Dynein and dynactin colocalize with AQP2 water channels in intracellular vesicles from kidney collecting duct
| Title: | Dynein and dynactin colocalize with AQP2 water channels in intracellular vesicles from kidney collecting duct |
|---|---|
| Authors: | Marples, David; Knepper, Mark; Nielsen, Soren; Schroer, Trina; Ahrens, Nikolai; Taylor, Ann |
| Publisher: | American Journal of Physiology |
| Date Published: | February 01, 1998 |
| Reference Number: | 193 |
We investigated whether the motor protein cytoplasmic dynein and dynactin, a protein complex thought to link dynein with vesicles, are present in rat renal collecting ducts and associated with aquaporin-2 (AQP2)-bearing vesicles. Immunoblotting demonstrated cytoplasmic dynein heavy and intermediate chains in kidney, with relative expression levels of inner medulla > outer medulla > cortex. In addition to being present in cytoplasmic fractions, dynein was abundant in membrane fractions enriched for intracellular vesicles. Dynactin was also abundant in membrane fractions enriched for intracellular vesicles. Furthermore, both dynactin and dynein were present in vesicles specifically immunoisolated using anti-AQP2 antibodies. Immunocytochemistry revealed labeling for dynein in the collecting duct principal cells with a pattern consistent with labeling of intracellular vesicles. Moreover, quantitative double immunogold labeling confirmed colocalization of AQP2 and dynein in the same vesicles at the electron microscopic level. Thus the microtubule-associated motor protein dynein and the associated dynactin complex are present in rat renal collecting duct principal cells and are associated with intracellular vesicles, including those bearing AQP2, consistent with the view that dynein and dynactin are involved in vasopressin-regulated trafficking of AQP2-bearing vesicles.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
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Marples, et al., wanted to know what it was that shuttled these AQP2-bearing vesicles to and from the collecting duct cell membrane. Recent studies had demonstrated that intracellular trafficking of vesicles can occur along microtubules, slender tubular structures within cell cytoplasm. But what moves the vesicles along the microtubules? Research indicated that the movers are microtubule-associated mechanoenzymes (often simply referred to as "motors").
Microtubules have a positively charged end (the plus end) and a negatively charged end (the minus end). The minus end is rooted in organizing centers within the cell. The plus end projects away from the organizing centers. One group of motors move vesicles toward the organizing centers at the minus end, and another group move vesicles away from the organizing centers toward the plus end.
In collecting duct cells, many organizing centers are near the apex of the cell, and microtubules project from them down toward the base and sides of the cell. In these cells, a motor which moves along the microtubule toward the minus end in the apex of the cell would be expected to be the motor which moves the vesicle-encased AQP2 to the membrane at the apex of the cell.
One candidate motor is dynein, a large protein with the collecting duct cell. Research suggests that dynein needs help to move vesicles along the microtubule. Specifically, it needs the protein complex dynactin, which may help dynein link with vesicles.
Marples, et al, sought to confirm whether or not dynein and dynactin were associated with AQP2-bearing vesicles. To do so, they examined rat kidney tissue, first to see if dynein and dynactin were present and then to see if they were associated with AQP2. The authors used analytical techniques such as immunoblotting, immunoisolation, immunocytochemistry and immunoelectron microscopy, all of which identify proteins by means of antigen-antibody specific reactions. Their analyses was able to demonstrate that cellular dynein was present in the rat kidney, being most abundant in the kidney's inner medulla, next most abundant in the outer medulla and least abundant in the kidney cortex. Dynein was also abundant in that part of the kidney tissue rich in intracellular vesicles (such as those that transport AQP2). Dynactin was also abundant in vesicle-rich kidney tissue.
The authors refined their findings by analyses showing dynein located in the collecting duct principal cells. Finally, by using electron microscopy, they found both dynein and AQP2 in the same vesicles. The authors concluded that the motor protein dynein and the dynactin complex associated with it are associated with intracellular vesicles bearing AQP2. This finding strengthens the view that dynein and dynactin are both involved in moving AQP2-bearing vesicles to the apex of the kidney collecting duct cells.