Intracellular trafficking of bile salt export pump (ABCB11) in polarized hepatic cells: constitutive cycling between the canalicular membrane and rab11-positive endosomes - PubMed (original) (raw)

Intracellular trafficking of bile salt export pump (ABCB11) in polarized hepatic cells: constitutive cycling between the canalicular membrane and rab11-positive endosomes

Yoshiyuki Wakabayashi et al. Mol Biol Cell. 2004 Jul.

Abstract

The bile salt export pump (BSEP, ABCB11) couples ATP hydrolysis with transport of bile acids into the bile canaliculus of hepatocytes. Its localization in the apical canalicular membrane is physiologically regulated by the demand to secrete biliary components. To gain insight into how such localization is regulated, we studied the intracellular trafficking of BSEP tagged with yellow fluorescent protein (YFP) in polarized WIF-B9 cells. Confocal imaging revealed that BSEP-YFP was localized at the canalicular membrane and in tubulo-vesicular structures either adjacent to the microtubule-organizing center or widely distributed in the cytoplasm. In the latter two locations, BSEP-YFP colocalized with rab11, an endosomal marker. Selective photobleaching experiments revealed that single BSEP-YFP molecules resided in canalicular membranes only transiently before exchanging with intracellular BSEP-YFP pools. Such exchange was inhibited by microtubule and actin inhibitors and was unaffected by brefeldin A, dibutyryl cyclic AMP, taurocholate, or PI 3-kinase inhibitors. Intracellular carriers enriched in BSEP-YFP elongated and dissociated as tubular elements from a globular structure adjacent to the microtubule-organizing center. They displayed oscillatory movement toward either canalicular or basolateral membranes, but only fused with the canalicular membrane. The pathway between canalicular and intracellular membranes that BSEP constitutively cycles within could serve to regulate apical pools of BSEP as well as other apical membrane transporters.

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Figures

Figure 1.

Figure 1.

BSEP-YFP targeted to the canalicular membrane in polarized WIF-B9 cells. (A) BSEP-YFP colocalized with cCAM105, a bile canalicular membrane marker. WIF-B9 cells infected with BSEPYFP adenovirus were fixed and stained with antibody for cCAM105. BSEP and cCAM105 were imaged separately and merged. BSEPYFP occurs in two hemi-canaliculi of a single cell (asterisk) because adjacent cells were not infected. Antibody to cCAM105 reacted with the entire canalicular membrane and the basolateral membrane (gray line). In the merged image, BSEP-YFP (green) and cCAM105 (red) overlapped at the canalicular membrane (yellow) but not at the basolateral membrane. (B) BSEP-YFP localized in the canalicular membrane and intracellular sites. WIF-B9 cells infected with BSEPYFP adenovirus were labeled with sNHS-LC biotin at 4°C and incubated with unconjugated streptavidin before fixation. After permeabilization, the biotinylated canalicular membrane was detected with Cy3-conjugated streptavidin. Bar, 10 μm. BSEP-YFP and streptavidin were imaged separately and merged. Gray line denotes basolateral membrane. One cell expressed BSEP-YFP, which visualized half of a bile canaliculus (asterisk). Streptavidin shows canalicular membrane. In the merged image, BSEP-YFP (green) and streptavidin (red) overlapped at the right half of the canalicular membrane (yellow). Several BSEP-YFP intracellular sites were distinct from the canalicular membrane (arrowhead and lower enlarged image). (C) Schematic picture of polarized WIF-B9 cells. BC, bile canaliculus; BL, basolateral membrane; N, nucleli; TJ, tight junction. (D) BSEP-YFP remains at the canalicular membrane. After canalicular membrane biotinylation at 4°C, WIF-B9 cells infected with BSEP-YFP adenovirus were washed, lysed, and immunoprecipitated with antibody for BSEP. Biotinylated BSEP-YFP was detected by SDS-PAGE and Western blotting with HRP-conjugated streptavidin or antibody for BSEP. Immunoprecipitated BSEP-YFP was recognized by HRP-conjugated streptavidin indicating that BSEP-YFP remains at the canalicular membrane.

Figure 2.

Figure 2.

BSEP distributed with endosomal markers in subcellular fractions. WIF-B9 cells infected with BSEP-YFP adenovirus were labeled with sNHS-LC biotin at 4°C, washed, homogenized, and postnuclear supernatants were separated on sucrose step gradients. Aliquots of each fraction were analyzed by SDS-PAGE and Western blotting with antibodies against BSEP, rab4, 5, and 11, and EEA-1 or streptavidin. Fraction 1, homogenization buffer/8% sucrose interphase; fraction 2, 8%/12% interphase; fraction 3, 12%/16% interphase; fraction 4, 16%/19% interphase; fraction 5, 19%/23% interphase; fraction 6, 23%/27% interphase; fraction 7, 27%/31% interphase; fraction 8, 35%/43% interphase; and fraction 9, pelleted material. Bands on gels were quantified by densitometry, and results are expressed as percentage of total amount of each marker in the gradient.

Figure 3.

Figure 3.

BSEP colocalized with rab11-positive endosomes. After fixation, WIF-B9 cells infected with BSEP-YFP adenovirus were stained with antibody for rab11, TGN38, mannosidase II, γ-tubulin, or p150Glued. Specimens were imaged separately and merged. (A and C) Cell on the right side expressed BSEP-YFP. In addition to the canalicular membrane (asterisk), BSEP-YFP was identified in vesicular structures (D) and perinuclear globular structures (E). (D and E) Enlargements of corresponding areas in C. (B) rab11 distribution in WIF-B9 cells with or without BSEP-YFP expression. (C) A merge of A and B shows that BSEP-YFP (green) localized with rab11 (red) at vesicular tubular structures (D) and perinuclear globular structures (E). (F and G) BSEPYFP globular structure (green) is separated from Golgi (red), which reacted with antibody for TGN38 (F) and mannosidase II (G). (H and I) BSEP-YFP globular structure (green) is adjacent to the MTOC (red), which stained with antibody for γ-tubulin (H) and p150Glued (I). Bile canaliculus is indicated by asterisk. Gray line shows basolateral membrane.

Figure 4.

Figure 4.

Canalicular BSEP pool constitutively exchanges with other intracellular pools. Also see Videos 1 and 4. WIF-B9 cells infected with BSEP-YFP adenovirus were used for live cell imaging by confocal or conventional fluorescent microscopy at 37°C. (A) Canalicular membrane region BSEP-YFP recovers after photobleach. The area enclosed by a white line was photobleached. Time in the next two panels denotes minutes postbleach. Gray line shows basolateral membrane. (B) Quantitation of BSEP-YFP recovery in the canalicular membrane region after photobleaching (as in A). Mean fluorescence intensity was determined at the times indicated. n = 6 ± SD. (C) Synthesis and degradation of BSEP-YFP. To determine BSEP-YFP synthesis rate, WIF-B9 cells infected with BSEP-YFP adenovirus were photobleached throughout the cell. Fluorescence images were captured at 30-min intervals (solid circles, n = 6 ± SD). To determined BSEP-YFP degradation rate, WIF-B9 cells infected with BSEP-YFP adenovirus were treated with cycloheximide (50 μg/ml). Fluorescence images were captured at 30-min intervals (open circles, n = 6 ± SD). (D) Tannic acid blocked basolateral endocytosis. WIF-B9 cells were incubated with (bottom) or without (top) tannic acid (0.5%) in serum-free medium for 5 min at 37°C. Cells were washed and assayed for uptake of Cy5-conjugated transferrin (Tf) in serum-free medium after 30-min incubation at 0°C (center) or 37°C (right), or uptake of fluorescein isothiocyanate conjugated dextran (mol. wt. 580,000) after 10-min incubation at 37°C. Bile canaliculus is indicated by asterisk. Transferrin and dextran were endocytosed from basolateral membrane in control cells. Tannic acid treatment impaired transferrin and dextran endocytosis from the basolateral membrane. Bile canaliculus is indicated by asterisk. White line shows basolateral membrane. (E) WIF-B9 cells infected with BSEP-YFP adenovirus were incubated with tannic acid (0.5%) in serum-free medium for 5 min at 37°C. The cells were washed and the area enclosed by the white line was photobleached. Time in the second two panels denotes minutes postbleach. Gray line shows basolateral membrane. (F) Quantitation of BSEP-YFP recovery in the canalicular BSEP pool after tannic acid treatment. The canalicular membrane region was photobleached as shown in E. Fluorescence intensity was determined at the times indicated. Open circles denote control values and solid circles are values after tannic acid treatment. (E and F) Tannic acid did not alter BSEP-YFP recovery in the canalicular region.

Figure 5.

Figure 5.

BSEP-YFP constitutively cycles between the canalicular membrane, globular structure and transport carriers. Also see Videos 2 and 3. WIF-B9 cells infected with BSEP-YFP adenovirus were used for live cell imaging by confocal or conventional fluorescence microscopy at 37°C. (A) Globular pool of BSEP-YFP recovered after photobleaching. The area in the box was photobleached. Time in the next two panels denotes minutes postbleach. Gray line shows basolateral membrane. Asterisk shows bile canaliculus. (B) Quantitation of BSEP-YFP recovery in the globular structure pool. The pool was photobleached as shown in A. Mean fluorescence intensity was determined as indicated. n = 6 ± SD. (C) BSEP-YFP loss in the globular pool after photobleaching of the canalicular pool. The canalicular area (red) was photobleached at 0 and again at 10 min. Time in the other panels denotes minutes postbleach. Blue circle indicates the globular pool. Gray line shows basolateral membrane. (D) Quantitation of BSEP-YFP loss in the globular pool. The canalicular area was photobleached at 0 and again at 10 min as shown in C. Blue circles denote globular structure pool, and red squares are canalicular membrane pool values. (E) BSEP-YFP loss in the canalicular pool after photobleaching the entire intracellular BSEP-YFP pool (yellow line). Time in subsequent two panels denotes minutes postbleach. Blue line indicates the globular region and red is the canalicular region. (F) Quantitation of BSEP-YFP fluorescence in the canalicular membrane and globular structure pools as described for E. Blue denotes globular structure pool and red is the canalicular membrane pool. (G) The canalicular membrane domain (red) and globular structure (blue) were photobleached simultaneously. Time in subsequent two panels denotes minutes postbleach. Gray line shows basolateral membrane. (H) Quantitation of BSEP-YFP recovery in the canalicular membrane and globular structure pools as described for G. Blue denotes globular structure pool and red is the canalicular membrane region.

Figure 6.

Figure 6.

Dynamics of BSEP-YFP transport carrier segregation and fusion in the globular structure and canalicular region. Also see Video 5. (A) WIF-B9 cells infected with BSEPYFP adenovirus were used for live cell imaging at 37°C with a conventional fluorescence microscope equipped with CCD camera. Bar, 5 μm. The areas in A designated B and C are placed in the corresponding panels. (B) BSEPYFP transport carriers elongate from the globular structure. Times are relative to the first image in the series. Arrowhead shows BSEP elongation sites. (C) Small transport carriers fuse with globular structure at other sites (arrowhead). Times are relative to the first image in the series. (D) Velocity of two exocytic transport carriers from globular structure. Velocity was measured as the distance between pixel coordinates in consecutive images.

Figure 7.

Figure 7.

BSEP transport carriers also traffic toward the basolateral membrane. Also see Video 6. WIF-B9 cells infected with BSEP-YFP adenovirus were used for live cell imaging at 37°C with conventional fluorescence microscope equipped with CCD camera. (A) BSEP transport carrier distribution in WIF-B9 cell. Bar, 2 μm. Areas in A designated B and C are placed in the corresponding panels. (B) BSEP-YFP transport carriers traffic toward the basolateral membrane, denoted by black arrowhead. Times shown are relative to the first image in the series. Bar, 2 μm. (C) BSEP-YFP transport carriers traffic along the basolateral membrane which they leave and return to the region of the globular structure. BSEP-YFP transport carriers denoted by black arrowhead. Times are relative to the first image in the series. Bar, 2 μm.

Figure 8.

Figure 8.

Requirement of microtubules for constitutive BSEP cycling. After treatment with nocodazole (33 μM) for 60 min at 37°C, WIF-B9 cells infected with BSEP-YFP adenovirus were used for live cell imaging by confocal or conventional fluorescence microscopy. (A) Microtubule disruption perturbs recovery of globular structure BSEP pool. After treatment with nocodazole for 60 min at 37°C, globular structure pool enclosed by the white line was photobleached. Time in the subsequent two panels denotes minutes postbleach. Gray line shows basolateral membrane. (B) Quantitation of BSEP-YFP recovery in globular structure pool, which was photobleached as in A. Fluorescence intensity was determined as indicated. Solid circles denote nocodazole treatment and open circles are control values. (C) Microtubule disruption perturbs recovery of the canalicular BSEP pool. After treatment with nocodazole for 60 min at 37°C, canalicular area enclosed by the white line was photobleached. Time in the subsequent two panels denotes minutes postbleach. Gray line shows basolateral membrane. (D) Quantitation of BSEP-YFP recovery in the canalicular membrane pool which was photobleached as in C. Fluorescence intensity was determined as indicated. Solid circles indicate nocodazole treatment and open circles indicate control values. (E) Microtubule disruption perturbs BSEP-YFP loss in the canalicular pool after photobleaching the entire intracellular pool. Time in the subsequent two panels denotes minutes postbleach. Gray line shows basolateral membrane. (F) Quantitation of BSEP-YFP fluorescence change in the canalicular membrane pool. The entire cell except the canalicular membrane region was photobleached as in E. Solid circles denote nocodazole treatment and open circles are control values.

Figure 9.

Figure 9.

Requirement of actin for constitutive BSEP cycling. After treatment with cytocholasin D (1 μg/ml) for 60 min at 37°C, WIF-B9 cells infected with BSEP-YFP adenovirus were used for live cell imaging by confocal microscopy at 37°C. (A) WIF-B9 cells infected with BSEP-YFP adenovirus were imaged pre- and postcytocholasin D treatment. Cytocholasin D treatment increased the canalicular BSEP pool. Gray line shows basolateral membrane. (B) Cytocholasin D treatment does not perturb BSEP recovery in the canalicular pool. After treatment with cytocholasin D for 60 min at 37°C, the canalicular region (white line) was photobleached. Time in the second two panels denotes minutes postbleach. Gray line shows basolateral membrane. (C) Quantitation of BSEP-YFP recovery in the canalicular membrane pool. The canalicular pool was photobleached as in C. Fluorescence intensity was determined as indicated. Solid squares denote cytocholasin D treatment and open circles denote control values. (D) Cytocholasin D perturbs BSEP retrieval from the canalicular pool. After treatment with cytocholasin D for 60 min at 37°C, the entire cell except the canalicular region was photobleached. Time in the second two panels denotes minutes postbleach. Gray line shows basolateral membrane. (E) Quantitation of BSEP-YFP fluorescence change in the canalicular membrane pool. The entire cell except canalicular region was photobleached as in D. Fluorescence intensity was determined as indicated. Solid squares denote cytocholasin D treatment and open circles denote control values.

Figure 10.

Figure 10.

Mechanism of constitutive BSEP cycling in canalicular membrane. (A) After treatment with BFA (5 μg/ml) for 30 min at 37°C, WIF-B9 cells infected with BSEP-YFP adenovirus were used for live cell imaging by confocal microscopy at 37°C. B (bright field) and C (BSEP-YFP) show that LY294002 (200 μM) treatment for 60 min at 37°C produced vacuoles (closed arrowhead in B), which did not contain BSEP-YFP in C. (D) Quantitation of BSEP-YFP recovery in the canalicular membrane pool after various treatments. After treatment, canalicular BSEP-YFP pool was photobleached as in Figure 4A. Mean fluorescence intensity was determined as indicated. Open circles are control; solid circles are BFA (5 μg/ml); open squares are cyclic AMP (250 μM) + taurocholate (100 μM); solid squares are LY294002 (200 μM).

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