Myosin VI mediates the movement of NHE3 down the microvillus in intestinal epithelial cells

Abstract

The intestinal brush border Na + /H + exchanger NHE3 is tightly regulated through changes in its endocytosis and exocytosis. Myosin VI, a minus-end-directed actin motor, has been implicated in endocytosis at the inter-microvillar cleft and during vesicle remodeling in the terminal web. Here, we asked whether myosin VI also regulates NHE3 movement down the microvillus. The basal NHE3 activity and its surface amount, determined by fluorometry of the ratiometric pH indicator BCECF and biotinylation assays, respectively, were increased in myosin-VI-knockdown (KD) Caco-2/ Bbe cells. Carbachol (CCH) and forskolin (FSK) stimulated NHE3 endocytosis in control but not in myosin VI KD cells. Importantly, immunoelectron microscopy results showed that NHE3 was preferentially localized in the basal half of control microvilli but in the distal half in myosin VI KD cells. Treatment with dynasore duplicated some aspects of myosin VI KD: it increased basal surface NHE3 activity and prevented FSK-induced NHE3 endocytosis. However, NHE3 had an intermediate distribution along the microvillus (between that in myosin VI KD and untreated cells) in dynasore-treated cells. We conclude that myosin VI is required for basal and stimulated endocytosis of NHE3 in intestinal cells, and suggest that myosin VI also moves NHE3 down the microvillus.

Myosin-7b Promotes Distal Tip Localization of the Intermicrovillar Adhesion Complex

“…However, myosin-6 has been implicated in cargo retention and/or movement towards the pointed-ends of microvillar actin bundles, which are embedded in the terminal web at the base of the BB [12, 13]. Unconventional myosin motors have also been implicated in tipward transport in other actin-supported protrusions, playing roles in the formation and maintenance of these structures.…”

Regulation of Transporters and Channels by Membrane-Trafficking Complexes in Epithelial Cells

“…Signaling pathways that raise intracellular Ca 2þ , intracellular cAMP, or intracellular cGMP activate the trafficking of NHE3 down microvilli, in a clathrin-associated myosin VI -dependent fashion (Hegan et al 2012;Chen et al 2014), to the intermicrovillar cleft (region) where it may be endocytosed into endosomal compartments (Donowitz and Li 2007). Thus, its surface expression is reduced, lowering its V max at the cell surface.…”

Mechanisms of Cell Polarity–Controlled Epithelial Homeostasis and Immunity in the Intestine

“…The motor activity of myosin-Ia and myosin-VI controls the microvillus tip-and base-directed movement, respectively, of the plasma membrane along the actin filaments that make up the microvillus core (McConnell and Tyska 2007). As such, these myosins regulates the intramicrovillus (that is, microvillus tip versus microvillus base) distribution of brush-border enzymes (Tyska and Mooseker 2004;Hegan et al 2012;Chen et al 2014). Interestingly, the tips of the apical microvilli give rise to vesicles that are shed into the gut lumen (McConnell et al 2009).…”