Biomedical subjects

T S Reese

Publications and source records attributed to T S Reese.

At least 37 records · Page 2Linked to original sources

PubMed1997

Phosphorylation and subunit organization of axonal neurofilaments determined by scanning transmission electron microscopy.

Phosphorylation plays a critical role in controlling the function of cytoskeletal assemblies but no direct method yet exists to measure the phosphorylation state of proteins at the level of individual molecules and assemblies. Herein, we apply scanning transmission electron microscopy in combination with electron energy loss spectroscopy to measure the distributions of mass and phosphorus in neurofilaments (NFs) isolated from the squid giant axon. We find that native squid NFs, in contrast to typical reconstituted intermediate filaments, are a relatively homogeneous population containing only eight coiled-coil dimers per cross section. The measured stoichiometry of approximately 1:1 for light/heavy peptides strongly suggests that squid NFs are composed of heterodimers. Furthermore, each heavy chain of the dimers carries at least 100 phosphate groups and is, therefore, near-maximally phosphorylated. These results also demonstrate that scanning transmission electron microscopy combined with electron energy loss spectroscopy at the nanometer scale is capable of characterizing the level and distribution of phosphorylation in individual mass-mapped protein assemblies.

Animals↗

PubMed1997

Organization of the cortical endoplasmic reticulum in the squid giant axon.

The organization of the cortical endoplasmic reticulum in the squid giant axon was investigated by rapid freeze and freeze-substitution electron microscopy, thereby eliminating the effects of fixatives on this potentially labile structure. Juvenile squid, which have thinner Schwann sheaths, were used in order to achieve freezing deep enough to include the entire axonal cortex. The smooth endoplasmic reticulum is composed of subaxolemmal and deeper cisternae, tubules, tethers and vesicles. The subaxolemmal cisternae make junctional contacts with the axolemma which are characterized by filamentous-granular bridging structures approximately 3 nm in diameter. The subaxolemmal junctions with the axolemma resemble the coupling junctions between the sarcoplasmic reticulum and the T-tubules in muscle. Reconstruction of short series of sections showed that a number of the elements of the endoplasmic reticulum were continuous but numerous separate vesicles were present as well. The morphology of endoplasmic reticulum as described here suggests that it is a highly dynamic entity as well as a Ca2+ sequestering organelle.

Animals↗

PubMed1996

FliN is a major structural protein of the C-ring in the Salmonella typhimurium flagellar basal body.

The Salmonella typhimurium FliN protein has been proposed to form a mutually interacting complex with FliG and FliM, the switch complex, that is required for flagellar morphogenesis and function. We have used affinity chromatography for purification of extended flagellar basal bodies sufficient for quantitative analysis of their protein composition. The belled, extended structure is predominantly comprised of the switch complex proteins; with FliN present in the most copies (111 +/- 13). This explains why single, missense fliN, fliG or fliM mutations, found in many non-motile strains, can alter the belled morphology. Cell lysates from these strains contained the wild-type complement of FliG, FliM and FliN; but the basal bodies lacked the outer, cytoplasmic(C)-ring of the bell and were separated by sedimentation from FliM and FliN. The amount of FliG present in basal bodies from wild-type and one such mutant, FliN100LP, was comparable. These data show that: (1) the mutations define a FliG and FliMFliN multiple contact interface important for motility. (2) FliG is responsible for the increased size of the membrane-embedded MS-ring complex of belled relative to acid-treated basal bodies. (3) FliN, together with FliM, account for most of the C-ring. As a major component of the C-ring, FliN is distinct from the other proteins implicated in axial flagellar protein export. Inner, cytoplasmic rod basal substructure, seen by negative-stain and quick-freeze replica electron microscopy, may gate such export. Lack of connectivity between the cytoplasmic rod and ring substructures places contacts between FliG and FliMFliN at the periphery of the basal body, proximal to the flagellar intramembrane ring particles. This topology is consistent with models where torque results from interaction of circumferential arrays of the switch complex proteins with the ring particles.

Bacterial Proteins↗

PubMed1996

An axoplasmic myosin with a calmodulin-like light chain.

Organelles in the axoplasm from the squid giant axon move along exogenous actin filaments toward their barbed ends. An approximately 235-kDa protein, the only band recognized by a pan-myosin antibody in Western blots of isolated axoplasmic organelles, has been previously proposed to be a motor for these movements. Here, we purify this approximately 235-kDa protein (p235) from axoplasm and demonstrate that it is a myosin, because it is recognized by a pan-myosin antibody and has an actin-activated Mg-ATPase activity per mg of protein 40-fold higher than that of axoplasm. By low-angle rotary shadowing, p235 differs from myosin II and it does not form bipolar filaments in low salt. The amino acid sequence of a 17-kDa protein that copurifies with p235 shows that it is a squid optic lobe calcium-binding protein, which is more similar by amino acid sequence to calmodulin (69% identity) than to the light chains of myosin II (33% identity). A polyclonal antibody to this light chain was raised by using a synthetic peptide representing the calcium binding domain least similar to calmodulin. We then cloned this light chain by reverse transcriptase-PCR and showed that this antibody recognizes the bacterially expressed protein but not brain calmodulin. In Western blots of sucrose gradient fractions, the 17-kDa protein is found in the organelle fraction, suggesting that it is a light chain of the p235 myosin that is also associated with organelles.

Amino Acid Sequence↗

PubMed1996

Freeze-substitution as a preparative technique for immunoelectronmicroscopy: evaluation by atomic force microscopy.

Cryofixation followed by freeze substitution in osmium tetroxide was evaluated as a method for preparing biological specimens for immunoelectronmicroscopy. Samples were rapidly frozen by impact onto a sapphire block cooled with liquid nitrogen, substituted at -80 degrees C in acetone containing osmium tetroxide, and embedded in epoxy resin. With this protocol, excellent ultrastructure can be combined with localization of antigens that otherwise would be inactivated by the osmium, but labeling may need to be enhanced by chemically etching the sections prior to staining. The effects of etching on various structures in the sections were investigated by examining the sections with atomic force microscopy, an approach that yields three-dimensional views of the surface of the section. A considerable part of the section was removed or collapsed by the etching, and these effects occurred differentially in several components of the tissue and with different etching protocols. Nevertheless, the results suggest that the partial removal of the plastic by etching of freeze-substituted tissue can be explored as a method for exposing fine biological structures for observation with atomic force microscopy.

Animals↗

PubMed1996

Actin-based motility of isolated axoplasmic organelles.

We previously showed that axoplasmic organelles from the squid giant axon move toward the barbed ends of actin filaments and that KI-washed organelles separated from soluble proteins by sucrose density fractionation retain a 235-kDa putative myosin. Here, we examine the myosin-like activities of KI-washed organelles after sucrose density fractionation to address the question whether the myosin on these organelles is functional. By electron microscopy KI-washed organelles bound to actin filaments in the absence of ATP but not in its presence. Analysis of organelle-dependent ATPase activity over time and with varying amounts of organelles revealed a basal activity of 350 (range: 315-384) nmoles Pi/mg/min and an actin-activated activity of 774 (range: 560-988) nmoles/mg/min, a higher specific activity than for the other fractions. By video microscopy washed organelles moved in only one direction on actin filaments with a net velocity of 1.11 +/- .03 microns/s and an instantaneous velocity of 1.63 +/- 0.29 microns/s. By immunogold electronmicroscopy, 7% of KI-washed organelles were decorated with an anti-myosin antibody as compared to 0.5% with non-immune serum. Thus, some axoplasmic organelles have a tightly associated myosin-like activity.

Actins↗

PubMed1996

Plus-end motors override minus-end motors during transport of squid axon vesicles on microtubules.

Plus- and minus-end vesicle populations from squid axoplasm were isolated from each other by selective extraction of the minus-end vesicle motor followed by 5'-adenylyl imidodiphosphate (AMP-PNP)-induced microtubule affinity purification of the plus-end vesicles. In the presence of cytosol containing both plus- and minus-end motors, the isolated populations moved strictly in opposite directions along microtubules in vitro. Remarkably, when treated with trypsin before incubation with cytosol, purified plus-end vesicles moved exclusively to microtubule minus ends instead of moving in the normal plus-end direction. This reversal in the direction of movement of trypsinized plus-end vesicles, in light of further observation that cytosol promotes primarily minus-end movement of liposomes, suggests that the machinery for cytoplasmic dynein-driven, minus-end vesicle movement can establish a functional interaction with the lipid bilayers of both vesicle populations. The additional finding that kinesin overrides cytoplasmic dynein when both are bound to bead surfaces indicates that the direction of vesicle movement could be regulated simply by the presence or absence of a tightly bound, plus-end kinesin motor; being processive and tightly bound, the kinesin motor would override the activity of cytoplasmic dynein because the latter is weakly bound to vesicles and less processive. In support of this model, it was found that (a) only plus-end vesicles copurified with tightly bound kinesin motors; and (b) both plus- and minus-end vesicles bound cytoplasmic dynein from cytosol.

Adenylyl Imidodiphosphate↗

PubMed1995

Transport of cytoskeletal elements in the squid giant axon.

In order to explore how cytoskeletal proteins are moved by axonal transport, we injected fluorescent microtubules and actin filaments as well as exogenous particulates into squid giant axons and observed their movements by confocal microscopy. The squid giant axon is large enough to allow even cytoskeletal assemblies to be injected without damaging the axon or its transport mechanisms. Negatively charged, 10- to 500-nm beads and large dextrans moved down the axon, whereas small (70 kDa) dextrans diffused in all directions and 1000-nm beads did not move. Only particles with negative charge were transported. Microtubules and actin filaments, which have net negative charges, made saltatory movements down the axon, resulting in a net rate approximating that previously shown for slow transport of cytoskeletal elements. The present observations suggest that particle size and charge determine which materials are transported down the axon.

Actins↗

PubMed1995

Effect of calpain on the composition and structure of postsynaptic densities.

Endogenous calcium-activated proteases, the calpains, are thought to play a role in the regulation of postsynaptic function. Here we characterize some biochemical and morphological effects of calpain on isolated postsynaptic densities (PSDs). When a PSD preparation from rat forebrain was treated with exogenous calpain, many proteins, including spectrin, tubulin and the alpha-subunit of calcium calmodulin-dependent protein kinase (alpha-CaM kinase), were proteolyzed at varying rates, while another major protein, actin, remained intact. The selectivity of calpain action became more apparent in experiments designed to achieve limited proteolysis by using a lower calpain-to-protein ratio; it was possible to obtain extensive breakdown of spectrin with no decrease in the levels of either tubulin, alpha-CaM kinase, or actin. Electron microscopy of freeze-substituted preparations showed that limited calpain action caused a partial unraveling of the PSD, in which the characteristic central dense lamina became wider and less dense. We interpret these changes as due to calpain-mediated breakdown of cross-bridging elements, leading to a partial unraveling of the central PSD lamina. Opening up of the PSD structure following limited calpain action could facilitate exposure of previously occluded functional sites within the PSD and contribute to the modification of the synaptic function.

Animals↗

PubMed1995

Cytoplasmic constriction and vesiculation after axotomy in the squid giant axon.

The squid giant axon responded to a transection injury by producing a gradient of cytoplasmic and vesicular changes at the cut end. At the immediate opening of the cut axon the cytoplasm was fragmented and dispersed and the vesicles in this region were in rapid Brownian movement. Approximately 0.1 mm further in, at the site of maximal axonal constriction, the axoplasm was condensed into a compact, constricted mass containing many large vesicles. The axoplasm was normal a few millimetres beyond this constricted, vesiculated end. It appears that transection triggered the transformation of normal axoplasm into a tightly constricted, highly vesiculated structure. This modified axoplasm at the cut end may slow the spread of damage and degeneration by preventing the bulk outflow of axoplasm, by slowing down the loss of intracellular molecules and by slowing down the influx of destructive extracellular ions (like calcium and chloride).

Animals↗

PubMed1995

Conserved machinery of the bacterial flagellar motor.

Novel periplasmic and cytoplasmic structural modules of the bases of bacterial flagella have been observed in situ and isolated using new biochemical protocols. Flagellar rotation may depend upon interactions of these modules with the intramembrane particle rings, a ubiquitous feature of flagellar bases necessary for torque generation. The outer membrane-associated basal disk of the Wolinella succinogenes polar flagellum has architecture well suited for interaction with the ring particles. However, antibody against the main W. succinogenes basal disk protein did not cross-react with flagella-enriched fractions from Salmonella typhimurium and Bacillus firmus; nor have such structures been observed in these species thus far. Antibodies against two S. typhimurium proteins, FliG and FliM, known to be involved in motor function and part of the cytoplasmic module in this species cross-reacted with flagella-enriched fractions from both W. succinogenes and B. firmus. In addition, flagellar cytoplasmic structure could be isolated from B. firmus. The basal disk may anchor the flagellar motor to the cell wall in some polar bacteria, but this does not seem to be a unique strategy. In contrast, the data indicate that the cytoplasmic module is conserved.

Antigens, Bacterial↗

PubMed1994

Continuous network of endoplasmic reticulum in cerebellar Purkinje neurons.

Purkinje neurons in rat cerebellar slices injected with an oil drop saturated with 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC16(3) or DiI] to label the endoplasmic reticulum were observed by confocal microscopy. DiI spread throughout the cell body and dendrites and into the axon. DiI spreading is due to diffusion in a continuous bilayer and is not due to membrane trafficking because it also spreads in fixed neurons. DiI stained such features of the endoplasmic reticulum as densities at branch points, reticular networks in the cell body and dendrites, nuclear envelope, spines, and aggregates formed during anoxia nuclear envelope, spines, and aggregates formed during anoxia in low extracellular Ca2+. In cultured rat hippocampal neurons, where optical conditions provide more detail, DiI labeled a clearly delineated network of endoplasmic reticulum in the cell body. We conclude that there is a continuous compartment of endoplasmic reticulum extending from the cell body throughout the dendrites. This compartment may coordinate and integrate neuronal functions.

Animals↗

PubMed1994

Interactions among endoplasmic reticulum, microtubules, and retrograde movements of the cell surface.

Relationships among the endoplasmic reticulum (ER), microtubules, and bead movements on the cell surface were investigated in the thin peripheral region of A6 cells, a frog kidney cell line. ER tubules were often aligned with microtubules, as shown by double-labeling with DiOC6(3) and anti-tubulin in fixed cells. In living cells stained with DiOC6(3) and observed in time lapse, there were frequent extensions, but few retractions, of ER tubules. In addition, there was a steady retrograde (towards the cell center) movement of all of the ER at approximately 0.3 microns/min. Since microtubules are often aligned with the ER, microtubules must also be moving retrogradely. By simultaneous imaging, it was found that the ER moves retrogradely at the same rate as aminated latex beads on the cell surface. This indicates that the mechanisms for ER and bead movement are closely related. Cytochalasin B stopped bead and ER movement in most of the cells, providing evidence that actin is involved in both retrograde movements. The ER retracted towards the cell center in nocodazole while both ER and microtubules retracted in taxol. Time lapse observations showed that for both drugs, the retraction of the ER is the result of retrograde movement in the absence of new ER extensions. Presumably, ER extensions do not occur in nocodazole because of the absence of microtubules, and do not occur in taxol because taxol-stabilized microtubules move retrogradely and there is no polymerization of new microtubule tracks for ER elongation.

Actins↗

PubMed1993

Evidence for myosin motors on organelles in squid axoplasm.

Squid axoplasm has proved a rich source for the identification of motors involved in organelle transport. Recently, squid axoplasmic organelles have been shown to move on invisible tracks that are sensitive to cytochalasin, suggesting that these tracks are actin filaments. Here, an assay is described that permits observation of organelles moving on unipolar actin bundles. This assay is used to demonstrate that axoplasmic organelles move on actin filaments in the barbed-end direction, suggesting the presence of a myosin motor on axoplasmic organelles. Indeed, axoplasm contains actin-dependent ATPase activity, and a pan-myosin antibody recognized at least four bands in Western blots of axoplasm. An approximately 235-kDa band copurified in sucrose gradients with KI-extracted axoplasmic organelles, and the myosin antibody stained the organelle surfaces by immunogold electron microscopy. The myosin is present on the surface of at least some axoplasmic organelles and thus may be involved in their transport through the axoplasm, their movement through the cortical actin in the synapse, or some other aspect of axonal function.

Acrosome↗

PubMed1993

Single kinesin molecules crossbridge microtubules in vitro.

Kinesin is a cytoplasmic motor protein that moves along microtubules and can induce microtubule bundling and sliding in vitro. To determine how kinesin mediates microtubule interactions, we determined the shapes and mass distributions of squid brain kinesin, taxol-stabilized microtubules (squid and bovine), and adenosine 5'-[beta, gamma-imido]triphosphate-stabilized kinesin-microtubule complexes by high-resolution metal replication and by low-temperature, low-dose dark-field scanning transmission electron microscopy of unfixed, directly frozen preparations. Mass mapping by electron microscopy revealed kinesins loosely attached to the carbon support as asymmetrical dumbbell-shaped molecules, 40-52 nm long, with a mass of 379 +/- 15 kDa. The mass distribution and shape of these molecules suggest that these images represent kinesin in a shortened conformation. Kinesin-microtubule complexes were organized as bundles of linearly arrayed microtubules, stitched together at irregular intervals by cross-bridges typically < or = 25 nm long. The crossbridges had a mass of 360 +/- 15 kDa, consistent with one kinesin per crossbridge. These results suggest that kinesin has a second microtubule binding site in addition to the known site on the motor domain of the heavy chain; this second site may be located near the C terminus of the heavy chains or on the associated light chains. Thus, kinesin could play a role in either crosslinking or sliding microtubules.

Animals↗

PubMed1993

Inhibition of endogenous phosphatase in a postsynaptic density fraction allows extensive phosphorylation of the major postsynaptic density protein.

The major postsynaptic density protein, proposed to be a calcium/calmodulin-dependent protein kinase, becomes phosphorylated when a postsynaptic density preparation from rat cerebral cortex is incubated in medium containing calcium and calmodulin. Upon longer incubation, however, the level of phosphorylation declines, suggesting the presence of a phosphatase activity. When Microcystin-LR, a phosphatase inhibitor, is included in the phosphorylation medium, the decline in phosphorylation is prevented and a higher maximal level of phosphorylation can be achieved. Under these conditions, the maximal phosphorylation of major postsynaptic density protein is accompanied by a nearly complete shift in its electrophoretic mobility from 50 kDa to 54 kDa, similar to that described for the alpha subunit of the soluble calcium/calmodulin-dependent protein kinase II. Of the four major groups of serine/threonine protein phosphatases, the enzyme responsible for the dephosphorylation of major postsynaptic density protein is neither type 2C, which is insensitive to Microcystin-LR, nor type 2B, which is calcium-dependent. As Microcystin-LR is much more potent than okadaic acid in inhibiting the dephosphorylation of major postsynaptic density protein, it is likely that the postsynaptic density-associated phosphatase is a type 1. The above results indicate that the relatively low level of phosphorylation of the major postsynaptic density protein observed in preparations containing postsynaptic densities is not due to a difference between the cytoplasmic and postsynaptic density-associated calcium/calmodulin-dependent kinases as previously proposed, but to a phosphatase activity, presumably belonging to the type 1 group.

Animals↗

PubMed1993

Multiple structural types of gap junctions in mouse lens.

Gap junctions in the epithelium and superficial fiber cells from young mice were examined in lenses prepared by rapid-freezing, and processed for freeze-substitution and freeze-fracture electron microscopy. There appeared to be three structural types of gap junction: one type between epithelial cells and two types between fiber cells. Epithelial gap junctions seen by freeze-substitution were approximately 20 nm thick and consistently associated with layers of dense material lying along both cytoplasmic surfaces. Fiber gap junctions, in contrast, were 15-16 nm (type 1) or 17-18 nm thick (type 2), and had little associated cytoplasmic material. Type 1 fiber gap junctions were extensive in flat expanses of cell membrane and had a thin, discontinuous central lamina, whereas type 2 fiber gap junctions were associated with the ball-and-socket domains and exhibited a dense, continuous central lamina. Both types of fiber gap junction had a diffuse arrangement of junctional intramembrane particles, whereas particles and pits of epithelial gap junctions were in a tight, hexagonal configuration. The type 2 fiber gap junctions, however, had a larger particle size (approximately 9 nm) than the type 1 (approximately 7.5 nm). In addition, a large number of junctional particles typified the E-faces of both fiber types but not the epithelial type of gap junction. Gap junctions between fiber and epithelial cells had structural features of type 1 fiber gap junctions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

The cytoplasmic component of the bacterial flagellar motor.

We have used electron microscopy to examine freshly isolated Salmonella typhimurium and Escherichia coli basal flagellar fragments, purified without resort to extremes of pH or ionic strength. Such fragments contain the large bell-like basal structures visualized recently in freeze-substituted or fixed preparations. We have found mot (non-motile) mutants produced by lesions in fli genes (G, M, N) in which the bell structures do not coisolate with the flagellar basal body. The coisolation of the bell with the flagellar basal body was unaffected in strains lacking the genes for the motility-associated Mot proteins or for the Che family of proteins, which are necessary for chemotaxis. Proper assembly and interaction of the cytoplasmically located bell with the membrane-associated flagellar basal structures appears to be necessary for motor function. The FliG, FliM, and FliN proteins are thought to form a structural complex responsible for energization and switching of the flagellar motor. Our findings are consistent with the existence of such a complex and imply that it forms part of the flagellar bell.

Cell Fractionation↗