Nonmuscle myosin II proteins, Myh9 and Myh10, interacts with unconventional myosin Myo18A in immortalized mouse thick ascending limb epithelial cells

Abstract

Conventional nonmuscle myosin II (NM2) and unconventional myosin 18A (Myo18A) belong to an evolutionarily conserved family of molecular motor proteins that slide or walk on the actin cytoskeleton to facilitate a myriad of cellular functions including cargo transport pathways. NM2 and Myo18A are known to copolymerize into filaments in vitro in cell-free systems and in cells. Our previous work demonstrated the crucial role for Myh9&10 proteins in regulating transport of uromodulin (UMOD) and the sodium, potassium, chloride cotransporter (NKCC2) in the thick ascending limb (TAL) of mouse kidney using a Myh9&10 conditional knockout mouse model (Otterpohl, et al; 2020). Myo18A is expressed in the proximal tubule and TAL cells in the murine kidney and localizes to the apical membrane. Our results show reduced expression of Myo18A in the TAL cells of the Myh9&10 cKO mouse kidney sections. Furthermore, we identified Myo18A as a major interaction partner to the Myh10 heavy chain in a BioID screen performed in renal epithelial cells. Given that Myo18A alpha is the only myosin with a PDZ domain, we hypothesize that Myo18A coordinates with Myh9&10 in the TAL cells to regulate membrane remodeling during vesicular transport, including the targeting and stabilization of TAL proteins like UMOD and NKCC2. To test this hypothesis, we stably expressed GFP-tagged Myo18A alpha construct in immortalized, mouse thick ascending limb (imTAL) cells generated in the lab. We performed coimmunoprecipitation (Co-IP) experiments that showed interaction between Myo18A alpha, Myh9 and Myh10 proteins. Immunostaining experiments show colocalization of GFP-Myo18A alpha with Myh9 and Myh10 in imTAL cells. These results strongly support the coordinated functions of Myo18A and Myh9&10 proteins in TAL epithelia and warrants further experiments to determine their role in regulating UMOD and NKCC2 transport in the TAL cells. 1R01DK131020-01A1 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.