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

Tiane Chen, Ann Hubbard, Rakhilya Murtazina, Jennifer Price, Jianbo Yang, Boyoung Cha, Rafiquel Sarker, Mark Donowitz

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)3535-3545
Number of pages11
JournalJournal of cell science
Volume127
Issue number16
DOIs
StatePublished - 2014

Keywords

  • Brush border
  • Endocytosis
  • Myosin VI
  • Trafficking

ASJC Scopus subject areas

  • Cell Biology

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