Vault localization has been determined by isolating cellular fractions (subcellular fractionation) and by staining cells with antibodies attached to fluorescent dyes (immunocytochemistry). Utilizing both techniques, the bulk of vaults were found to be in the cytoplasm in all cell types examined with most cells containing thousands of vaults. However, vault concentration within different rat cell types shows marked diferences. The cell at left is called a fibroblast. It has been stained with an antibody that binds to vaults and a second antibody linked to the red fluorescent dye rhodamine. Each punctate red spot is likely a vault. Vaults are seen at the leading edge of the cell at structures called lamellapodia and at spots where the cell attaches to the culture dish (adhesion placques).
Below is a fibroblast stained for vaults (left panel) and for actin (right panel). Actin is a protein in long cables (filaments) which traverse the cell and give it structure (like a scaffold). At this high magnification (100X), you can see individual vaults decorating the ends of actin filaments (arrow). This is the location of adhesion placques.
Although present in all cells, vaults are most abundant in macrophages and epithelial cells. Vault enrichment in microglia (the brain macrophage) relative to other cells of the brain, allowed Diane Chugani to examine the developmental profile of microglia in rat brain and provided new insights regarding the origin of these cells. Vault-positive microglia (see figure above) were seen to enter the brain in two migrations, rather than one recognized by other macrophage markers. Vault antiserum reveals an early migration of ramified microglia entering from blood vessels before embryonic day 15 and subsiding between postnatal days 7 and 14.
The second migration consists of amoeboid microglia which appear in the corpus callosum and other large fiber tracks in the first postnatal week. These cells differentiate into ramified microglia between postnatal days 4 and 14.
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