Browsing by Subject "Microscopy, Electron"

Now showing 1 - 9 of 9
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    Association of an axonally transported polypeptide (H) with 100-A filaments. Use of immunoaffinity electron microscope grids.
    (The Journal of cell biology, 1980-06) Willard, M; Simon, C; Baitinger, C; Levine, J; Skene, P
    Polypeptide H (mol wt 195,000) is axonally transported in rabbit retinal ganglion cells at a velocity of 0.7--1.1 mm/d, i.e., in the most slowly moving of the five transport groups described in these neurons. To identify the organelle with which H is associated, we purified H, prepared antibodies directed against it, and adsorbed the antibodies onto Formvar-coated electron microscope grids. When the resulting "immuno-affinity grids" were incubated with extracts of spinal cord and then examined in the electron microscope, they contained as many as 100 times more 100-A filaments than did grids coated similarly with nonimmune IgG. The ability of the anti-H IgG to specifically adsorb filaments to grids was completely blocked by incubating the IgG with polypeptide H. The 100-A filaments adsorbed to anti-H immunoaffinity grids could be specifically decorated by incubating them with anti-H IgG. These observations demonstrate that H antigens (and most likely H itself) are associated with 100-A neurofilaments. In addition, they suggest that the use of immunoaffinity grids may be a useful approach for determining the organelle associations of polypeptides.
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    Conformational changes of FtsZ reported by tryptophan mutants.
    (Biochemistry, 2011-05-03) Chen, Yaodong; Erickson, Harold P
    E. coli FtsZ has no native tryptophan. We showed previously that the mutant FtsZ L68W gave a 2.5-fold increase in trp fluorescence when assembly was induced by GTP. L68 is probably buried in the protofilament interface upon assembly, causing the fluorescence increase. In the present study we introduced trp residues at several other locations and examined them for assembly-induced fluorescence changes. L189W, located on helix H7 and buried between the N- and C-terminal subdomains, showed a large fluorescence increase, comparable to L68W. This may reflect a shift or rotation of the two subdomains relative to each other. L160W showed a smaller increase in fluorescence, and Y222W a decrease in fluorescence, upon assembly. These two are located on the surface of the N and C subdomains, near the domain boundary. The changes in fluorescence may reflect movements of the domains or of nearby side chains. We prepared a double mutant Y222W/S151C and coupled ATTO-655 to the cys. The Cα of trp in the C-terminal subdomain was 10 Å away from that of the cys in the N-terminal subdomain, permitting the ATTO to make van der Waals contact with the trp. The ATTO fluorescence showed strong tryptophan-induced quenching. The quenching was reduced following assembly, consistent with a movement apart of the two subdomains. Movements of one to several angstroms are probably sufficient to account for the changes in trp fluorescence and trp-induced quenching of ATTO. Assembly in GDP plus DEAE dextran produces tubular polymers that are related to the highly curved, mini-ring conformation. No change in trp fluorescence was observed upon assembly of these tubes, suggesting that the mini-ring conformation is the same as that of a relaxed, monomeric FtsZ.
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    Epithelial injury and interstitial fibrosis in the proximal alveolar regions of rats chronically exposed to a simulated pattern of urban ambient ozone.
    (Toxicology and applied pharmacology, 1992-08) Chang, LY; Huang, Y; Stockstill, BL; Graham, JA; Grose, EC; Menache, MG; Miller, FJ; Costa, DL; Crapo, JD
    Electron microscopic morphometry was used to study the development of lung injury during and after chronic (78 weeks) exposure to a pattern of ozone (O3) designed to simulate high urban ambient concentrations that occur in some environments. The daily exposure regimen consisted of a 13-hr background of 0.06 ppm, an exposure peak that rose from 0.06 to 0.25 ppm, and returned to the background level over a 9-hr period, and 2-hr downtime for maintenance. Rats were exposed for 1, 3, 13, and 78 weeks. Additional groups of rats exposed for 13 or 78 weeks were allowed to recover in filtered clean air for 6 or 17 weeks, respectively. Rats exposed to filtered air for the same lengths of time were used as controls. Samples from proximal alveolar regions and terminal bronchioles were obtained by microdissection. Analysis of the proximal alveolar region revealed a biphasic response. Acute tissue reactions after 1 week of exposure included epithelial inflammation, interstitial edema, interstitial cell hypertrophy, and influx of macrophages. These responses subsided after 3 weeks of exposure. Progressive epithelial and interstitial tissue responses developed with prolonged exposure and included epithelial hyperplasia, fibroblast proliferation, and interstitial matrix accumulation. The epithelial responses involved both type I and type II epithelial cells. Alveolar type I cells increased in number, became thicker, and covered a smaller average surface area. These changes persisted throughout the entire exposure and did not change during the recovery period, indicating the sensitivity of these cells to injury. The main response of type II epithelial cells was cell proliferation. The accumulation of interstitial matrix after chronic exposure consisted of deposition of both increased amounts of basement membrane and collagen fibers. Interstitial matrix accumulation underwent partial recovery during follow-up periods in air; however, the thickening of the basement membrane did not resolve. Analysis of terminal bronchioles showed that short-term exposure to O3 caused a loss of ciliated cells and differentiation of preciliated and Clara cells. The bronchiolar cell population stabilized on continued exposure; however, chronic exposure resulted in structural changes, suggesting injury to both ciliated and Clara cells. We conclude that chronic exposure to low levels of O3 causes epithelial inflammation and interstitial fibrosis in the proximal alveolar region and bronchiolar epithelial cell injury.
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    FtsZ Protofilament Curvature Is the Opposite of Tubulin Rings.
    (Biochemistry, 2016-07) Housman, Max; Milam, Sara L; Moore, Desmond A; Osawa, Masaki; Erickson, Harold P
    FtsZ protofilaments (pfs) form the bacterial cytokinetic Z ring. Previous work suggested that a conformational change from straight to curved pfs generated the constriction force. In the simplest model, the C-terminal membrane tether is on the outside of the curved pf, facing the membrane. Tubulin, a homologue of FtsZ, also forms pfs with a curved conformation. However, it is well-established that tubulin rings have the C terminus on the inside of the ring. Could FtsZ and tubulin rings have the opposite curvature? In this study, we explored the FtsZ curvature direction by fusing large protein tags to the FtsZ termini. Thin section electron microscopy showed that the C-terminal tag was on the outside, consistent with the bending pf model. This has interesting implications for the evolution of tubulin. Tubulin likely began with the curvature of FtsZ, but evolution managed to reverse direction to produce outward-curving rings, which are useful for pulling chromosomes.
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    Functional elastic hydrogel as recyclable membrane for the adsorption and degradation of methylene blue.
    (PloS one, 2014-01) Bao, Song; Wu, Dongbei; Wang, Qigang; Su, Teng
    Developing the application of high-strength hydrogels has gained much attention in the fields of medical, pharmacy, and pollutant removal due to their versatility and stimulus-responsive properties. In this presentation, a high-strength freestanding elastic hydrogel membrane was constructed by clay nanosheets, N, N-dimethylacrylamide and 2-acrylamide-2-methylpropanesulfonic acid for adsorption of methylene blue and heavy metal ions. The maximum values of elongation and Young's modulus for 0.5% AMPSNa hydrogel were 1901% and 949.4 kPa, respectively, much higher than those of traditional hydrogels. The adsorptions were confirmed to follow pseudo-second kinetic equation and Langmuir isotherm model fits the data well. The maximum adsorption capacity of hydrogel towards methylene blue was 434.8 mg g(-1). The hydrogel also exhibited higher separation selectivity to Pb(2+) than Cu(2+). The methylene blue adsorbed onto the hydrogel membrane can be photocatalytically degraded by Fenton agent and the hydrogel membrane could be recycled at least five times without obvious loss in mechanical properties. In conclusion, this presentation demonstrates a convenient strategy to prepare tough and elastic clay nanocomposite hydrogel, which can not only be applied as recyclable membrane for the photocatalytic degradation of organic dye, but also for the recovery of valuables.
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    Negative-stain electron microscopy of inside-out FtsZ rings reconstituted on artificial membrane tubules show ribbons of protofilaments.
    (Biophysical journal, 2012-07) Milam, Sara L; Osawa, Masaki; Erickson, Harold P
    FtsZ, the primary cytoskeletal element of the Z ring, which constricts to divide bacteria, assembles into short, one-stranded filaments in vitro. These must be further assembled to make the Z ring in bacteria. Conventional electron microscopy (EM) has failed to image the Z ring or resolve its substructure. Here we describe a procedure that enabled us to image reconstructed, inside-out FtsZ rings by negative-stain EM, revealing the arrangement of filaments. We took advantage of a unique lipid that spontaneously forms 500 nm diameter tubules in solution. We optimized conditions for Z-ring assembly with fluorescence light microscopy and then prepared specimens for negative-stain EM. Reconstituted FtsZ rings, encircling the tubules, were clearly resolved. The rings appeared as ribbons of filaments packed side by side with virtually no space between neighboring filaments. The rings were separated by variable expanses of empty tubule as seen by light microscopy or EM. The width varied considerably from one ring to another, but each ring maintained a constant width around its circumference. The inside-out FtsZ rings moved back and forth along the tubules and exchanged subunits with solution, similarly to Z rings reconstituted outside or inside tubular liposomes. FtsZ from Escherichia coli and Mycobacterium tuberculosis assembled rings of similar structure, suggesting a universal structure across bacterial species. Previous models for the Z ring in bacteria have favored a structure of widely scattered filaments that are not in contact. The ribbon structure that we discovered here for reconstituted inside-out FtsZ rings provides what to our knowledge is new evidence that the Z ring in bacteria may involve lateral association of protofilaments.
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    Postsynaptic positioning of endocytic zones and AMPA receptor cycling by physical coupling of dynamin-3 to Homer.
    (Neuron, 2007-09) Lu, Jiuyi; Helton, Thomas D; Blanpied, Thomas A; Rácz, Bence; Newpher, Thomas M; Weinberg, Richard J; Ehlers, Michael D
    Endocytosis of AMPA receptors and other postsynaptic cargo occurs at endocytic zones (EZs), stably positioned sites of clathrin adjacent to the postsynaptic density (PSD). The tight localization of postsynaptic endocytosis is thought to control spine composition and regulate synaptic transmission. However, the mechanisms that situate the EZ near the PSD and the role of spine endocytosis in synaptic transmission are unknown. Here, we report that a physical link between dynamin-3 and the postsynaptic adaptor Homer positions the EZ near the PSD. Disruption of dynamin-3 or its interaction with Homer uncouples the PSD from the EZ, resulting in synapses lacking postsynaptic clathrin. Loss of the EZ leads to a loss of synaptic AMPA receptors and reduced excitatory synaptic transmission that corresponds with impaired synaptic recycling. Thus, a physical link between the PSD and the EZ ensures localized endocytosis and recycling by recapturing and maintaining a proximate pool of cycling AMPA receptors.
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    SulA inhibits assembly of FtsZ by a simple sequestration mechanism.
    (Biochemistry, 2012-04) Chen, Yaodong; Milam, Sara L; Erickson, Harold P
    We have investigated the inhibition by SulA of the assembly of Escherichia coli FtsZ. Using quantitative GTPase and fluorescence assays, we found that SulA inhibition resulted in an increase in the apparent critical concentration for FtsZ assembly. The increase in apparent critical concentration was always less than the total amount of SulA added, suggesting that the association of SulA and FtsZ was of modest affinity. Isothermal titration calorimetry gave a value of 0.78 μM for the dissociation constant of the FtsZ-SulA complex, similar in magnitude to the 0.72 μM critical concentration of FtsZ protofilament assembly at steady state. We modeled the reaction as an equilibrium competition between (a) FtsZ subunits assembling onto protofilaments or (b) binding SulA. When FtsZ was assembled in GMPCPP or in EDTA, the inhibition by SulA was reduced. The reduced inhibition could be explained by a 3- and 10-fold weaker binding of SulA to FtsZ. The mutant D212G, which has no GTPase activity and therefore minimal subunit cycling, was shown here to assemble one-stranded protofilaments, and the assembly was blocked by SulA. We also assayed the SulA and FtsZ proteins from Pseudomonas. The SulA inhibition was stronger than with the E. coli proteins, and the model indicated a 5-fold higher affinity of Pseudomonas SulA for FtsZ.
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    UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling.
    (Proc Natl Acad Sci U S A, 2013-08-20) Moore, Carlene; Cevikbas, Ferda; Pasolli, H Amalia; Chen, Yong; Kong, Wei; Kempkes, Cordula; Parekh, Puja; Lee, Suk Hee; Kontchou, Nelly-Ange; Yeh, Iwei; Jokerst, Nan Marie; Fuchs, Elaine; Steinhoff, Martin; Liedtke, Wolfgang B
    At our body surface, the epidermis absorbs UV radiation. UV overexposure leads to sunburn with tissue injury and pain. To understand how, we focus on TRPV4, a nonselective cation channel highly expressed in epithelial skin cells and known to function in sensory transduction, a property shared with other transient receptor potential channels. We show that following UVB exposure mice with induced Trpv4 deletions, specifically in keratinocytes, are less sensitive to noxious thermal and mechanical stimuli than control animals. Exploring the mechanism, we find that epidermal TRPV4 orchestrates UVB-evoked skin tissue damage and increased expression of the proalgesic/algogenic mediator endothelin-1. In culture, UVB causes a direct, TRPV4-dependent Ca(2+) response in keratinocytes. In mice, topical treatment with a TRPV4-selective inhibitor decreases UVB-evoked pain behavior, epidermal tissue damage, and endothelin-1 expression. In humans, sunburn enhances epidermal expression of TRPV4 and endothelin-1, underscoring the potential of keratinocyte-derived TRPV4 as a therapeutic target for UVB-induced sunburn, in particular pain.