Novel electron and super resolution microscopy techniques reveal unique cellular and organelle architecture regulated by myosin motors within murine thick ascending limb

We recently confirmed that myosin motor proteins Myh9 and Myh10 ( Myh9&10­ ) are crucial for proper cargo protein trafficking in the thick ascending limb (TAL) of adult mouse kidney. TAL-specific conditional knockout of Myh9&10 results in significant reduction of the TAL ion cotransporter protein NKCC2, transient accumulation of the glycoprotein uromodulin (UMOD) in the endoplasmic reticulum (ER) of TAL cells, and severe progressive kidney disease with early mortality (Otterpohl & Busselman et al. 2024). The TAL contributes to the reabsorption of ions and generation of the medullary osmotic gradient via active sodium transport. The ER, mitochondria, and plasma membrane (PM) of TAL cells are in close proximity within the basolateral region where highly differentiated infoldings of the PM partially encapsulate ER and mitochondria as well as interdigitate with neighboring cells. Given the impact of Myh9&10 loss on ER homeostasis and the complex and elaborate organellar architecture of TAL cells, we have sought to explore the role of Myh9&10 in facilitating interorganelle contact. By employing a novel serial section-electron microscopy method (ATUM-SEM) and super resolution confocal microscopy, we have carefully reconstructed TAL subcellular membrane architecture in three dimensions and correlated the resulting models with the localization of numerous proteins of interest via immunostaining. High relative expression of the basolateral sodium potassium pump (Na + K + ATPase) is observed in the TAL where it localizes to basolateral PM infoldings. The outer mitochondrial membrane protein TOM20 is seen in apposition to Na + K + ATPase-positive infoldings, while ER proteins calreticulin, calnexin, RTN4, and STIM1 localize to adjacent structures. MYH9 and β-actin localization is also observed within basolateral PM infoldings and in close proximity to ER proteins. Interestingly, claudin-10 localizes in distinct patterns along the basolateral infoldings with the actin cytoskeleton. Importantly, UMOD protein consistently localizes along basolateral PM infoldings; aberrant UMOD trafficking upon loss of Myh9&10 may be the result of disruption of a novel protein transport pathway involving movement of protein along infoldings and/or the fission of transport vesicles from the apical aspect of infoldings. Furthermore, our results provide experimental evidence and expand upon the structural details of TAL cell interdigitation models described by others (Welling et al. 1978). Cellular structure appears to have a profound influence on the ion transport capacity of the TAL, enabling it to act as an “ion sponge” by leveraging ample Na + K + ATPase-rich basolateral surface area and the energy from adjacent cristae-dense mitochondria. Ultimately, Myh9&10 serve to influence key aspects of kidney function by regulating protein trafficking in the TAL, which is a critical nephron segment for ion and water homeostasis. NIDDK R01DK131020 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.