• Organization of the intact human erythrocyte cytoskeleton and its attachment to the membrane: Ultrastructural studies

      Ursitti, Jeanine Anne; Wade, James B. (1992)
      The cytoskeleton of the human erythrocyte is responsible for the remarkable ability of this cell to change shape. In order to study the organization of this structure in the intact human erythrocyte, I attached paraformaldehyde-fixed ghosts to coated coverslips and sheared them to expose the cytoskeleton. Quick-freeze, deep-etch, rotary-replication with Pt/C (QFDERR), or tannic acid/osmium fixation and plastic embedding, revealed the cytoskeleton as a dense network of intersecting straight filaments. I found 3-4 filaments at each intersection and 40% more intersections/{dollar}\mu{dollar}m{dollar}\sp2{dollar} of membrane than predicted on the basis of actin junctions alone. Immunogold labeling verified that the filaments were spectrin, but their lengths (29-37 nm) were approximately one-third that of extended spectrin dimers. The length and diameter of the filaments were sufficient to accommodate spectrin dimers, but not spectrin tetramers. These results suggested that, in situ, spectrin dimers may associate as hexamers and octamers, rather than tetramers. I then isolated the skeleton from the membrane and visualized it by either negative staining or QFDERR. Negatively stained isolated skeletons had 5-7 filaments at an intersection, whereas isolated skeletons visualized by QFDERR had 3-5 filaments at an intersection. This difference appears to arise from filaments unraveling to reveal two strands in negatively stained skeletons. Immunogold labeling revealed that spectrin tetramers and hexamers were the predominant species in the isolated skeletons after QFDERR. Unlabeled skeletons also showed evidence of laterally associated spectrin filaments. Similar associations in the intact cytoskeleton could account for the discrepancy in the number of apparent spectrin hexamers and octamers. Extracting sheared erythrocyte ghosts with solutions of low ionic strength removed the cytoskeleton to reveal projections from the cytoplasmic surface of the membrane. These projections contained band 3, as shown by immunogold labeling, and they aggregated to a similar extent as intramembrane particles (IMP) when the cytoskeleton was removed, suggesting a direct relationship between these structures. Quantification indicated a stoichiometry of 2 IMP for each cytoplasmic projection. Cytoplasmic projections presumably contain other proteins besides band 3 since further treatment with high ionic strength solutions extracts peripheral proteins and reduces the diameter of projections by {dollar}\sp\sim{dollar}3 nm.
    • Vesicular traffic in the ADH-sensitive toad urinary bladder

      Bellve, Karl Dean; Wade, James B. (1996)
      Water channels and fluid-phase markers are retrieved from the apical membrane of the toad bladder upon removal of antidiuretic hormone (ADH). These cells maintain high ADH responses even after repeated stimulation, suggesting a high recycling rate of vesicles carrying water channel proteins. Alternatively, there could be a large population of vesicles with only a small fraction responding. The number of retrieved labeled vesicles with single or multiple markers were counted by custom image analysis programs that extracted novel information for statistical analysis. The programs included median and brightness thresholds and an algorithm that counted vesicles by color and size. Recycling was evaluated by loading the retrieved vesicles with Texas Red dextran or biotin/avidin-fluorescein and evaluating the loss of these vesicles due to ADH restimulation. Tissue was stimulated with ADH and then surface proteins were covalently labeled with NHS-SS-Biotin followed by avidin-fluorescein. The tissue was then exposed to Texas Red dextran for 10 min during washout of ADH. ADH restimulation caused a significant reduction in vesicle number with a maximal drop of 22%. Biotin-labeled vesicles were also significantly reduced by 25% after ADH restimulation but did not show preferential sorting and recycling back to the membrane in comparison to vesicles with fluid-phase marker. To study marker retrieved from both the apical and basolateral membranes, samples were exposed to FITC dextran in the serosal bath for 60 min, followed by addition of ADH and Texas Red dextran to the mucosal bath. Cells were examined for colocalization of both markers at various time points following ADH washout. No significant colocalization was seen at the 0 time point but colocalization increased by 60 and 105 mins. After ADH restimulation, vesicles with marker only from the basolateral or apical bath were reduced but vesicles with both markers remained unchanged. Additionally, the basolateral marker uptake was stimulated by a transepithelial osmotic gradient compared to ADH stimulation alone. This work provides evidence that in native ADH-responsive tissue the majority of vesicles labeled from the apical membrane do not respond to ADH. Additionally, apical marker can enter ADH-insensitive vesicles with marker retrieved from the basolateral membrane.