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Am J Physiol Renal Physiol (November 13, 2001). doi:10.1152/ajprenal.00287.2001
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Articles in PresS, published online ahead of print November 13, 2001
Am J Physiol Renal Physiol, 10.1152/ajprenal.00287.2001
Submitted on September 14, 2001
Accepted on November 7, 2001

Expression of myosin VI within the early endocytic pathway in the adult and developing proximal tubule

Daniel Biemesderfer1*, Sue Ann Mentone2, Mark Mooseker3, and Tama Hasson4

1 Internal Medicine, Yale University School of Medicine, New Haven, CT, USA
2 Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA
3 Molecular, Cellular and Developmental Biology, Yale University School of Medicine, New Haven, CT, USA; Cell Biology, Yale University School of Medicine, New Haven, CT, USA; Pathology, Yale University School of Medicine, New Haven, CT, USA
4 Section of Cell and Developmental Biology, University of California at San Diego, La Jolla, CA, USA

* To whom correspondence should be addressed. E-mail: daniel.biemesderfer{at}yale.edu.

Myosin-VI is a reverse-direction molecular motor implicated in membrane transport events. Since myosin-VI is most highly expressed in the kidney we investigated its renal localization using high resolution immunocytochemical and biochemical methods. Indirect immunofluorescence microscopy revealed myosin-VI at the base of the brush border in proximal tubule cells. HRP uptake studies, which labeled endosomes, and double staining for the clathrin adapter protein, AP-2, showed that myosin VI was closely associated with the intermicrovillar (IMV) coated pit region of the brush border. Localization of myosin-VI to the IMV region was confirmed at the EM level by colloidal gold labeling of ultrathin cryosections. In addition, antigen retrieval demonstrated a small but significant pool of myosin-VI on the microvilli. To confirm myosin-VI's association with the IMV compartment, these membranes were separated from other membrane compartments using 15%-25% OptiPrep density gradients. Immunoblotting of the gradient fractions confirmed that myosin-VI was enriched with markers for the IMV microdomain of the brush border, suggesting that myosin-VI associates with proteins in this compartment. Finally, we examined the expression of myosin-VI during nephron development. We found myosin-VI present in a diffuse cytoplasmic pattern at stage II (S-shaped body phase) and only redistributed fully to the brush border in the stage IV nephron. These studies support a model for myosin-VI function in the endocytic process of the proximal tubule.




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