes, loops, and crescents, which are structurally related to the agglutinins. Four non-agglutinating mutant strains are found to produce the three latter fibrils but not canes, supporting our identification of the cane-shaped molecule as the agglutinin. The heads of the plus and minus canes are shown to differ in morphology. Moreover, two treatments that inactivate the plus agglutinin in vitro--thermolysin digestion and disulfide reduction/alkylation--bring about detectable structural changes only in the head domain of the cane, suggesting that the head may play an indispensible role in affecting gametic recognition/adhesion. We also present quick-freeze, deep-etch images of the flagellar surfaces of gametic, vegetative, and mutant cells of Chlamydomonas reinhardi. The gametic flagella are shown to carry the canes, short canes, loops, and crescents present in in vitro preparations. The cane and crescent proteins self-associate on the flagellar surface into stout fibers of uniform caliber, and they align along the longitudinal axis of the flagellum. The short canes and loops co-purify with flagella but, in the presence of mica, dissociate so that they lie to the sides of the flagella. The agglutinin canes of both mating types are oriented with their hooks at the membrane surface and their heads directed outward, where they are positioned to participate in the initial events of sexual agglutination.

Agglutinins↗
PubMed1985

The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella.

Outer-arm dynein from the sperm of the sea urchin S. purpuratus was adsorbed to mica flakes and visualized by the quick-freeze, deep-etch technique. Replicas reveal particles comprised of two globular heads joined by two irregularly shaped stems which make contact along their length. One head is pear-shaped (18.5 X 12.5 nm) and the other is spherical (14.5-nm diam). The stems are decorated by a complex of bead-like subunits. The same two-headed protein is found in the 21S dynein-1 fraction of sucrose gradients. The beta-heavy chain/intermediate chain 1 (beta/IC-1) dynein subfraction, produced by low-salt dialysis and zonal centrifugation of the high-salt-extracted dynein-1, contains only single-headed molecules with single stems. These heads are predominantly pear-shaped (18.5 X 12.5 nm). Since 21S dynein-1 contains two heavy chains (alpha and beta), and the beta/IC-1 subfraction is comprised of only the beta-heavy chain (Tang et al., 1982, J. Biol. Chem. 257: 508-515), we conclude that each head is formed by a heavy chain, that the pear-shaped head contains the beta-heavy chain, and that the spherical head contains the alpha-heavy chain. The in situ outer dynein arms of demembranated sperm were also studied by the quick-freeze, deep-etch method. When frozen in reactivation buffer devoid of ATP, each arm consists of a large globular head that attaches to the A-microtubule by distally skewed subunits and attaches to the B-microtubule by a slender stalk. In ATP, this head shifts its orientation such that it can be seen to be constructed from two globular domains. We offer possible correlates between the in situ and the in vitro images, and we compare the structure of sea-urchin dynein with dynein previously described from Chlamydomonas and Tetrahymena.

Adenosine Triphosphatases↗
PubMed1985

The Chlamydomonas cell wall and its constituent glycoproteins analyzed by the quick-freeze, deep-etch technique.

Using the quick-freeze, deep-etch technique, we have analyzed the structure of the intact cell wall of Chlamydomonas reinhardi, and have visualized its component glycoproteins after mechanical shearing and after depolymerization induced by perchlorate or by the wall-disrupting agent, autolysin. The intact wall has previously been shown in a thin-section study (Roberts, K., M. Gurney-Smith, and G. J. Hills, 1972, J. Ultrastruct. Res. 40:599-613) to consist of a discrete central triplet bisecting a meshwork of fibrils. The deep-etch technique provides additional information about the architecture of each of these layers under several different experimental conditions, and demonstrates that each layer is constructed from a distinct set of components. The innermost layer of the central triplet proves to be a fibrous network which is stable to perchlorate but destabilized by autolysin, disassembling into fibrillar units we designate as "fishbones." The medial layer of the triplet is a loose assemblage of large granules. The outer layer is a thin, crystalline assembly that is relatively unaffected by autolysin. It depolymerizes into two glycoprotein species, one fibrous and one globular. The wall glycoproteins prove to be structurally similar to two fibrous proteins that associate with the flagellar membrane, namely, the sexual agglutinins and the protomers of a structure we designate a "hammock." They are also homologous to some of the fibrous components found in the extracellular matrices of multicellular plants and animals. The quick-freeze, deep-etch technique is demonstrated to be a highly informative way to dissect the structure of a fibrous matrix and visualize its component macromolecules.

Cell Wall↗
PubMed1984

Arrest of pigment granule motion in erythrophores by quick-freezing.

We report the use of quick-freezing, as an alternative to conventional chemical fixation, to arrest the movement of pigment granules at various stages of the dispersion-aggregation cycle in Holocentrus erythrophores. During pigment aggregation, the granules in these cells move at up to 20 microns/sec, hence the structural changes underlying the movement are likely to be too fleeting to be captured faithfully by conventional aldehyde fixation. On the other hand, quick-frozen cells, when examined by freeze-etch electron microscopy, provide novel views of certain cytoplasmic components which appear to be involved in pigment granule movement, namely, fine (2- to 6-nm diameter) fibrils which link the granules to each other and to the radial array of microtubules. These fine crosslinking fibrils can be distinguished from thicker (8- to 15-nm diameter) strands of coherent granular material which pervade the cytoplasm of pigment-dispersed as well as pigment-aggregated cells. This granular matrix is removed by detergent permeabilization, after which it becomes apparent that the fine fibrils are insoluble and are distributed both within and distal to the aggregated pigment mass. The diameter of the specific fibrils does not change during pigment motion, which indicates that they are not contractile.

Animals↗
PubMed1984

Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction.

Frog nerve-muscle preparations were quick-frozen at various times after a single electrical stimulus in the presence of 4-aminopyridine (4-AP), after which motor nerve terminals were visualized by freeze-fracture. Previous studies have shown that such stimulation causes prompt discharge of 3,000-6,000 synaptic vesicles from each nerve terminal and, as a result, adds a large amount of synaptic vesicle membrane to its plasmalemma. In the current experiments, we sought to visualize the endocytic retrieval of this vesicle membrane back into the terminal, during the interval between 1 s and 2 min after stimulation. Two distinct types of endocytosis were observed. The first appeared to be rapid and nonselective. Within the first few seconds after stimulation, relatively large vacuoles (approximately 0.1 micron) pinched off from the plasma membrane, both near to and far away from the active zones. Previous thin-section studies have shown that such vacuoles are not coated with clathrin at any stage during their formation. The second endocytic process was slower and appeared to be selective, because it internalized large intramembrane particles. This process was manifest first by the formation of relatively small (approximately 0.05 micron) indentations in the plasma membrane, which occurred everywhere except at the active zones. These indentations first appeared at 1 s, reached a peak abundance of 5.5/micron2 by 30 s after the stimulus, and disappeared almost completely by 90 s. Previous thin-section studies indicate that these indentations correspond to clathrin-coated pits. Their total abundance is comparable with the number of vesicles that were discharged initially. These endocytic structures could be classified into four intermediate forms, whose relative abundance over time suggests that, at this type of nerve terminal, endocytosis of coated vesicles has the following characteristics: (a) the single endocytotic event is short lived relative to the time scale of two minutes; (b) earlier forms last longer than later forms; and (c) a single event spends a smaller portion of its lifetime in the flat configuration soon after the stimulus than it does later on.

Animals↗
PubMed1984

Quick-freeze, deep-etch rotary replication of Trypanosoma cruzi and Herpetomonas megaseliae.

The fine structure of epimastigotes of Trypanosoma cruzi and promastigotes of Herpetomonas megaseliae was analysed in replicas of quick-frozen, freeze-fractured, deeply etched and rotary-replicated cells. Using control cells and cells treated with Triton X-100 before glutaraldehyde fixation, images were obtained that showed connections of the sub-pellicular microtubules with each other, with the plasma membrane, and with the endoplasmic reticulum. Images were also obtained that showed the DNA network in the kinetoplast. Filamentous structures were found to connect the kinetoplast to the basal body, and to connect the main basal body to the accessory one. In addition, deep-etch images of detergent-extracted flagella display dynein arm substructure and the filamentous architecture of the paraxial structures.

Animals↗
PubMed1983

Actin-myosin interactions visualized by the quick-freeze, deep-etch replica technique.

A new method of preparing biological samples for electron microscopy has been used to re-examine the structure of actin filaments, actin filaments decorated by myosin subfragment-1 (S1), and insect flight muscles. Samples were quick-frozen by contact with a block of copper cooled to approximately 4 K; then were freeze-fractured, deep-etched, rotary-replicated with platinum, and viewed in a transmission electron microscope. By this approach, actin filaments display prominent transverse bands whose repeat (approximately 5.5 nm) and pitch (approximately 15 to 20 degrees) fit with the expected left-handed "genetic" helix. Freeze-etched actin filaments do not, however, display the usual two-start helix as prominently as is seen after negative staining, and they also appear substantially thicker than after negative staining (9 to 10 nm versus 8 nm). The latter two-start helix appears very clearly after S1 decoration. Nevertheless, freeze-etched acto-S1 does not display the "arrowheads" that are seen after negative staining. Instead it displays the outer envelope of the helically deployed S1, and as would be expected from current models derived from optical reconstruction of negatively stained samples, this surface view looks only slightly polarized. Finally, the quick-freeze, deep-etch approach provides particularly distinct images of the crossbridges in insect flight muscles. These are plentiful and regularly arranged in rigor muscles, but rare in muscles relaxed with ATP before freezing. In rigor muscles fixed with aldehydes, these crossbridges assume a broad distribution of inclination, ranging from 45 degrees to 90 degrees with a mean of approximately 80 degrees, which is less tilt than has been seen before in thin-sectioned muscles. However, when aldehyde fixation is followed by exposure to tannic acid with or without uranyl acetate block-staining, crossbridges assume a more acute angle with respect to the fiber axis, centering around 45 degrees. This is associated with a commensurate reduction in interfilament spacing within the muscle fibers, such that tilted crossbridges are not any longer than untilted ones (both measuring approximately 15 nm). At the opposite extreme, crossbridges often become stretched in unfixed muscles, owing to an unnatural increase in interfilament spacing that occurs during sample preparation; in such regions, crossbridges display narrow "stalks", which invariably emerge from the thick filaments at close to 90 degrees. We conclude that crossbridge shape and orientation is strongly affected by different methods of sample preparation, and this will make it difficult to visualize natural crossbridge movements by electron microscopy.

Actins↗
PubMed1983

Structure of the myosin crossbridge lattice in insect flight muscle.

Freeze fracture and deep-etching of quick-frozen insect flight muscles provides unusually clear views of thick filament projections in rigor and relaxed states. In rigor, these projections form crossbridges that are deployed helically. The tracks of these helices are left-handed, repeat every approximately 38 nm, tilt at approximately 42 degrees to the muscle axis, and, when viewed on edge, produce the unique "double chevron" pattern of crossbridges that characterizes this muscle type in thin sections (Reedy, 1968). These helical parameters substantiate Reedy's earlier deduction that rigor crossbridges form two-start helices in this muscle. On the other hand, deep-etchings of insect flight muscles relaxed with Mg-ATP before freezing do not fit with earlier results. Contrary to earlier thin section views and the expectations of X-ray diffraction, thick filaments in such relaxed muscles display no hint of a 14.5 nm axial periodicity; instead, their projections appear to be very disordered. This suggests that when crossbridges are detached, they are free to "wobble" by at least +/- 7 nm in the axial direction and thus obscure their points of origin from the thick filaments. With the images of detached crossbridges in mind, closer inspection of rigor thick filaments yields no indication of any "extra" projections between the helically deployed ones, i.e. there is no indication of any detached crossbridges in rigor muscles. Thus in this type of muscle, at least, the establishment of a rigor pattern may not involve a "selection" of suitably located myosin heads from a larger population, as is generally thought, but may instead involve a systematic redistribution of the whole population of heads until all of them became crossbridges.

Actins↗