Biomedical subjects

T S Reese

Publications and source records attributed to T S Reese.

At least 55 records · Page 3Linked to original sources

PubMed1992

Structural domains of the tight junctional intramembrane fibrils.

Freeze-fracture reveals intramembrane fibrils lying along the intermembrane contacts that characterize tight junctions. Tight junctions from a variety of species are reexamined here by rapid freezing prior to freeze-fracture. The tight junction fibril is uprooted alternatively from either the cytoplasmic or the exoplasmic hemibilayer during freeze-cleavage, exposing two distinct but complementary views of its hybrid structure within the same replica. When the transmembrane fibril is uprooted from the exoplasmic hemibilayer it appears on the P-fracture face as a smooth-surfaced cylinder which is sometimes resolved into periodic globular structures. The lack of indication that the P-face cylinder has been pulled out through the opposite membrane half indicates that this domain of the fibril is, in large part, buried in the hydrophobic interior of the membrane. However, when the transmembrane fibril is uprooted from the cytosolic hemibilayer it appears on the E-fracture face as a row of irregular intramembrane particles. The irregular particles on the E-face aspect of the fibril are interpreted as corresponding to transmembrane protein segments that may very well make projections onto the cytosolic surface of the bilayer. En face views of the outermost junction strand between adjacent epithelial cells show periodic lines on the bilayer on each side of the junction which are interpreted as periodic transmembrane protein segments arising from the core structure of the tight junction fibril. If the backbone of the tight junction strand is an inverted cylindrical micelle, it must typically include proteins, which might anchor it to structures outside the membrane bilayer.

Animals↗

PubMed1992

Kinesin is bound with high affinity to squid axon organelles that move to the plus-end of microtubules.

This paper addresses the question of whether microtubule-directed transport of vesicular organelles depends on the presence of a pool of cytosolic factors, including soluble motor proteins and accessory factors. Earlier studies with squid axon organelles (Schroer et al., 1988) suggested that the presence of cytosol induces a > 20-fold increase in the number of organelles moving per unit time on microtubules in vitro. These earlier studies, however, did not consider that cytosol might nonspecifically increase the numbers of moving organelles, i.e., by blocking adsorption of organelles to the coverglass. Here we report that treatment of the coverglass with casein, in the absence of cytosol, blocks adsorption of organelles to the coverglass and results in vigorous movement of vesicular organelles in the complete absence of soluble proteins. This technical improvement makes it possible, for the first time, to perform quantitative studies of organelle movement in the absence of cytosol. These new studies show that organelle movement activity (numbers of moving organelles/min/micron microtubule) of unextracted organelles is not increased by cytosol. Unextracted organelles move in single directions, approximately two thirds toward the plus-end and one third toward the minus-end of microtubules. Extraction of organelles with 600 mM KI completely inhibits minus-end, but not plus-end directed organelle movement. Upon addition of cytosol, minus-end directed movement of KI organelles is restored, while plus--end directed movement is unaffected. Biochemical studies indicate that KI-extracted organelles attach to microtubules in the presence of AMP-PNP and copurify with tightly bound kinesin. The bound kinesin is not extracted from organelles by 1 M KI, 1 M NaCl or carbonate (pH 11.3). These results suggest that kinesin is irreversibly bound to organelles that move to the plus-end of microtubules and that the presence of soluble kinesin and accessory factors is not required for movement of plus-end organelles in squid axons.

Animals↗

PubMed1992

Characterization of endoplasmic reticulum by co-localization of BiP and dicarbocyanine dyes.

The original concept of endoplasmic reticulum derived from the observation of a reticular network in cultured fibroblasts by electron microscopy of whole cells. It was previously reported that the fluorescent dye, DiOC6(3), stains a similar network as well as mitochondria and other organelles in living cells. Here, we investigate the significance of the structures labeled by DiO6(3) in CV-1 cells, a monkey epithelial cell line. First, we show that the network stained in living CV-1 cells is preserved by glutaraldehyde fixation and then we co-label it with an antibody against BiP (immunoglobulin binding protein), a protein commonly accepted to be present in the endoplasmic reticulum. Anti-BiP labeled the same network as that labeled by DiOC6(3), so this network now is identified as being part of the endoplasmic reticulum. DiOC6(3) labels many other membrane compartments in addition to the endoplasmic reticulum. This, along with its lipophilic properties, suggests that DiOC6(3) stains all intracellular membranes. However, the extensive reticular network in the thin peripheral regions of cultured cells is easily distinguished from these other membranes. Thus, staining by DiOC6(3) is a useful method for localizing the endoplasmic reticulum, particularly in thin peripheral regions of cultured cells.

Animals↗

PubMed1991

Pertussis toxin-sensitive G proteins are transported toward synaptic terminals by fast axonal transport.

We find that half of the pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) in the squid (Loligo pealei) giant axon is cytoplasmic and that this species of G protein is intermediate in size between the two forms present in axolemma. This G protein is transported toward synaptic terminals at 44 mm/day. Moreover, these data are consistent with there being two additional steps leading to the maturation of G proteins: (i) association with and transport on intracellular organelles and (ii) modification at the time of transfer to the plasmalemma resulting in a molecular weight shift. Since the other two components of G protein-mediated signal transduction pathways, receptors and effector enzymes, are known to be delivered to the synaptic terminals by fast axonal transport, our findings introduce the possibility that these three macromolecules are assembled as a complex in the cell body and delivered together to the plasma membrane of the axon and synaptic terminals.

Adenosine Diphosphate Ribose↗

PubMed1991

New structural features of the flagellar base in Salmonella typhimurium revealed by rapid-freeze electron microscopy.

The structure of the flagellar base in Salmonella typhimurium has been studied by rapid-freeze techniques. Freeze-substituted thin sections and freeze-etched replicas of cell envelope preparations have provided complementary information about the flagellar base. The flagellar base has a bell-shaped extension reaching as far as 50 nm into the bacterial cytoplasm. This structure can be recognized in intact bacteria but was studied in detail in cell envelopes, where some flagella lacking parts of the bell were helpful in understanding its substructure. Structural relationships may be inferred between this cytoplasmic component of the flagellum and the recently described flagellar intramembrane particle rings as well as the structures associated with the basal body in isolated, chemically fixed flagella.

Flagella↗

PubMed1990

Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy.

Cells can be transiently permeabilized by exposing them briefly to an intense electric field (a process called "electroporation"), but it is not clear what structural changes the electric field induces in the cell membrane. To determine whether membrane pores are actually created in the electropermeabilized cells, rapid-freezing electron microscopy was used to examine human red blood cells which were exposed to a radio-frequency electric field. Volcano-shaped membrane openings appeared in the freeze-fracture faces of electropermeabilized cell membranes at intervals as short as 3 ms after the electrical pulse. We suggest that these openings represent the membrane pathways which allow entry of macromolecules (such as DNA) during electroporation. The pore structures rapidly expand to 20-120 nm in diameter during the first 20 ms of electroporation, and after several seconds begin to shrink and reseal. The distribution of pore sizes and pore dynamics suggests that interactions between the membrane and the submembrane cytoskeleton may have an important role in the formation and resealing of pores.

Electric Stimulation↗

PubMed1990

Delayed start-up of kinesin-driven microtubule gliding following inhibition by adenosine 5'-[beta,gamma-imido]triphosphate.

Kinesin is a microtubule-activated ATPase that moves objects toward the plus end of microtubules and makes microtubules glide along a glass surface. Here we investigate a remarkable effect of the nonhydrolyzable analogue of ATP, adenosine 5'-[beta,gamma-imido]triphosphate (p[NH]ppA), on kinesin-driven microtubule gliding. Microtubule gliding that has been blocked by rapid replacement of ATP with p[NH]ppA requires 1-2 min of exposure to ATP before microtubule gliding resumes. This latency is not shortened by prolonged washing of p[NH]ppA-blocked microtubules in nucleotide-free buffer for up to 15 min, suggesting that ATP binding to a second nucleotide binding site on kinesin triggers the release of bound p[NH]ppA. To test this hypothesis, the release of [3H]p[NH]ppA from kinesin-microtubule complexes was followed in parallel biochemical assays. In nucleotide-free buffer, the bound p[NH]ppA was released over several hours from the complexes. However, addition of ATP caused the release of p[NH]ppA from the kinesin-microtubule complexes within 2 min, which was similar to the latent period for start-up of microtubule gliding after p[NH]ppA inhibition. The stoichiometry of p[NH]ppA bound per kinesin heavy chain at saturation was estimated to be approximately 1:2. These results suggest a model in which each molecule of kinesin has at least two nucleotide binding sites that alternately bind nucleotide.

Adenosine Triphosphatases↗

PubMed1989

New structural features of synapses in the anteroventral cochlear nucleus prepared by direct freezing and freeze-substitution.

The rostral anteroventral cochlear nucleus (AVCN) of the chinchilla provides a preparation in which neuronal cell bodies and synapses in the mammalian central nervous system can be examined after direct freezing and freeze-substitution of rapidly excised brain stem slices. Cell bodies and synapses in the freeze-substituted AVCN differed from those in perfusion-fixed AVCN in several interesting respects. Despite of these differences, four types of synaptic terminal were distinguished in freeze-substituted AVCN and correlated with the four well-known types of perfusion-fixed terminal. Since the transmitter at each of the four types of terminal has been tentatively identified, the structure of synaptic vesicles and junctions in the freeze-substituted terminals could be related to transmitter type. Synaptic vesicles were uniformly round, but their diameters, deployment, and related cytoskeletal elements near the synaptic junction differed in each chemical type of synapse; the synapses thought to be cholinergic, for instance, had only a few vesicles clustered at their presynaptic junctions while the rest of the vesicles were separated from the junction by a network of fine filaments. Two types of filamentous components, short vertical projections from the postsynaptic membrane and thin filaments protruding from these projections, comprised the basic structure of the postsynaptic specialization, but their sizes and distribution differed at each chemical type of terminal. For instance, the postsynaptic specialization at the glycine terminal was distinguished by numerous thin filaments which curved sideways to run parallel to the plasmalemma. Thus, freeze-substitution gives new information about structural differences between chemically different types of synapses, which may reflect differences in their transmitter storage, release, and reception. In addition, the AVCN preparation is of general interest in making cell bodies in the mammalian central nervous system available to the various structural and analytical techniques which depend on direct, rapid freezing.

Animals↗

PubMed1989

Substructure in the assemblies of intramembrane particles in astrocytic membranes.

Assemblies are a specialization of intramembrane structure in astrocytes which is concentrated in astrocytic processes at the interface with vascular and cerebrospinal fluid compartments. When astrocytic processes are rapidly frozen and then freeze-fractured at very low stage temperature, the constituent particles of assemblies fracture in at least two planes in the lipid bilayer. The true outer surface of the astrocytic membrane can be exposed by etching rapidly frozen tissue and in such preparations the assemblies are seen to extend through the extracellular half of the membrane to be exposed on the surface to the extracellular fluid. The dimensions of the particles, their tendency to fracture at several levels and their exposure to the extracellular space all indicate that they are not composed solely of lipid. We conclude that assemblies represent a protein which partially or entirely bridges the membrane and which may serve a transport function.

Animals↗

PubMed1989

Dynein is the motor for retrograde axonal transport of organelles.

Vesicular organelles in axons of nerve cells are transported along microtubules either toward their plus ends (fast anterograde transport) or toward their minus ends (retrograde transport). Two microtubule-based motors were previously identified by examining plastic beads induced to move along microtubules by cytosol fractions from the squid giant axon: (i) an anterograde motor, kinesin, and (ii) a retrograde motor, which is characterized here. The retrograde motor, a cytosolic protein previously termed HMW1, was purified from optic lobes and extruded axoplasm by nucleotide-dependent microtubule affinity and release; microtubule gliding was used as the assay of motor activity. The following properties of the retrograde motor suggest that it is cytoplasmic dynein: (i) sedimentation at 20-22 S with a heavy chain of Mr greater than 200,000 that coelectrophoreses with the alpha and beta subunits of axonemal dynein, (ii) cleavage by UV irradiation in the presence of ATP and vanadate, and (iii) a molecular structure resembling two-headed dynein from axonemes. Furthermore, bead movement toward the minus end of microtubules was blocked when axoplasmic supernatants were treated with UV/vanadate. Treatment of axoplasmic supernatant with UV/vanadate also blocks the retrograde movement of purified organelles in vitro without changing the number of anterograde moving organelles, indicating that dynein interacts specifically with a subgroup of organelles programmed to move toward the cell body. However, purified optic lobe dynein, like purified kinesin, does not by itself promote the movement of purified organelles along microtubules, suggesting that additional axoplasmic factors are necessary for retrograde as well as anterograde transport.

Adenosine Triphosphatases↗

PubMed1988

Effects of mot gene expression on the structure of the flagellar motor.

Direct freezing procedures have enabled us to visualize distinctive intramembrane particle ring structures in the cytoplasmic membranes of peritrichously flagellated bacteria by means of freeze-fracture electron microscopy. These structures were identified as flagellar motor components because their distribution matched that of flagella, and because they were absent in non-flagellated mutants of Escherichia coli. Particle rings were present in both the Gram-positive Streptococcus and the Gram-negative E. coli. In E. coli, a non-functional mocha operon produced flagellated but immotile cells lacking the particle rings. Simultaneous introduction of the motA and motB genes, led to recovery of both motility and the ring structures but neither gene alone was sufficient. The concomitant loss of the rings and motility is consistent with the ring particles having a central role in the flagellar motor.

Escherichia coli↗

PubMed1988

Three-dimensional organization of the plasmalemmal vesicular system in directly frozen capillaries of the rete mirabile in the swim bladder of the eel.

Several recent studies comparing chemically fixed and cryofixed endothelium have indicated that glutaraldehyde fixation may result in increases in the population of "vesicles" in the cytoplasm. Other reports based on ultrathin serial-section reconstruction of chemically fixed endothelium have revealed that the vesicular system is comprised of interconnected membranous compartments, which are ultimately continuous with either cell surface but do not extend across the endothelial cell. In this study, we have investigated the three-dimensional organization of the vesicular system in directly frozen, freeze-substituted capillaries of the rete mirabile from the swim bladder of the eel, specifically using the same block of embedded capillaries in which frozen capillaries had previously been found to contain less "vesicles" than chemically fixed capillaries. The results show that essentially all vesicles remain interconnected with each other and are part of two separate sets of invaginations from the luminal and abluminal cell surface like in chemically fixed tissue. Any increase in vesicle number resulting from glutaraldehyde fixation does not affect the overall three-dimensional organization of the vesicular system in these endothelial cells.

Air Sacs↗

PubMed1988

The organization of cytoplasm at the presynaptic active zone of a central nervous system synapse.

The axoplasm at the presynaptic active zone of excitatory synapses between parallel fibers and Purkinje cell spines contains a meshwork of distinct filaments intermingled with synaptic vesicles, seen most clearly after the rapid freezing, freeze-etch technique of tissue preparation. One set of filaments extends radially from synaptic vesicles and intersects similar filaments associated with vesicles as well as larger filaments arising from the presynaptic membrane. The small, vesicle-associated filaments appear to link synaptic vesicles to one another and to enmesh them in the vicinity of the synaptic junction. The vesicle-associated filaments could be synapsin I because they have the same molecular dimensions and are distributed in the same pattern as synapsin I immunoreactivity.

Animals↗

PubMed1988

Activity-dependent accumulation of calcium in Purkinje cell dendritic spines.

The calcium content of synapses of parallel fibers on Purkinje cell dendritic spines was determined by electron probe x-ray microanalysis of freeze-dried cryosections from directly frozen slices of mouse cerebellar cortex. In fresh slices frozen within 20-30 sec of excision, calcium concentrations ranging from 0.8 to 18.6 mmol/kg of dry weight were measured in cisterns of smooth endoplasmic reticulum within Purkinje cell dendritic spines. The average calcium content of spine cisterns in rapidly excised slices (6.7 +/- 0.6 mmol/kg of dry weight +/- SEM) was higher than the average calcium content of spine cisterns in brain slices incubated without stimulation for 1-2 hr before direct freezing (2.5 +/- 0.4 mmol/kg of dry weight). Depolarization of incubated cerebellar slices by isotonic 55 mM KCl resulted in the accumulation within spine cisterns of very high amounts of calcium or isotonically substituted strontium, both derived from the extracellular fluid. These results suggest that one function of spine cisterns is to sequester free calcium that enters the spine through ligand-gated or voltage-gated channels during synaptic transmission.

Animals↗

PubMed1988

The mechanism of cytoplasmic streaming in characean algal cells: sliding of endoplasmic reticulum along actin filaments.

Electron microscopy of directly frozen giant cells of characean algae shows a continuous, tridimensional network of anastomosing tubes and cisternae of rough endoplasmic reticulum which pervade the streaming region of their cytoplasm. Portions of this endoplasmic reticulum contact the parallel bundles of actin filaments at the interface with the stationary cortical cytoplasm. Mitochondria, glycosomes, and other small cytoplasmic organelles enmeshed in the endoplasmic reticulum network display Brownian motion while streaming. The binding and sliding of endoplasmic reticulum membranes along actin cables can also be directly visualized after the cytoplasm of these cells is dissociated in a buffer containing ATP. The shear forces produced at the interface with the dissociated actin cables move large aggregates of endoplasmic reticulum and other organelles. The combination of fast-freezing electron microscopy and video microscopy of living cells and dissociated cytoplasm demonstrates that the cytoplasmic streaming depends on endoplasmic reticulum membranes sliding along the stationary actin cables. Thus, the continuous network of endoplasmic reticulum provides a means of exerting motive forces on cytoplasm deep inside the cell distant from the cortical actin cables where the motive force is generated.

Actins↗

PubMed1988

The role of kinesin and other soluble factors in organelle movement along microtubules.

Kinesin is a force-generating ATPase that drives the sliding movement of microtubules on glass coverslips and the movement of plastic beads along microtubules. Although kinesin is suspected to participate in microtubule-based organelle transport, the exact role it plays in this process is unclear. To address this question, we have developed a quantitative assay that allows us to determine the ability of soluble factors to promote organelle movement. Salt-washed organelles from squid axoplasm exhibited a nearly undetectable level of movement on purified microtubules. Their frequency of movement could be increased greater than 20-fold by the addition of a high speed axoplasmic supernatant. Immunoadsorption of kinesin from this supernatant decreased the frequency of organelle movement by more than 70%; organelle movements in both directions were markedly reduced. Surprisingly, antibody purified kinesin did not promote organelle movement either by itself or when it was added back to the kinesin-depleted supernatant. This result suggested that other soluble factors necessary for organelle movement were removed along with kinesin during immunoadsorption of the supernatant. A high level of organelle motor activity was recovered in a high salt eluate of the immunoadsorbent that contained only little kinesin. On the basis of these results we propose that organelle movement on microtubules involves other soluble axoplasmic factors in addition to kinesin.

Animals↗

PubMed1988

Compartmentalization of anterogradely and retrogradely transported organelles in axons and growth cones from chick optic tectum.

Previous work suggests that organelles contacting microtubules in axons are in fast transport. Here, we examine the distribution of organelles contacting microtubules in growing axons and growth cones from chick optic tectum. Five axon segments, each 10 microns long, and 4 entire growth cones were reconstructed from serial electron micrographs of quick-frozen, freeze-substituted chick optic tectum. Organelles contacting microtubules in axons are evenly distributed along all microtubules. Smaller organelles, presumably in anterograde transport, are enclosed in fascicles of microtubules, while larger organelles in retrograde transport lie outside the fascicles. In contrast, organelles contacting microtubules are prevalent only in the most proximal parts of the growth cone, before the microtubule fascicles splay out more distally. The distance between noncontacting organelles and microtubules also becomes progressively greater, reaching a maximum in the mid- and more distal region of the growth cone. Contacts with microtubules of both the smaller, presumably anterogradely transported organelles, as well as the larger, presumably retrogradely transported organelles, abruptly become less frequent in the proximal midregion of the growth cone. It is therefore of possible significance in stopping and starting microtubule-based organelle transport that microtubules change from a straight to an undulating configuration in the midregion of the growth cone. The decrease in organelle binding to microtubules at the demarcations between the straight and undulating microtubule segments may depend on proteins or other local factors as well as the splaying out of the microtubule bundles.

Animals↗

PubMed1987

Substructure in the postsynaptic density of Purkinje cell dendritic spines revealed by rapid freezing and etching.

In tissue prepared by rapid freezing, freeze fracture, and shallow etching, the postsynaptic density of Purkinje cell dendritic spines has a substructure consisting of fine filaments and irregular, globular adherent proteins. The number and packing density of the globular proteins vary from region to region within a single density and are even more variable when different junctions are compared. Whereas actinlike microfilaments and spectrinlike filaments are juxtaposed to the postsynaptic density, they do not appear to be continuous with the constituent filaments of the density. We suggest that the postsynaptic density at this class of synapse is composed of fine filamentous proteins that insert on the postsynaptic membrane and serve as a supporting framework for a variety of globular proteins. The globular proteins may vary qualitatively and quantitatively from junction to junction, and are positioned in the region of the spine that has the greatest concentration of ionized calcium entering with the synaptic current, and the greatest extent of postsynaptic depolarization.

Animals↗