Biochemical and microscopic analysis of inflammasome complex formation

Author(s):  
Patrycja Swacha ◽  
Nelson O. Gekara ◽  
Saskia F. Erttmann
2005 ◽  
Vol 79 (13) ◽  
pp. 8535-8544 ◽  
Author(s):  
Ying-Ju Lin ◽  
Suh-Chin Wu

ABSTRACT The formation of the flavivirus prM-E complex is an important step for the biogenesis of immature virions, which is followed by a subsequent cleavage of prM to M protein through cellular protease to result in the production and release of mature virions. In this study, the intracellular formation of the prM-E complex of Japanese encephalitis virus was investigated by baculovirus coexpression of prM and E in trans in Sf9 insect cells as analyzed by anti-E antibody immunoprecipitation and sucrose gradient sedimentation analysis. A series of carboxyl-terminally truncated prM mutant baculoviruses was constructed to demonstrate that the truncations of the transmembrane (TM) region resulted in a reduction of the formation of the stable prM-E complex by approximately 40% for the TM1 (at residues 130 to 147 [prM130-147]) truncation and 20% for TM2 (at prM153-167) truncation. Alanine-scanning site-directed mutagenesis on the prM99-103 region indicated that the His99 residue was the critical prM binding element for stable prM-E heterodimeric complex formation. The single amino acid mutation at the His99 residue of prM abolishing the prM-E interaction was not due to reduced expression or different subcellular location of the mutant prM protein involved in prM-E interactions as characterized by pulse-chase labeling and confocal scanning microscopic analysis. Recombinant subviral particles were detected in the Sf9 cell culture supernatants by baculovirus coexpression of prM and E proteins but not with the prM H99A mutant. Sequence alignment analysis was further conducted with different groups of flaviviruses to show that the prM H99 residues are generally conserved. Our findings are the first report to characterize the minimum binding elements of the prM protein that are involved in prM-E interactions of flaviviruses. This information, concerning a molecular framework for the prM protein, is considered to elucidate the structure/function relationship of the prM-E complex synthesis and provide the proper trajectory for flavivirus assembly and maturation.


Author(s):  
F.J. Sjostrand

In the 1940's and 1950's electron microscopy conferences were attended with everybody interested in learning about the latest technical developments for one very obvious reason. There was the electron microscope with its outstanding performance but nobody could make very much use of it because we were lacking proper techniques to prepare biological specimens. The development of the thin sectioning technique with its perfectioning in 1952 changed the situation and systematic analysis of the structure of cells could now be pursued. Since then electron microscopists have in general become satisfied with the level of resolution at which cellular structures can be analyzed when applying this technique. There has been little interest in trying to push the limit of resolution closer to that determined by the resolving power of the electron microscope.


Author(s):  
Charlotte L. Ownby ◽  
David Cameron ◽  
Anthony T. Tu

In the United States the major health problem resulting from snakebite poisoning is local tissue damage, i.e. hemorrhage and myonecrosis. Since commercial antivenin does not usually prevent such damage to tissue, a more effective treatment of snakebite-induced myonecrosis is needed. To aid in the development of such a treatment the pathogenesis of myonecrosis induced by a pure component of rattlesnake venom was studied at the electron microscopic level.The pure component, a small (4,300 mol. wt.), basic (isoelectric point of 9.6) protein, was isolated from crude prairie rattlesnake (Crotalus viridis viridis) venom by gel filtration (Sephadex G-50) followed by cation exchange chromatography (Sephadex C-25), and shown to be pure by electrophoresis. Selection of the myotoxic component was based on light microscopic observations of injected mouse muscle.


Author(s):  
W. K. Jones ◽  
J. Robbins

Two myosin heavy chains (MyHC) are expressed in the mammalian heart and are differentially regulated during development. In the mouse, the α-MyHC is expressed constitutively in the atrium. At birth, the β-MyHC is downregulated and replaced by the α-MyHC, which is the sole cardiac MyHC isoform in the adult heart. We have employed transgenic and gene-targeting methodologies to study the regulation of cardiac MyHC gene expression and the functional and developmental consequences of altered α-MyHC expression in the mouse.We previously characterized an α-MyHC promoter capable of driving tissue-specific and developmentally correct expression of a CAT (chloramphenicol acetyltransferase) marker in the mouse. Tissue surveys detected a small amount of CAT activity in the lung (Fig. 1a). The results of in situ hybridization analyses indicated that the pattern of CAT transcript in the adult heart (Fig. 1b, top panel) is the same as that of α-MyHC (Fig. 1b, lower panel). The α-MyHC gene is expressed in a layer of cardiac muscle (pulmonary myocardium) associated with the pulmonary veins (Fig. 1c). These studies extend our understanding of α-MyHC expression and delimit a third cardiac compartment.


Author(s):  
Ralph M. Albrecht ◽  
Scott R. Simmons ◽  
Marek Malecki

The development of video-enhanced light microscopy (LM) as well as associated image processing and analysis have significantly broadened the scope of investigations which can be undertaken using (LM). Interference/polarization based microscopies can provide high resolution and higher levels of “detectability” especially in unstained living systems. Confocal light microscopy also holds the promise of further improvements in resolution, fluorescence studies, and 3 dimensional reconstruction. Video technology now provides, among other things, a means to detect differences in contrast difficult to detect with the human eye; furthermore, computerized image capture, processing, and analysis can be used to enhance features of interest, average images, subtract background, and provide a quantitative basis to studies of cells, cell features, cell labelling, and so forth. Improvements in video technology, image capture, and cost-effective computer image analysis/processing have contributed to the utility and potential of the various interference and confocal microscopic instrumentation.Electron microscopic technology has made advances as well. Microprocessor control and improved design have contributed to high resolution SEMs which have imaging capability at the molecular level and can operate at a range of accelerating voltages starting at 1KV. Improvements have also been seen in the HVEM and IVEM transmission instruments. As a whole, these advances in LM and EM microscopic technology provide the biologist with an array of information on structure, composition, and function which can be obtained from a single specimen. Corrrelative light microscopic analysis permits examination of living specimens and is critical where the “history” of a cell, cellular components, or labels needs to be known up to the time of chemical or physical fixation. Features such as cytoskeletal elements or gold label as small as 0.01 μm, well below the 0.2 μm limits of LM resolution, can be “detected” and their movement followed by VDIC-LM. Appropriate identification and preparation can then lead to the examination of surface detail and surface label with stereo LV-HR-SEM. Increasing the KV in the HR-SEM while viewing uncoated or thinly coated specimens can provide information from beneath the surface as well as increasing Z contrast so that positive identification of surface and subsurface colloidal gold or other heavy metal labelled/stained material is possible. Further examination of the same cells using stereo HVEM or IVEM provides information on internal ultrastructure and on the relationship of labelled material to cytoskeletal or organellar distribution, A wide variety of investigations can benefit from this correlative approach and a number of instrumentational configurations and preparative pathways can be tailored for the particular study. For a surprisingly small investment in time and technique, it is often possible to clear ambiguities or questions that arise when a finding is presented in the context of only one modality.


Author(s):  
William Theurkauf

Cell division in eucaryotes depends on coordinated changes in nuclear and cytoskeletal components. In Drosophila melanogaster embryos, the first 13 nuclear divisions occur without cytokinesis. During the final four divisions, nuclei divide in a uniform monolayer at the surface of the embryo. These surface divisions are accompanied by dramatic changes in cortical actin and microtubule structure (Karr and Alberts, 1986), and inhibitor studies indicate that these changes are essential to orderly mitosis (Zalokar and Erk, 1976). Because the early embryo is syncytial, fluorescent probes introduced by microinjection are incorporated in structures associated with all of the nuclei in the blastoderm. In addition, the nuclei divide synchronously every 10 to 20 min. These characteristics make the syncytial blastoderm embryo an excellent system for the analysis of mitotic reorganization of both nuclear and cytoskeletal elements. However, the Drosophila embryo is a large cell, and resolution of cytoskeletal filaments and nuclear structure is hampered by out-of focus signal.


Sign in / Sign up

Export Citation Format

Share Document