Characterization and application of a novel immortalized murine thick ascending limb (imTAL) cell line to investigate the role of myosin II motor proteins Myh9 and Myh10 in TAL cargo transport

Abstract

Conditional genetic inactivation of Myh9and Myh10, the genes which encode the heavy chains of nonmuscle myosin IIA and IIB, respectively, in adult murine renal epithelium leads to aberrant trafficking of the thick ascending limb‐specific GPI‐anchored glycoprotein uromodulin (UMOD) and its subcellular accumulation in cells of the thick ascending limb of Henle’s loop (Otterpohl et al. 2020). A novel immortalized murine thick ascending limb (imTAL) cell line harboring doxycycline inducible Myh9and Myh10conditional knockout (cKO) capability was established in our lab to facilitate long term in vitrostudies. We utilized biotinylated anti‐uromodulin antibody to isolate cells from collagenase digested Myh9&10cKO whole mouse kidneys. Characterization of these cells has revealed endogenous expression of TAL‐specific protein UMOD and NKCC2. RT‐qPCR detected mRNA expression of TAL specific proteins. We generated imTAL cells stably expressing GFP‐tagged UMOD using retroviral transduction. GFP‐UMOD expressing cells underwent 72‐hour doxycycline treatment and, along with untreated control cells, were fixed and analyzed via confocal microscope. While GFP‐UMOD is enriched along the plasma membrane in control cells, we observe accumulation of GFP‐UMOD within vesicular structures upon doxycycline‐induced loss of Myh9 and Myh10 proteins. We also utilized imTAL cells stably expressing an GFP‐tagged thermosensitive variant of vesicular stomatitis virus glycoprotein (VSV‐GtsO45‐GFP), which has been widely employed in studying the secretory pathway. The temperature sensitive nature of VSV‐GtsO45 enabled us to perform pulse‐chase type experiments, following its anterograde transport through the ER, Golgi, and ultimately to the plasma membrane. Following incubation at the restrictive temperature (40°C) overnight, cells were moved to permissive temperature (32°C) and followed until 45 minutes. VSV‐GtsO45‐GFP in control cells undergoes ER export immediately (5 minutes) after incubation at the permissive temperature and is transported along the secretory pathway, arriving at the plasma membrane 30‐40 minutes later. Inactivation of Myh9and Myh10 results in sequestration of VSV‐GtsO45‐GFP in perinuclear vesicular and tubular structures. Immunolocalization studies are being performed to identify the vesicular compartment(s) in which VSV‐GtsO45‐GFP is sequestered in the absence of Myh9 and Myh10 proteins. imTAL cells stably expressing NKCC2‐GFP are also being analyzed to assess the anterograde transport of NKCC2 in doxycycline‐treated cells. Taken together, we have established a novel, immortalized, inducible, Myh9&10 cKO murine TAL cell line to enable long term in vitro studies and have expanded the molecular toolbox to elucidate the mechanisms underlying NM2 motor mediated TAL cargo transport.