Investigating novel role(s) for myosin motor proteins, Myo18A, Myh9 and Myh10 in maintaining thick ascending limb cellular architecture and trafficking of UMOD and NKCC2

Kidney function is dependent on coordinated trafficking of ion channels and transporters to maintain blood electrolyte and pH homeostasis within the renal epithelial cells. Our previous work showed that loss of Myh9 and Myh10 in the kidney leads to abnormal trafficking of thick ascending limb (TAL)-specific proteins uromodulin (UMOD) and sodium potassium chloride cotransporter 2 (NKCC2) and progressive kidney disease in mice. Interestingly, in Myh9<10 cKO mouse kidneys we observe loss of unconventional Myo18A from the TAL apical membrane, Myo18A is a unique unconventional myosin motor that contains a PDZ domain that facilitates its interaction with the plasma membrane. Myh9&10 proteins and Myo18A have been implicated in ER-Golgi transport and can copolymerize to form filaments in vitro and in cells. BioID experiments using Myh9 and Myo18A heavy chains independently identified each other as strong interacting candidates, along with a few other actin associated proteins. We hypothesize that Myh9, Myh10 and Myo18A copolymerize to mediate the trafficking of cargo proteins in the kidney epithelium. To test this, we have isolated two different immortalized, doxycycline (DOX)-inducible Myh9&10 knockout and Myo18A knockout TAL (imTAL) cell lines from our mouse models. Loss of Myh9, Myh10 and Myo18A were confirmed in imTAL cells after DOX treatment. Traditional pulse-chase, trafficking experiment using GFP-tagged temperature sensitive vesicular stomatitis virus G glycoprotein (VSVG) which misfolds at 41°C and retained in the endoplasmic reticulum (ER) shows defective trafficking of cargo with loss of Myh9, Myh10 and Myo18A in imTAL cells. Coimmunoprecipitation experiments using whole kidney lysates confirm Myh9 and Myo18A interaction in murine kidneys along with a few ER-Golgi proteins. Super resolution microscopy to uncover the localization and expression of Myo18A is being attempted using lab designed antibodies to correlate with UMOD, NKCC2 and ER-Golgi protein localization. Given the complex organelle architecture of the TAL epithelium, and its physiological role in maintaining ion transport and osmotic gradient along the corticomedullary axis, identifying roles for these novel myosin motors will uncover important details of the TAL cell architecture and their unique cargo trafficking pathways. NIDDK RO1DK131020 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.