Rab11a and myosin Vb are required for bile canalicular formation in WIF-B9 cells

Abstract

Hepatocytes polarize by forming functionally distinct sinusoidal (basolateral) and canalicular (apical) plasma membrane domains. Two distinct routes are used for delivery of membrane proteins to the canaliculus. Proteins having glycosylphosphatidylinositol anchors or single transmembrane domains are targeted to the sinusoidal plasma membrane from where they transcytose to the canalicular domain. In contrast, apical ATP-binding-cassette (ABC) transporters, which are required for energy-dependent biliary secretion of bile acids (ABCB11), phospholipids (ABCB4), and nonbile acid organic anions (ABCC2), lack initial residence in the basolateral plasma membrane and traffic directly from Golgi membranes to the canalicular membrane. While investigating mechanisms of apical targeting in WIF-B9 cells, a polarized hepatic epithelial cell line, we observed that rab11a is required for canalicular formation. Knockdown of rab11a or overexpression of the rab11a-GDP locked form prevented canalicular formation as did overexpression of the myosin Vb motorless tail domain. In WIF-B9 cells, which lack bile canaliculi, apical ABC transporters colocalized with transcytotic membrane proteins in rab11a-containing endosomes and, unlike the transcytotic markers, did not distribute to the plasma membrane. We propose that polarization of hepatocytes (i.e., canalicular biogenesis) requires recruitment of rab11a and myosin Vb to intracellular membranes that contain apical ABC transporters and transcytotic markers, permitting their targeting to the plasma membrane. In this model, polarization is initiated upon delivery of rab11a-myosin Vb-containing membranes to the surface, which causes plasma membrane at the site of delivery to differentiate into apical domain (bile canaliculus).

Fig. 1.

Rab11a requires constitutive apical cycling in polarized WIF-B9 cells. (A) BSEP-YFP localized canalicular membrane and rab11a-positive endosomes. WIF-B9 cells infected with BSEP-YFP were immunostained with rab11a antibody. BSEP localized in canalicular membrane (asterisk) and rab11a-positive endosomes (arrowhead). Gray line shows the basolateral membrane. (B) Rab11aS25N inhibit constitutive BSEP apical cycling. On the third day in culture, WIF-B9 cells were infected with adenovirus encoding BSEP-YFP, which was abundantly expressed in the bile canaliculus within 3 days, at which time the cells were infected with or without adenovirus that encoded rab11aS25N-CFP. WIF-B9 cells infected with adenovirus, which encoding BSEP-YFP and/or rab11aS25N-CFP, were used for live cell imaging by confocal fluorescent microscopy at 37°C. The area enclosed by the red line was photobleached. Time in Center Left, Center Right, and Right denotes minutes after bleach. Gray line identifies the basolateral membrane. (C) Quantification of BSEP-YFP recovery in the canalicular membrane region after photobleaching. Mean fluorescence intensity was determined at the times indicated. n = 10 ± SD. Fluorescence recovery after photobleaching was abolished by rab11aS25N-CFP expression in polarized WIF-B9 cells.

Fig. 2.

Rab11a is required for canalicular formation. (A) Effect of rab11aS25N expression on canalicular formation. At day 3 in culture, WIF-B9 cells were infected with adenovirus encoding rab11aS25N-CFP or BSEP-YFP. At day 7 in culture, the cells were immunostained with ZO-1, a tight junction marker. White arrowheads denote bile canaliculi in cells infected with adenovirus encoding rab11aS25N-CFP or BSEP-YFP. BSEP-YFP expression was a control for adenoviral infection. Percentage of WIF-B9 cells expressing rab11aS25N or BSEP-YFP ranged from ≈30% to ≈50% and averaged ≈40%. (B) Rab11aS25N expression reduced canalicular formation. The percentage of WIF-B9 cells manifesting bile canaliculi on day 7 after infection with adenovirus-encoding rab11aS25N-CFP or BSEP-YFP was determined. BSEP-YFP expression was a control for adenoviral infection. Percentage of cells forming BC refers to all cells in the control and only infected cells expressing rab11aS25N or BSEP. n = 6 ± SD. (C) WIF-B9 cells transfected with rab11a RNA interference (RNAi) vector were cloned. Equal protein loadings of homogenates from control cells or cells expressing short hairpin RNAs directed against rab11a were analyzed by SDS/PAGE before immunoblotting with the indicate antibody. β-actin served as the internal control. (D) The effect of rab11a knockdown on canalicular formation. WIF-B9 control and rab11a knockdown cells were cultured for 7 days and immunostained for ZO-1. Canalicular formation was evident in control cells but was rarely detected in rab11a knockdown cells. (E) Rab11a knockdown cells have decreased canalicular formation. WIF-B9 control and rab11a knockdown cells were immunostained for ZO-1 at day 1, 3, 5, and 7. Percentage of cells forming BC refers to all cells.

Fig. 3.

Before polarization, apical ABC transporters reside in rab11a-positive endosomes. (A) Localization of MRP2, 5′NT, and HA321 in polarized WIF-B9 cells. WIF-B9 cells were immunostained for endogenous MRP2, HA321, and 5′NT. MRP2 localized to the canalicular membrane and rab11a-positive endosomes, whereas HA321 localized in the basolateral membrane. 5′NT localized in both canalicular and basolateral membrane and rab11a-positive endosomes. Asterisks indicate bile canaliculus. Gray lines show basolateral membrane. (B) Localization of MRP2, 5′NT, and HA321 in nonpolarized WIF-B9 cells. MRP2 was localized in rab11a-positive endosomes. HA321 was distributed in the plasma membrane. 5′NT was distributed in plasma membrane and rab11a-positive endosomes. Gray line shows basolateral membrane.

Fig. 4.

Expression of myosin Vb tail domain inhibited canalicular formation. Distribution of endogenous myosin Vb in nonpolarized (A) and polarized (B) WIF-B9 cells. WIF-B9 cells infected with adenovirus encoding BSEP-YFP were immunostained with myosin Vb antibody. (A) In nonpolarized cells, endogenous myosin Vb localized with BSEP-YFP in perinuclear endosomes (arrowhead). Gray line shows plasma membrane. Nu, nuclei. (B) In polarized cells, endogenous myosin Vb colocalized with BSEP-YFP in perinuclear (arrowhead) and pericanalicular endosomes (arrow). Gray line shows basolateral membrane. Nu, nuclei. (C) The effect of myosin Vb tail expression on canalicular formation. At day 3 in culture, WIF-B9 cells were infected with adenovirus encoding myosin Vb tail-CFP. At day7in culture, the cells were immunostained with ZO-1. Percentage of WIF-B9 cells expressing myosin Vb tail or BSEP-YFP ranged from ≈30% to ≈50% and averaged ≈40%. Myosin Vb tail expressing cells did not form canalicular domains. (D) Myosin Vb tail expression reduced canalicular formation. The percentage of WIF-B9 cells manifesting bile canaliculi on day 7 after infection with adenovirus encoding myosin Vb-CFP or BSEP-YFP was determined. BSEP-YFP expression was a control for adenoviral infection. Myosin Vb tail expression decreased canalicular formation. Percentage of cells forming BC refers to all cells in the control and only infected cells expressing myosin Vb tail or BSEP. n = 6 ± SD. (E) Distribution of rab11a, MRP2, 5′NT, and HA321 in myosin Vb tail expressing WIF-B9 cells. Cells were immunostained for endogenous rab11a, MRP2, 5′NT, and HA321. Myosin Vb tail expression resulted in accumulation of perinuclear rab11a-positive endosomes. MRP2 and 5′NT colocalized with myosin Vb tail in perinuclear endosomes. 5′NT was also present in the plasma membrane. HA321 was restricted to the plasma membrane.