Increase In Number Of Vesicles Research Articles

Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae.

Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here we demonstrate, using quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs. We conclude that phosphorylation of Cav1 leads to separation or "spreading" of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane.

Electron Microscopy of Rhamnolipid (Biosurfactant) Morphology: Effects of pH, Cadmium, and Octadecane

A rhamnolipid biosurfactant produced by Pseudomonas aeruginosa ATCC 9027 is reported to increase the aqueous dispersion and biodegradation of petroleum hydrocarbons and to complex heavy metals. These reports indicate the potential for application of rhamnolipids in remediation of contaminated sites. Effective use of rhamnolipids will require understanding of rhamnolipid morphology and the effects of pH and organic and inorganic contaminants on that morphology. We used cryo-transmission electron microscopy to investigate the morphology of vitrified, frozen hydrated suspensions of rhamnolipid over a pH range of 5.5 to 8.0, and to determine the effect of a model alkane, octadecane, and a model heavy metal, cadmium, on rhamnolipid morphology. Micrographs clearly showed that rhamnolipid morphology was a function of pH, changing from lamellar, to vesicular, to micellar as pH increased. The effect of cadmium and octadecane on rhamnolipid morphology was determined at pH 6.8 and 7.0, where maximum cadmium complexation and maximum octadecane dispersion occurs. Cadmium seemed to stabilize rhamnolipid vesicle structures as shown by an increase in vesicle number and a decrease in vesicle diameter. In contrast, octadecane favored the micellar structure as shown by the complete absence of vesicles.

Evidence for 5-HT1B/1D receptors mediating the antimigraine effect of sumatriptan and dihydroergotamine.

Neurogenic plasma extravasation, endothelial cell activation (increase in vesicle number and vacuole formation), platelet aggregation and adhesion, and mast cell degranulation occur selectively in post-capillary venules of the dura mater following electrical trigeminal ganglion stimulation, and are mediated by release of neuropeptides from perivascular unmyelinated C fibres. Pre-treatment with the antimigraine drugs dihydroergotamine and sumatriptan, two drugs that bind with high affinity to 5-HT1B/1D receptors, markedly attenuated plasma protein extravasation induced by electrical trigeminal ganglion stimulation. Trigeminal stimulation increased plasma calcitonin gene-related peptide levels in rat superior sagittal sinus. Pre-treatment with dihydroergotamine and, to a lesser extent, sumatriptan, attenuated this increase. Both drugs reduced morphological changes in post-capillary venules and mast cells within dura mater following electrical trigeminal ganglion stimulation. Plasma protein extravasation was selectively blocked in dura mater (but not in extracranial tissues) by pre-treatment with those receptor agonists showing a rank order of potency suggesting a 3-HT1B/1D interaction (5-CT greater than 5-BT greater than DHE greater than sumatriptan greater than 8-OH-DPAT). Pre-treatment with 5-HT2 and 5-HT3 antagonists was not effective. Taken together, these data are consistent with the interpretation that putative 5-HT-1B/1D receptors located on sensory fibres are coupled to inhibition of peptide release and blockade of neurogenic inflammation. An important therapeutic action of ergot alkaloids and sumatriptan in migraine headaches is so defined.

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.

Ionoregulatory and toxicological responses of stonefly nymphs (Plecoptera) to acidic and alkaline pH

The acute toxicities of acidic and alkaline pH to nymphs of the stonefliesPteronarcys dorsata, P. proteus, andTallaperla maria were determined in 96-hr static bioassays. The acidic and alkaline 96-hr LC50 values were 2.8 to 3.3 and 12.1 to 10.3, respectively. Exposure to pH 3.0 for 72 hr or longer caused a significant loss of sodium from nymphs ofP. proteus. Morphological changes, including distension of cuticular disk and increased number of vesicles, were observed in gill tissue from nymphs ofP. dorsata exposed to pH 2.5 for 9 hr while minor changes were observed in nymphs exposed to pH 4.0 for 96 hr. Changes in gill tissue ultrastructure included an increase in number of vesicles and a decrease in number and size of mitochondria in nymphs exposed to alkaline pH of 11.75.

Acute NO2 effects on penetration and transport of horseradish peroxidase in hamster respiratory epithelium.

Male Golden Syrian hamsters were exposed to 28 ppm NO2 for 48 h. Intratracheal instillations of 25 mg horseradish peroxidase (HRP) in 0.2 ml saline followed exposure. After 10-to 30-min incubation periods, lungs were perfusion-fixed and excised, and bronchi, bronchioles, and alveolar tissues were isolated. A second group of NO2-exposed hamsters was allowed a 48-h recovery period prior to HRP instillations, and a third group served as air controls. Analysis by transmission electron microscopy of airways showed penetration by tracer material of 75% of bronchiolar tight junctions (TJ) examined after 48-h exposures. Only 15% of bronchial TJ were penetrated. No bronchial TJ were compromised after the recovery period, but 20% of bronchiolar TJ remained permeable. The TJ from bronchi and bronchioles of control animals showed no penetration of HRP; HRP penetration of interalveolar TJ was not seen in control animals or in “recovery” animals, but it was observed in 30% of the TJ after 48-h exposures. All penetrated junctions were between adjacent type I pneumocytes. Many pinocytotic vesicles in alveolar type I pneumocytes containing HRP could be seen 10 and 30 min after instillation in animals exposed to NO2. Control animals showed a similar pattern; however, fewer vesicles were present. The number of pinocytotic vesicles was analyzed statistically for each exposure and control group. The data indicated a 500% increase in vesicle number after 48 h of NO2 exposure. After 48 h of recovery the number of vesicles was 300% greater than that present in the control animals. These data were not altered by peroxidase stain. Vesicle size was found to be the same (80 nm in diameter) in NO2-exposed and control specimens. Our results suggest differing effects of NO2 on bronchial, bronchiolar, and alveolar epithelium. This observation confirms previous studies that the TJ of bronchiolar epithelium are most sensitive to NO2-induced injury. Further, the data indicated that NO2 injury increases endocytic activity of the alveolar type I cells.

Changes in the ultrastructure of the caecum of chickens caused by Trichomonas gallinarum.

The ultrastructure of the normal caecum of chickens and of chickens infected withTrichomonas gallinarumhas been investigated. In infected caeca some villi and crypts of LieberkÜhn appear normal whereas others show marked changes in structure of the epithelial cells. The most obvious changes are loss of microvilli and glycocalyx and an increase in number of vesicles which stain metachromatically with Azur II.T. gallinarumhas been detected between epithelial cells and in the lamina propria. Bacteria often, but not always, accompanied the protozoa in the tissues.

Studies of cerebral edema III

Normal perivascular glial cells have an abundance of paired fibrillar structures which run in bundles to the region of the basement membrane. Ferritin passes through the endothelium of white matter of cat brain and into both the extracellular space and the perivascular glial cells. The passage through the endothelium is by pinocytosis, the passage across the basement membrane is direct, the passage into the perivascular glial cells is by pinocytosis. In cerebral edema, there is an exaggeration of pinocytotic activity in the endothelial cells, with the contents of these vesicles being poured into the region of the basement membrane. Next, one notes an increase in the extracellular space, and then an increase in number of vesicles in the perivascular glial cells. (The passage through the endothelium is by pinocytosis, the passage across the basement membrane is direct, the passage into the perivascular glial cells is by pinocytosis.) Alteration of the fine structure of the perivascular glial cells follows, with the appearance of protein globules in these cells. The changes in the glial cells peripheral to the vessels occur after those in the extracellular space adjacent to the vessel and in the perivascular glial cells.