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Biomedical subjects

J E Heuser

Publications and source records attributed to J E Heuser.

At least 73 records · Page 4Linked to original sources
PubMed1983

Procedure for freeze-drying molecules adsorbed to mica flakes.

The quick-freeze, deep-etch, rotary-replication technique is useful for visualizing cells and cell fractions but does not work with suspensions of macromolecules. These inevitably clump or collapse during deep-etching, presumably due to surface tension forces that develop during their transfer from ice to vacuum. Previous protocols have attempted to overcome such forces by attaching macromolecules to freshly cleaved mica before drying and replication. I describe here an adaptation of this procedure to the deep-etch technique as otherwise practiced. My innovation is to mix the molecules with an aqueous suspension of tiny flakes of mica and then to quick-freeze and freeze-fracture the suspension exactly as if one were dealing with cells. The fracture inevitably strikes the surfaces of many mica flakes and thereby cleaves the adsorbed macromolecules cleanly enough to reveal interesting substructure within them. The subsequent step of deep-etching exposes large expanses of unfractured mica and thus reveals intact macromolecules. These macromolecules are not obscured by salt deposits, even if they were frozen in hypertonic solutions, apparently because the fracturing step removes nearly all of the overlying electrolyte. Moreover, these macromolecules are minimally freeze-dried (since exposure is sufficient after only 3 min of etching at -102 degrees C) so they retain their three-dimensional topology. I show that molluscan hemocyanin is a good internal standard for this new technique. It is available commercially in stable solutions, mixes well with all sizes of macromolecules, and consists of particles that display distinct five-start surface helices, which have been measured carefully in the past and which possess a known handedness, useful for determining the orientation of micrographs when examining the various helical patterns possessed by most types of extended macromolecules. The fractured hemocyanin particles also display characteristic internal structures, which permit determination of the elevation of the "molecular cleavage" described above. Finally, molluscan hemocyanin is delicate enough to reflect bad freezing or poor replication, if these steps become a problem. A survey of several macromolecules is presented, including soluble enzymes, antibodies, filamentous proteins and nucleoproteins. These images, for the most part, correspond to those previously obtained by negative staining. New details of their structures are noted, and the images are used to illustrate both the advantages and drawbacks of the new procedure.

Adsorption↗
PubMed1983

Chlamydomonas agglutinin is a hydroxyproline-rich glycoprotein.

The mt(+) sexual agglutinin from Chlamydomonas reinhardi is shown to contain approximately 12% hydroxyproline, and two inhibitors of hydroxyproline formation, alpha,alpha'-dipyridyl and 3,4-dehydroproline, are shown to block the production of agglutinin activity in an in vivo bioassay system. These results indicate that the agglutinin glycoprotein may be related to a class of hydroxyproline-rich glycoproteins found in the extracellular matrix of higher plants, several of which have been shown to have lectin activity.

Journal Article↗
PubMed1982

Membrane specializations in the outer plexiform layer of the turtle retina.

The internal organization of the plasma membrane at specialized contacts in the outer plexiform layer of the turtle, Pseudemys scripta elegans, was analyzed with the aid of the freeze-fracturing technique. In the invaginating synapse of cone pedicles the plasma membrane of the photoreceptor ending contains an aggregate of P-face particles, images of synaptic vesicle exocytosis, and rows of forming coated vesicles which are arranged in sequence from apex to base of the synaptic ridge. Thus, freeze-fracturing provides positive evidence that the synaptic ridge represents the active zone at the surface of the photoreceptor endings. Horizontal cell processes of dyads and triads have an aggregate of P-face particles opposite the apex of the ridge, but lack images of vesicle exocytosis. Deep-etching and rotary-shadowing demonstrate that an array of minute protrusions decorates the true outer surface of the horizontal cell membrane at the site of the intramembrane particle aggregate. The membrane of the invaginating bipolar dendrite is unspecialized. At basal junctions, the cone pedicle membrane has a sparse complement of P-face particles, but images of vesicle exocytosis are absent. The adjoining bipolar membrane is characterized by a prominent aggregate of E-face particles, often arranged in an orthogonal lattice. The freeze-fracture profile therefore suggests the existence of (1) a sign-conserving cone-to-horizontal cell synapse; (2) a sign-inverting synapse between cones and invaginating bipolar dendrites; and (3) a sign-conserving synapse between cones and bipolar dendrites at basal junctions. No freeze-fracture evidence was found for a horizontal-to-cone or horizontal-to-bipolar cell synapse within the synaptic invaginations.

Animals↗
PubMed1982

Internal and external differentiations of the postsynaptic membrane at the neuromuscular junction.

Frog, snake and rat neuromuscular junctions were prepared for electron microscopy by the quick-freeze, deep-etch, rotary replication procedure. The postsynaptic membrane was exposed by treating muscles with 1 mg/ml collagenase to remove the basal lamina. Present on the apices of the postsynaptic folds are regular arrays of 8-9 nm protrusions. These are not seen in the depths of the folds nor elsewhere on the muscle surface, thus they presumably represent the heads of cholinergic receptor molecules. These protrusions tend to be arranged in parallel rows two-abreast. Their high concentration (10 000/microns2) and their orderly arrangement is basically similar to the receptors seen in Torpedo postsynaptic membrane. Their distribution did not appear to change after denervation. Efforts were made to expose possible anchoring structures of these receptors, by treating muscles with 0.1% Saponin immediately before and/or during fixation in 1% formaldehyde, or by homogenizing muscles after brief formaldehyde fixation. This washed most soluble protein out of the cytoplasm and exposed a submembraneous meshwork just beneath the postsynaptic membrane. This meshwork appears to connect the membrane to underlying bundles of intermediate filaments which course through the postsynaptic processes that border each fold. This meshwork is presumably equivalent to the postsynaptic 'density' seen in thin sections. Its three-dimensional structure suggests that it could anchor receptor molecules to underlying cytoskeletal elements and thus immobilize receptors in the plane of the postsynaptic membrane.

Animals↗
PubMed1982

Organization of actin, myosin, and intermediate filaments in the brush border of intestinal epithelial cells.

Terminal webs prepared from mouse intestinal epithelial cells were examined by the quick-freeze, deep-etch, and rotary-replication method. The microvilli of these cells contain actin filaments that extend into the terminal web in compact bundles. Within the terminal web these bundles remain compact; few filaments are separated from the bundles and fewer still bend towards the lateral margins of the cell. Decoration with subfragment 1 (S1) of myosin confirmed that relatively few actin filaments travel horizontally in the web. Instead, between actin bundles there are complicated networks of the fibrils. Here we present two lines of evidence which suggest that myosin is one of the major cross-linkers in the terminal web. First, when brush borders are exposed to 1 mM ATP in 0.3 M KCl, they lose their normal ability to bind antimyosin antibodies as judged by immunofluorescence, and they lose the thin fibrils normally found in deep-etch replicas. Correspondingly, myosin is released into the supernatant as judged by SDS gel electrophoresis. Second, electron microscope immunocytochemistry with antimyosin antibodies followed by ferritin-conjugated second antibodies leads to ferritin deposition mainly on the fibrils at the basal part of rootlets. Deep-etching also reveals that the actin filament bundles are connected to intermediate filaments by another population of cross-linkers that are not extracted by ATP in 0.3 M KCl. From these results we conclude that myosin in the intestinal cell may not only be involved in a short range sliding-filament type of motility, but may also play a purely structural role as a long range cross-linker between microvillar rootlets.

Actins↗
PubMed1982

Surfaces of rod photoreceptor disk membranes: integral membrane components.

The membrane surfaces within the rod outer segment of the toad, Bufo marinus, were exposed by rapid-freezing followed by freeze-fracture and deep-etching. Platinum-carbon replicas of disk membranes prepared in this way demonstrate a distinct sidedness. The membrane surface that faces the lumen of the disk shows a fine granularity; particles of approximately 6 nm are packed at a density of approximately 30,000/micron 2. These dimensions suggest that the particles represent protrusions of the integral membrane protein, rhodopsin, into the intradisk space. In addition, when rhodopsin packing is intentionally perturbed by exhaustive digestion with phospholipase C, a concomitant change is observed in the appearance of the luminal surface granularity. The cytoplasmic surface of the disk rarely displays this rough texture; instead it exhibits a collection of much larger particles (8-12 nm) present at approximately 10% of the concentration of rhodopsin. This is about the size and concentration expected for certain light-regulated enzymes, cGMP phosphodiesterase and GTP-binding protein, which are currently thought to localize on or near the cytoplasmic surface of the disk. The molecular identity of the 8-12-nm particles will be identified in the following companion paper. A further differentiation of the cytoplasmic surface can be seen around the very edge, or rim, of each disk. This rim has relatively few 8-12-nm particles and instead displays short filamentlike structures connecting it to other membranes. These filaments extend between adjacent disks, across disk incisures, and from disk rims to the nearby plasma membrane.

Animals↗
PubMed1982

Surfaces of rod photoreceptor disk membranes: light-activated enzymes.

The light-activated GTP-binding protein (GBP) in toad rod outer segments has been located on the cytoplasmic surface (CS) of rod disk membranes by correlating biochemical results with images of quick-frozen, freeze-fractured, and deep-etched rod outer segments. This has been accomplished by selectively removing and replacing the 8-12-nm particles that are found on the CS of disk membranes, exactly in parallel with the GBP. In contrast, the large particles are not correlated with another major disk enzyme, the light-activated cGMP phosphodiesterase. We have been unable to visualize this protein. The surface density of large particles, one particle per eleven rhodopsins in isolated rod outer segments and one particle per nine rhodopsins in intact retina, correlates well with previous biochemical estimates of GBP numbers based on enzyme activity. After the identification of the large particles, we tested the effects of light on the density of particles on the surface of disk membranes in intact retinas. Retinas quick-frozen at various intervals after a bright flash of light show a modest increase (approximately 30%) in particle density by 10 s after the flash but no increase before 1 s. The number of particles on the disk membrane returns to dark levels between 1 and 10 min after the flash. The 1-s latency in the change of particle binding would appear to rule out this process as a mechanism for initiating phototransduction in the rod.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1982

Substructure of the outer dynein arm.

The substructure of the outer dynein arm has been analyzed in quick-frozen deep-etch replicas of Tetrahymena and Chlamydomonas axonemes. Each arm is found to be composed of five morphologically discrete components: an elliptical head; two spherical feet; a slender stalk; and an interdynein linker. The feet make contact with the A microtubule of each doublet; the stalk contacts the B microtubule; the head lies between the feet and stalk; and the linker associates each arm with its neighbor. The spatial relationships between these five components are found to be distinctly different in rigor (ATP-depleted) versus relaxed (ATP- or vanadate plus ATP-treated) axonemes, and the stalk appears to alter its affinity for the B microtubule in the relaxed state. Images of living cilia attached to Tetrahymena cells show that the relaxed configuration is adopted in vivo. We relate our observations to morphological and experimental studies reported by others and propose several models that suggest how this newly described dynein morphology may relate to dynein function.

Adenosine Triphosphatases↗
PubMed1981

Orderly particle arrays on the mitochondrial outer membrane in rapidly-frozen sperm.

By deep-etching and rotary replication of unfixed, non-cryoprotected tissue frozen on a helium-cooled copper block, previously undemonstrable organellar surfaces and intramembranous structures can be examined. Among the more remarkable features of mammalian spermatozoa thus prepared are the highly ordered particulate arrays on the surface of the mitochondrial outer membrane. In the midpiece of the sperm, mitochondria curl around dense fibers and the axoneme. The surface of the mitochondrion that faces the plasmalemma carries closely packed rods in haphazard dispersement, composed of two to four 70-to 80-A particles, less than 20 A apart, while the concave aspect of the organelle contains rods in stepladder pattern. These ladders are parallel, with their particles in neighboring rungs apparently in register at a 40--45 degree angle relative to the mitochondrial axis. This organizational disparity between the convex and concave surfaces of the organelle not only affords evidence of a new mitochondrial substructure, but represents a type of topographical heterogeneity rarely found except within specialized areas of the plasma membrane. Other novel findings in the sperm cell include the observation of "lipid" tracts flanking intramembranous particle-strands in the plasmalemma of the cytoplasmic droplet, and a gridiron design on the cytoplasmic faces of the droplet's microcisternae, as well as both within and atop its plasma membrane--a motif consistent with the presence of exocytotic or endocytotic activity in this portion of the cell. Additional recent observations are the differing internal and external periodicities of axonemal microtubules and the subunit structure of rectangles on the tail surface overlying the intramembranous particles of the zipper.

Animals↗
PubMed1981

Structural evidence that botulinum toxin blocks neuromuscular transmission by impairing the calcium influx that normally accompanies nerve depolarization.

Taking advantage of the fact that nerve terminal mitochondria swell and sequester calcium during repetitive nerve stimulation, we here confirm that this change is caused by calcium influx into the nerve and use this fact to show that botulinum toxin abolishes such calcium influx. The optimal paradigm for producing the mitochondrial changes in normal nerves worked out to be 5 min of stimulation at 25 Hz in frog Ringer's solution containing five time more calcium than normal. Applying this same stimulation paradigm to botulinum-intoxicated nerves produced no mitochondrial changes at all. Only when intoxicated nerves were stimulated in 4-aminopyridine (which grossly exaggerates calcium currents in normal nerves) or when they were soaked in black widow spider venom (which is a nerve-specific calcium ionophore) could nerve mitochondria be induced to swell and accumulate calcium. These results indicate that nerve mitochondria are not damaged directly by the toxin and point instead to a primary inhibition of the normal depolarization-evoked calcium currents that accompany nerve activity. Because these currents normally provide the calcium that triggers transmitter secretion from the nerve, this demonstration of their inhibition helps to explain how botulinum toxin paralyzes.

Animals↗
PubMed1981

Structural changes after transmitter release at the frog neuromuscular junction.

The sequence of structural changes that occur during synaptic vesicle exocytosis was studied by quick-freezing muscles at different intervals after stimulating their nerves, in the presence of 4-aminopyridine to increase the number of transmitter quanta released by each stimulus. Vesicle openings began to appear at the active zones of the intramuscular nerves within 3-4 ms after a single stimulus. The concentration of these openings peaked at 5-6 ms, and then declined to zero 50-100 ms late. At the later times, vesicle openings tended to be larger. Left behind at the active zones, after the vesicle openings disappeared, were clusters of large intramembrane particles. The larger particles in these clusters were the same size as intramembrane particles in undischarged vesicles, and were slightly larger than the particles which form the rows delineating active zones. Because previous tracer work had shown that new vesicles do not pinch off from the plasma membrane at these early times, we concluded that the particle clusters originate from membranes of discharged vesicles which collapse into the plasmalemma after exocytosis. The rate of vesicle collapse appeared to be variable because different stages occurred simultaneously at most times after stimulation; this asynchrony was taken to indicate that the collapse of each exocytotic vesicle is slowed by previous nearby collapses. The ultimate fate of synaptic vesicle membrane after collapse appeared to be coalescence with the plasma membrane, as the clusters of particles gradually dispersed into surrounding areas during the first second after a stimulus. The membrane retrieval and recycling that reverse this exocytotic sequence have a slower onset, as has been described in previous reports.

Animals↗
PubMed1981

Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells.

The cytoskeleton that supports microvilli in intestinal epithelial cells was visualized by the quick-freeze, deep-etch, rotary-replication technique (Heuser and Salpeter. 1979. J. Cell Biol. 82: 150). Before quick freezing, cells were exposed to detergents or broken open physically to clear away the granular material in their cytoplasm that would otherwise obscure the view. After such extraction, cells still displayed a characteristic organization of cytoskeletal filaments in their interiors. Platinum replicas of these cytoskeletons had sufficient resolution to allow us to identify the filament types present, and to determine their characteristic patterns of interaction. The most important new finding was that the apical "terminal web" in these cells, which supports the microvilli via their core bundles of actin filaments, does not itself contain very much actin but instead is comprised largely of narrow strands that interconnect adjacent actin bundles with one another and with the underlying base of intermediate filaments. These strands are slightly thinner than actin, do not display actin's 53A periodicity, and do not decorate with myosin subfragment S1. On the contrary, two lines of evidence suggested that these strands, could include myosin molecules. First, other investigators have shown that myosin is present in the terminal web (Mooseker et al. 1978. J. Cell Biol. 79: 444-453), yet we could find no thick filaments in this area. Second, we found that the strands were removed completely in the process of decorating the core filament bundles with the myosin subfragment S1, suggesting that they had been competitively displaced by exogenous myosin. We conclude that myosin may play a structural role in these cells, via its cross-linking distribution, in addition to whatever role it plays in microvillar motility.

Actins↗
PubMed1980

Protease effects on the structure of acetylcholine receptor membranes from Torpedo californica.

Protease digestion of acetylcholine receptor-rich membranes derived from Torpedo californica electroplaques by homogenization and isopycnic centrifugation results in degradation of all receptor subunits without any significant effect on the appearance in electron micrographs, the toxin binding ability, or the sedimentation value of the receptor molecule. Such treatment does produce dramatic changes in the morphology of the normally 0.5- to 2-microns-diameter spherical vesicles when observed by either negative-stain or freeze-fracture electron microscopy. Removal of peripheral, apparently nonreceptor polypeptides by alkali stripping (Neubig et al. 1979, Proc. Natl. Acad. Sci. U. S. A. 76:690-694) results in increased sensitivity of the acetylcholine receptor membranes to the protease trypsin as indicated by SDS gel electrophoretic patterns and by the extent of morphologic change observed in vesicle structure. Trypsin digestion of alkali-stripped receptor membranes results in a limit degradation pattern of all four receptor subunits, whereupon all the vesicles undergo the morphological transformation to minivesicles. The protein-induced morphological transformation and the limit digestion pattern of receptor membranes are unaffected by whether the membranes are prepared so as to preserve the receptor as a disulfide bridged dimer, or prepared so as to generate monomeric receptor.

Animals↗
PubMed1980

Filament organization revealed in platinum replicas of freeze-dried cytoskeletons.

This report presents the appearance of rapidly frozen, freeze-dried cytoskeletons that have been rotary replicated with platinum and viewed in the transmission electron microscope. The resolution of this method is sufficient to visualize individual filaments in the cytoskeleton and to discriminate among actin, microtubules, and intermediate filaments solely by their surface substructure. This identification has been confirmed by specific decoration with antibodies and selective extraction of individual filament types, and correlated with light microscope immunocytochemistry and gel electrophoresis patterns. The freeze-drying preserves a remarkable degree of three-dimensionality in the organization of these cytoskeletons. They look strikingly similar to the meshwork of strands or "microtrabeculae" seen in the cytoplasm of whole cells by high voltage electron microscopy, in that the filaments form a lattice of the same configutation and with the same proportions of open area as the microtrabeculae seen in whole cells. The major differences between these two views of the structural elements of the cytoplasmic matrix can be attributed to the effects of aldehyde fixation and dehydration. Freeze-dried cytoskeletons thus provide an opportunity to study--at high resolution and in the absence of problems caused by chemical fixation--the detailed organization of filaments in different regions of the cytoplasm and at different stages of cell development. In this report the pattern of actin and intermediate filament organization in various regions of fully spread mouse fibroblasts is described.

Actins↗
PubMed1980

Arrest of membrane fusion events in mast cells by quick-freezing.

We have used quick-freezing and freeze-fracture to study early stages of exocytosis in rat peritoneal mast cells. Mast cells briefly stimulated with 48/80 (a synthetic polycation and well-known histamine-releasing agent) at 22 degrees C displayed single, narrow-necked pores (some as small as 0.05 micrometer in diameter) joining single granules with the plasma membrane. Pores that had become as large as 0.1 micrometer in diameter were clearly etchable and thus represented aqueous channels connecting the granule interior with the extracellular space. Granules exhibiting pores usually did not have wide areas of contact with the plasma membrane, and clearings of intramembrane particles, seen in chemically fixed mast cells undergoing exocytosis, were not present on either plasma or granule membranes. Fusion of interior granules later in the secretory process also appeared to involve pores; granules were often joined by one pore or a group of 2-4 pores. Also found were groups of extremely small, etchable pores on granule membranes that may represent the earliest aqueous communication between fusing granules.

Animals↗
PubMed1979

Morphological studies of stimulated adrenergic axon varicosities in the mouse vas deferens.

The postganglionic axons of sympathetic neurons innervating the mouse vas deferens were stimulated transmurally in vitro by passing square pulses between two platinum electrodes. The ultrastructural appearance of the adrenergic nerve terminals was compared to samples fixed immediately after 30 min of stimulation and in samples allowed to recover for 2 h before fixation. The contralateral vasa deferentia served as controls, and these were incubated in Krebs solution for the same period as stimulated muscles. For each of four experiments, the mean number of large and small dense-core vesicles per square micrometer was calculated, as were the mean area and perimeter of the axon varicosities in each group. It was found that the number of small vesicles per square micrometer decreased by 60% during the stimulation period, but returned almost to control levels 2 h later. Large vesicles did not change in number during the stimulation or recovery periods. The proportion of vesicles containing cores was also determined for each group and found to decline just after stimulation in the small vesicle population, but to remain constant in the large vesicle population. The core depletion was partly reversed after 2 h. The vesicle recovery process was studied by use of the extracellular tracer horseradish peroxidase (HRP). When HRP was present in the extracellular space during stimulation, large numbers of vesicles contained the marker after recovery from stimulation. Thus, it is proposed that adrenergic axon varicosities recycle vesicle membrane through the plasma membrane in a manner similar to that already described for cholinergic nerve terminals.

Animals↗
PubMed1979

Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release.

We describe the design and operation of a machine that freezes biological tissues by contact with a cold metal block, which incorporates a timing circuit that stimulates frog neuromuscular junctions in the last few milliseconds before thay are frozen. We show freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synpatic vesicles caught in the act of exocytosis. We use 4-aminopyridine (4-AP) to increase the number of transmitter quanta discharged with each nerve impulse, and show that the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that one vesicle undergoes exocytosis for each quantum that is discharged. We perform statistical analyses on the spatial distribution of synaptic vesicle discharge sites along the "active zones" that mark the secretory regions of these nerves, and show that individual vesicles fuse with the plasma membrane independent of one another, as expected from physiological demonstrations that quanta are discharged independently. Thus, the utility of quick-freezing as a technique to capture biological processes as evanescent as synaptic transmission has been established. An appendix describes a new capacitance method to measure freezing rates, which shows that the "temporal resolution" of our quick-freezing technique is 2 ms or better.

Aminopyridines↗