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991.
During the life cycle of a membrane protein its molecular structure may change and for aggregated proteins this process may be observed on the supramolecular level. Here we demonstrate that this is the case for gap junction channels which maintain cell-cell communication. Freshly synthesized connexins are integrated as hexamers (connexons) into the plasma membrane where they form plaques after pairing with connexons of an attached cell. We inhibited protein trafficking by applying the fungal metabolite brefeldin A (BFA), quantified cell-cell coupling by calcein transfer and fluorescence-activated flow cytometry, and examined the degradation and formation of gap junction plaques by indirect immunofluorescence and immunogold labeling. Under control conditions 50% of the detected plaques were ubiquitylated and less than 10% showed a two-dimensional crystalline packing. One hour after BFA reversal about 60% of the plaques were crystalline and ubiquitylation dropped to 14%. Label for ubiquitin was predominantly found on non-crystalline plaques. We, therefore, conclude that newly formed gap junction plaques are of crystalline morphology which changes to a pleomorphic structure when individual channels are modified during their aging process. This dynamic in plaque morphology correlates with channel inactivation and plaque ubiquitylation. 相似文献
992.
Mac Millan SV Ishiyama N White GF Palaniyar N Hallett FR Harauz G 《European journal of cell biology》2000,79(5):327-335
Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid. 相似文献
993.
Tracking chromaffin granules on their way through the actin cortex 总被引:13,自引:0,他引:13
Quantitative time-lapse evanescent-wave imaging of individual fluorescently labelled chromaffin granules was used for kinetic
analysis of granule trafficking through a ∼300-nm (1/e2) optical section beneath the plasma membrane. The mean squared displacement (MSD) was used to estimate the three-dimensional
diffusion coefficient (D
(3)). We calculated the granules' speed, frame-to-frame displacement and direction and their autocorrelation to identify different
stages of approach to the membrane. D
(3) was about 10,000 times lower than expected for free diffusion. Granules located ∼60 nm beneath the plasma membrane moved
on random tracks (D
(3)≈10−10 cm2 s−1) with several reversals in direction before they approached their docking site at angles larger than 45∘. Docking was observed as a loss of vesicle mobility by two orders of magnitude within <100 ms. For longer observation times
the MSD saturated, as if the granules' movement was confined to a volume only slightly larger than the granule. Rarely, the
local random motion was superimposed with a directed movement in a plane beneath the membrane. Stimulation of exocytosis selectively
depleted the immobile, near-membrane granule population and caused a recruitment of distant granules to sites at the plasma
membrane. Their absolute mobility levels were not significantly altered. Application of latrunculin or jasplakinolide to change
F-actin polymerisation caused a change in D
(3) of the mobile granule population as well as a reduction of the rate of release, suggesting that granule mobility is constrained
by the filamentous actin meshwork and that stimulation-dependent changes in actin viscosity propel granules through the actin
cortex.
Received: 18 November 1999 / Revised version: 26 January 2000 / Accepted: 2 February 2000 相似文献
994.
Santos MJ Henderson SC Moser AB Moser HW Lazarow PB 《Biology of the cell / under the auspices of the European Cell Biology Organization》2000,92(2):85-94
Peroxisome ghosts are aberrant peroxisomal structures found in cultured skin fibroblasts from patients affected by Zellweger Syndrome (ZS), a genetic disorder of peroxisomal assembly. They contain peroxisomal integral membrane proteins (PxIMPs) and they lack most of the matrix enzymes that should be inside the organelle (Santos et al., Science 239 (1988) 1536-1538). Considerable evidence indicates that these ghosts result from genetic defects in the cellular machinery for importing newly-synthesized peroxisomal proteins into the organelle. In contrast to these observations, (Heikoop et al., Eur. J. Cell Biol. 57 (1992) 165-171) report that in Zellweger Syndrome, peroxisomal membranes are located within lysosomes and/or contain lysosomal enzymes. We have undertaken a more detailed and systematic investigation of this matter, employing confocal laser scanning microscopy (CLSM). In fibroblasts derived from ZS patients belonging to different complementation groups, peroxisomes were labeled with antibodies against PxIMPs and lysosomes were labeled with an antibody against a lysosome associated membrane protein (LAMP-2) or with LysoTracker. The results unambiguously demonstrated no appreciable colocalization of PxIMPs and LAMPs (or LysoTracker), indicating that peroxisomal ghosts are distinct subcellular structures, occupying separate subcellular locations. 相似文献
995.
Pradal G Berreur M Pouyet B Riet A 《Biology of the cell / under the auspices of the European Cell Biology Organization》2000,92(7):545-554
Parietal cells of the gastric fundic mucosa are small and contain only a few tiny mitochondria when they begin to differentiate from mucous neck cells. The canalicular ATPase activity characteristic of mature parietal cells is discrete in these young cells, whereas areas of very high activity are apparent in the Golgi complex, reticulum, nuclear envelope, mitochondrial wall, and plasma membrane. Close relations and contacts occur between mitochondria and these organelles, and the size and number of mitochondria increase progressively. These relations, as well as mitochondrial ATPase activity (a true differentiation marker), cease once the mitochondria become as numerous and large as those of a mature parietal cell. Our observations suggest that a secondary form of mitochondrial biogenesis, involving the massive participation of other organelles and independent of the classical mechanisms inherent in mitosis, occurs in parietal cells at the beginning of G1 phase during the 6 days of their maturation. 相似文献
996.
Michailova L Markova N Radoucheva T Stoitsova S Kussovski V Jordanova M 《FEMS immunology and medical microbiology》2000,28(1):55-65
Groups of rats were injected intraperitoneally with cell wall-deficient (L) forms of Streptococcus pyogenes, with their parental (S) forms, as well as with a combined inoculum of both forms (S+L). Peritoneal exudate samples were harvested on days 1, 3, 7, 15 and 30 after challenge and were investigated by microbiological, electron microscopic, cytometric and biochemical methods. Parental S forms were isolated from peritoneal exudate samples up to day 15 post infection, while L form cultures were isolated until the end of the examined interval. Electron microscopic examination revealed continuous adhesion of L forms on the macrophage surface as well as intracellular persistence inside them. It was demonstrated that the intraperitoneal inflammatory response to L form infection was higher than to the other infections and the monocyte-macrophage populations were predominant. The established atypical behaviour and long survival of S. pyogenes L forms in the rat's peritoneum could explain some of the mechanisms of the pathogens' persistence as well as the reasons for chronic streptococcal infections. 相似文献
997.
In our study we investigated hemispherical phospholipid bilayer membranes and phospholipid vesicles made from hexadecaprenyl
monophosphate (C80-P), dioleoylphosphatidylocholine (DOPC) and their mixtures by voltammetric and transmission electron microscopy (TEM) techniques.
The current-voltage characteristics, the membrane conductance-temperature relationships and the membrane breakdown voltage
have been measured for different mixtures of C80-P/DOPC. The membrane hydrophobic thickness and the activation energy of ion migration across the membrane have been determined.
Hexadecaprenyl monophosphate decreased in comparison with DOPC bilayers, the membrane conductance, increased the activation
energy and the membrane breakdown voltage for the various value of C80-P/DOPC mole ratio, respectively. The TEM micrographs of C80-P, DOPC and C80-P/DOPC lipid vesicles showed several characteristic structures, which have been described. The data indicate that hexadecaprenyl
monophosphate modulates the surface curvature of the membranes by the formation of aggregates in liquid-crystalline phospholipid
membranes. We suggest that the dynamics and conformation of hexadecaprenyl monophosphate in membranes depend on the transmembrane
electrical potential. The electron micrographs indicate that polyprenyl monophosphates with single isoprenyl chains form lipid
vesicular bilayers. The thickness of the bilayer, evaluated from the micrographs, was 11 ± 1 nm. This property creates possibility
of forming primitive bilayer lipid membranes by long single-chain polyprenyl phosphates in abiotic conditions. It can be the
next step in understanding the origin of protocells.
Received: 10 January 2000/Revised: 7 June 2000 相似文献
998.
The advent of GFP imaging has led to a revolution in the study of live cell protein dynamics. Ease of access to fluorescently tagged proteins has led to their widespread application and demonstrated the power of studying protein dynamics in living cells. This has spurred development of next generation approaches enabling not only the visualization of protein movements, but correlation of a protein's dynamics with its changing structural state or ligand binding. Such methods make use of fluorescence resonance energy transfer and dyes that report changes in their environment, and take advantage of new chemistries for site-specific protein labeling. 相似文献
999.
The purpose of this paper was to study the effect of the isopropyl myristic acid ester (IPM) on the physicochemical characteristics
of etoposide-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres-specifically, the effects on the size and drug loading
of the microspheres, the polymer matrix and surface morphology, and the release of etoposide from the microspheres. The experiment
was structured to examine 2 IPM concentrations (25% and 50%) and 1 control (no IPM) at 2 different etoposide-loading percentages
(10% and 5%). The microspheres were prepared using a single-emulsion solvent-extraction procedure. Samples from each batch
of microspheres were then analyzed for size distribution. drug-loading efficiency, surface characteristics, in vitro release,
and in vitro microsphere degradation. The incorporation of 50% IPM significantly increased (P<05) the size of the microspheres when compared with the control and 25% IPM microspheres. However, incorporation of 25% or
50% IPM did not change (P>.05) the drug-loading efficiency in comparison with the microspheres prepared without IPM. The microspheres containing 50%
IPM were shown to significantly increase (P<.05) the release of etoposide from the microspheres at both etoposide concentrations. The microspheres prepared incorporating
25% IPM and 5% etoposide increased the in vitro release (P<.05) in comparison with the microspheres prepared without IPM. The 5% etoposide-PLGA microspheres showed a smooth, nonporous
surface that changed to a dimpled. nonporous surface after addition of 25% IPM. During the in vitro degradation study, the
IPM-containing microspheres slowly became porous but retained their structural integrity throughout the experiment. 相似文献
1000.
Vincenza Dolo Sandra D'Ascenzo Maurizio Sorice Antonio Pavan Mariateresa Sciannamblo Alessandro Prinetti Vanna Chigorno Guido Tettamanti Sandro Sonnino 《Glycoconjugate journal》2000,17(3-4):261-268
This paper is the first report on the use of the electron microscopy autoradiography technique to detect metabolically tritium labeled sphingolipids in intact cells in culture.To label cell sphingolipids, human fibroblasts in culture were fed by a 24 hours pulse, repeated 5 times, of 3×10–7 M [1-3H]sphingosine. [1-3H]sphingosine was efficently taken up by the cells and very rapidly used for the biosynthesis of complex sphingolipids, including neutral glycolipids, gangliosides, ceramide and sphingomyelin. The treatment with [1-3H]sphingosine did not induce any morphological alteration of cell structures, and well preserved cells, plasma membranes, and intracellular organelles could be observed by microscopy.Ultrathin sections from metabolic radiolabeled cells were coated with autoradiographic emulsion. One to four weeks of exposition resulted in pictures where the location of radioactive sphingolipids was evidenced by the characteristic appearance of silver grains as irregular coiled ribbons of metallic silver. Radioactive sphingolipids were found at the level of the plasma membranes, on the endoplasmic reticulum and inside of cytoplasmic vesicles. Thus, electron microscopy autoradiography is a very useful technique to study sphingolipid-enriched membrane domain organization and biosynthesis. 相似文献