Apical endocytosis in rat hepatocytes in situ involves clathrin, traverses a subapical compartment, and leads to lysosomes

doi:10.1053/gast.2000.20233

Gastroenterology

Volume 119, Issue 6, December 2000, Pages 1692-1707

Parts of this work have been presented in abstract form at the 38th Annual Meeting of the American Society for Cell Biology (Rahner C, Stieger B, Landmann L. Apical endocytosis in rat liver cells in situ. Mol Biol Cell 1997;9:343a).

https://doi.org/10.1053/gast.2000.20233 Get rights and content

Abstract

Background & Aims This study demonstrates and characterizes apical (canalicular) endocytic pathways in hepatocytes in situ. Methods: Endocytic markers were administered by retrograde infusion through the common bile duct. Colocalization with proteins that are specific for various endocytic compartments was performed on stacks of deconvoluted confocal immunofluorescence images. The subcellular distribution of marker proteins was assessed by electron microscopy (EM). Results: Bulk-phase, as well as membrane-associated markers, were internalized readily at the apical cell pole. At the EM level, marker was found initially in 60–100-nm tubulovesicular structures and 150–200-nm cup-shaped vesicles, whereas multivesicular bodies and lysosomes became labeled after longer time intervals. Apical endocytosis involved clathrin and delivered marker to late endosomes (rab7⁺, cathepsin D⁺), as well as lysosomes (rab7⁻, cathepsin D⁺). Simultaneous labeling of the basolateral endocytic route resulted in overlap of both pathways in the late endosomal and lysosomal compartments. In addition, apical endocytosis involved a subapical compartment (endolyn-78⁺, rab11⁺, polymeric IgA receptor [pIgA-R⁺]) that is passed by the transcytotic route, thus constituting a crossroads. pIgA-R immunoreactivity, probably reflecting the cleaved receptor fragment, was associated with apical endocytic marker and colocalized with clathrin and later with cathepsin D. Conclusions: Apical endocytosis involves coated pits/vesicles, leads to a subapical compartment, and plays a role in the retrieval of canalicular plasma membrane components for lysosomal degradation.

GASTROENTEROLOGY 2000;119:1692-1707

Section snippets

Materials

Pentobarbital (Nembutal; Abbot Laboratories, North Chicago, IL), promethazine (Phenergan; Rhône-Poulenc Pharma, Thalwil, Switzerland), dextran (M_r 70,000) conjugated to Texas Red (Dx-TxR; Molecular Probes, Eugene, OR), FITC-conjugated Ricinus communis lectin (RCA-II, 60 kilodaltons; EY Laboratories Inc., San Mateo, CA), horseradish peroxidase (HRP, type II; Sigma Chemical Co., St. Louis, MO), and 3,3'diaminobenzidine tetrahydrochloride (DAB; Polysciences Inc., Warrington, PA). All other

Assessment of infusion protocol

Direct application of markers for apical endocytosis in the liver is impossible because puncturing the small (approximately 1 μm diameter) canaliculi has never been achieved in situ. Therefore, we developed an infusion protocol for rat livers and applied marker compounds retrogradely through the catheterized common bile duct to get access to the apical (canalicular) PM of hepatocytes via the canaliculi. Although this experimental approach has been used earlier,41, 42 it inherently carries the

Discussion

In this study, we demonstrate and partially characterize the postulated⁵ apical (canalicular) endocytic pathway in rat hepatocytes in situ. Our experimental approach involved the direct administration of various endocytic markers to the apical PM domain by retrograde infusion through the common bile duct and the monitoring of their subsequent fate. Our data demonstrate that: (1) hepatocytes have the competence for apical endocytosis, a pathway that can be probed with fluid-phase, as well as PM

Acknowledgements

The authors thank Jean-Paul Boeglin, Ruth Münster, Rolf Plank, and Erika Weber for excellent technical assistance and Drs. I. Mellman (New Haven, CT), M. Rosenfeld (New York, NY), and M. Zerial (Heidelberg, Germany) for generously providing antibodies.

References (73)

J Gruenberg et al.
Membrane transport in the endocytic pathway
Curr Opin Cell Biol
(1995)
A Helenius et al.
Endosomes
Trends Biochem Sci
(1983)
SCD van Ijzendoorn et al.
The subapical compartment: a novel sorting centre?
Trends Cell Biol
(1999)
EI Christensen et al.
Membrane receptors for endocytosis in the renal proximal tubule
Intern Rev Cytol
(1998)
P Novick et al.
The diversity of rab proteins in vesicle transport
Curr Opin Cell Biol
(1997)
C Rahner et al.
Structure-function correlation of tight junctional impairment after intrahepatic and extrahepatic cholestasis in rat liver
Gastroenterology
(1996)
A Quaroni et al.
Study of intestinal cell differentiation with monoclonal antibodies to intestinal cell surface components
Dev Biol
(1985)
JK Petell et al.
Alteration in the regulation of plasma membrane glycoproteins of the hepatocyte during ontogeny
Exp Cell Res
(1990)
B Stieger et al.
In situ localization of the hepatocytic Na+/taurocholate cotransporting polypeptide in rat liver
Gastroenterology
(1994)
TC Brelje et al.
Multicolor laser scanning confocal immunofluorescence microscopy: practical application and limitation
Methods Cell Biol
(1993)

P Chavrier et al.

Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments

Cell

(1990)

K Sandvig et al.

Endocytosis and intracellular sorting of ricin and shiga toxin

FEBS Lett

(1994)

M Ishii et al.

Regional orientation of actin filaments in the pericanalicular cytoplasm of rat hepatocytes

Gastroenterology

(1991)

R Pohlmann et al.

The two mannose 6-phosphate receptors transport distinct complements of lysosomal proteins

J Biol Chem

(1995)

CJ Soroka et al.

Canalicular export pumps traffic with polymeric immunoglobulin A receptor on the same microtu-bule-associated vesicle in rat liver

J Biol Chem

(1999)

LJ Scott et al.

Dynamics of four rat liver plasma membrane proteins and polymeric IgA receptor rates of synthesis and selective loss into the bile

J Biol Chem

(1992)

B Stieger et al.

Bile acid and xenobiotic transporters in liver

Curr Opin Cell Biol

(1998)

JL Boyer et al.

Vesicle targeting to the apical domain regulates bile excretory function in isolated rat hepatocyte couplets

Gastroenterology

(1995)

R Kubitz et al.

Osmodependent dynamic localization of the multidrug resistance protein 2 in the rat hepatocyte canalicular membrane

Gastroenterology

(1997)

RB Sewell et al.

Pharmacologic perturbation of rat liver lysosomes: effects on release of lysosomal enzymes and of lipids into bile

Gastroenterology

(1988)

I. Mellman

Endocytosis and molecular sorting

Annu Rev Cell Dev Biol

(1996)

D Kornfeld et al.

The biogenesis of lysosomes

Annu Rev Cell Biol

(1989)

B Stieger et al.

Effects of cholestasis on membrane flow and surface polarity in hepatocytes

J Hepatol

(1996)

M Bomsel et al.

Endocytosis in filter-grown Madin-Darby canine kidney cells

J Cell Biol

(1989)

RG Parton et al.

Meeting of the apical and basolateral endocytic pathways of the Madin-Darby canine kidney cell in late endosomes

J Cell Biol

(1989)

M Cohen et al.

Convergence of apical and basolateral endocytic pathways for beta(2)-microglobulin in LLC-PK1 cells

Am J Physiol

(1995)

EJ Hughson et al.

Endocytic pathways in polarized Caco-2 cells: identification of an endosomal compartment accessible from both apical and basolateral surfaces

J Cell Biol

(1990)

M Fujita et al.

Convergence of apical and basolateral endocytic pathways at apical late endosomes in absorptive cells of suckling rat ileum in vivo

J Cell Sci

(1990)

KE Mostov

Transepithelial transport of immunoglobulins

Annu Rev Immunol

(1994)

A Knight et al.

Membrane protein trafficking through the common apical endosome compartment of polarized Caco-2 cells

Mol Biol Cell

(1995)

G Odorizzi et al.

Apical and basolateral endosomes of MDCK cells are interconnected and contain polarized sorting mechanisms

J Cell Biol

(1996)

G Apodaca et al.

Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes

J Cell Biol

(1994)

M Barroso et al.

Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome

J Cell Biol

(1994)

A Gibson et al.

Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells

J Cell Biol

(1998)

P Zacchi et al.

Rab17 regulates membrane trafficking through apical recycling endosomes in polarized epithelial cells

J Cell Biol

(1998)

S Mukherjee et al.

Endocytosis

Physiol Rev

(1997)

Cited by (43)

Annexin A6 in the liver: From the endocytic compartment to cellular physiology
2017, Biochimica et Biophysica Acta - Molecular Cell Research
Citation Excerpt :
From the CRE, proteins internalized from the basolateral domain are either re-directed there without a further intermediate or, when designated for basolateral-to-apical transcytosis, are targeted to the apical domain via an apical recycling endosome/subapical compartment (ARE/SAC) [20–22]. Based on studies in polarized HepG2 cells [23–26] and rat liver [27,28], the subapical compartment (SAC) represents an apically localized, tubular endosome containing apical and basolateral cargo. The SAC was also identified as a central compartment for the apical/basolateral sorting of sphingolipids.
Annexin A6 (AnxA6) belongs to the conserved annexin family — a group of Ca^2 +-dependent membrane binding proteins. AnxA6 is the largest of all annexins and highly expressed in smooth muscle, hepatocytes, endothelial cells and cardiomyocytes. Upon activation, AnxA6 binds to negatively charged phospholipids in a wide range of intracellular localizations, in particular the plasma membrane, late endosomes/pre-lysosomes, but also synaptic vesicles and sarcolemma. In these cellular sites, AnxA6 is believed to contribute to the organization of membrane microdomains, such as cholesterol-rich lipid rafts and confer multiple regulatory functions, ranging from vesicle fusion, endocytosis and exocytosis to programmed cell death and muscle contraction. Growing evidence supports that Ca^2 + and Ca^2 +-binding proteins control endocytosis and autophagy. Their regulatory role seems to operate at the level of the signalling pathways that initiate autophagy or at later stages, when autophagosomes fuse with endolysosomal compartments. The convergence of the autophagic and endocytic vesicles to lysosomes shares several features that depend on Ca^2 + originating from lysosomes/late endosomes and seems to depend on proteins that are subsequently activated by this cation. However, the involvement of Ca^2 + and its effector proteins in these autophagic and endocytic stages still remains poorly understood. Although AnxA6 makes up almost 0.25% of total protein in the liver, little is known about its function in hepatocytes. Within the endocytic route, we identified AnxA6 in endosomes and autophagosomes of hepatocytes. Hence, AnxA6 and possibly other annexins might represent new Ca^2 + effectors that regulate converging steps of autophagy and endocytic trafficking in hepatocytes. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
The unique polarity phenotype of hepatocytes
2014, Experimental Cell Research
Citation Excerpt :
Regardless of whether they are internalized via clathrin-dependent or -independent mechanisms from the basolateral domain, bile canaliculi proteins reach the bile canalicular-membrane in common transcytotic transport carriers after being segregated from basolateral proteins in sorting endosomes [49,50] The nature of the hepatocytic sorting endosomes, however, is still controversial. Hoekstra and colleagues as well as Landmann׳s group refer to an apically localized, tubular endosome that contains apical and basolateral cargo and thus would be the hepatocytic equivalent of the common recycling endosome in MDCK cells, as Sub-Apical Compartment (SAC) [51,52]. Their model describes a sub-domain of the SAC that contains sorted apically-destined proteins and is characterized by the presence of Rab11, Rab25 and Rab3 [52,53].
Hepatocytes, the main epithelial cell type of the liver, function like all epithelial cells to mediate the vectorial flow of macromolecules into and out of the organ they encompass. They do so by establishing polarized surface domains and by restricting paracellular flow via their tight junctions and cell–cell adhesion. Yet, the cell and tissue organization of hepatocytes differs profoundly from that of most other epithelia, including those of the digestive and urinary tracts, the lung or the breast. The latter form monolayered tissues in which the apical domains of individual cells align around a central continuous luminal cavity that constitutes the tubules and acini characteristic of these organs. Hepatocytes, by contrast, form capillary-sized lumina with multiple neighbors resulting in a branched, tree-like bile canaliculi network that spreads across the liver parenchyme. I will discuss some of the key molecular features that distinguish the hepatocyte polarity phenotype from that of monopolar, columnar epithelia.
A novel hypothesis for an alkaline phosphatase 'rescue' mechanism in the hepatic acute phase immune response
2013, Biochimica et Biophysica Acta - Molecular Basis of Disease
Citation Excerpt :
This suggests that the ASGP-R recycles through the TGN, much like another basolaterally-targeted, clathrin-dependent recycling receptor, the transferrin receptor (TfR) [52,134,148]. This may provide a location for ASGP-R switching between the State 2 and State 1 pathways, as may the sub-apical compartment, but details regarding this possible rerouting have not been established [149–152]. For many years after the ASGP-R's discovery, endogenous ligands for the receptor remained unidentified.
The liver isoform of the enzyme alkaline phosphatase (AP) has been used classically as a serum biomarker for hepatic disease states such as hepatitis, steatosis, cirrhosis, drug-induced liver injury, and hepatocellular carcinoma. Recent studies have demonstrated a more general anti-inflammatory role for AP, as it is capable of dephosphorylating potentially deleterious molecules such as nucleotide phosphates, the pathogenic endotoxin lipopolysaccharide (LPS), and the contact clotting pathway activator polyphosphate (polyP), thereby reducing inflammation and coagulopathy systemically. Yet the mechanism underlying the observed increase in liver AP levels in circulation during inflammatory insults is largely unknown. This paper hypothesizes an immunological role for AP in the liver and the potential of this system for damping generalized inflammation along with a wide range of ancillary pathologies. Based on the provided framework, a mechanism is proposed in which AP undergoes transcytosis in hepatocytes from the canalicular membrane to the sinusoidal membrane during inflammation and the enzyme's expression is upregulated as a result. Through a tightly controlled, nucleotide-stimulated negative feedback process, AP is transported in this model as an immune complex with immunoglobulin G by the asialoglycoprotein receptor through the cell and secreted into the serum, likely using the receptor's State 1 pathway. The subsequent dephosphorylation of inflammatory stimuli by AP and uptake of the circulating immune complex by endothelial cells and macrophages may lead to decreased inflammation and coagulopathy while providing an early upstream signal for the induction of a number of anti-inflammatory gene products, including AP itself.
Mathematical analysis of hepatic high density lipoprotein transport based on quantitative imaging data
2005, Journal of Biological Chemistry
Hepatocytes internalize high density lipoprotein (HDL) at the basolateral membrane. Most HDL is recycled while some is shuttled to the canalicular membrane by transcytosis. Here, transport of HDL was analyzed by mathematical modeling based on measurements in polarized hepatic HepG2 cells. Recycling of HDL from basolateral sorting endosomes was modeled by applying the rapid equilibrium approach. Analytical expressions were derived, which describe in one model the transport of HDL to the subapical compartment/apical recycling compartment, the biliary canaliculus (BC), and to late endosomes and lysosomes (LE/LYS). Apical endocytosis of HDL predicted by the model was confirmed for rhodamine-dextran and fluorescent asialoorosomucoid, markers for LE/LYS in living HepG2 cells. Budding of endocytic vesicles from the BC was directly observed by time lapse imaging of a fluorescent lipid probe. Based on fitted kinetic parameters and their covariance matrix a Monte Carlo simulation of HDL transport in hepatocytes was performed. The model was used to quantitatively assess release of HDL-associated free cholesterol by scavenger receptor BI. It is shown that only 6% of HDL-associated sterol reaches the BC as a constituent of the HDL particles, whereas the remaining sterol is rapidly released from HDL and shuttled to the BC by non-vesicular transport.
Oxidative Stress and Localization Status of Hepatocellular Transporters: Impact on Bile Secretion and Role of Signaling Pathways
2021, Antioxidants and Redox Signaling
A link between intrahepatic cholestasis and genetic variations in intracellular trafficking regulators
2021, Biology

View all citing articles on Scopus

^☆: Address requests for reprints to: Lukas Landmann, Ph.D., Department of Anatomy, University of Basel, Pestalozzistr. 20, CH-4056 Basel, Switzerland. e-mail: [email protected]; fax: (41) 61-2673959.

^☆☆: Supported by the Swiss National Science Foundation (grant 32-40486.94 to L. L.).

^★: Dr. Rahner's present address is Department of Internal Medicine, Section of Digestive Disease, Yale University Medical School, New Haven, Connecticut 06520-8019.

View full text

Liver, Pancreas, and Biliary TractApical endocytosis in rat hepatocytes in situ involves clathrin, traverses a subapical compartment, and leads to lysosomes☆,☆☆,★

Abstract

Section snippets

Materials

Assessment of infusion protocol

Discussion

Acknowledgements

Curr Opin Cell Biol

Trends Biochem Sci

Trends Cell Biol

Intern Rev Cytol

Curr Opin Cell Biol

Gastroenterology

Dev Biol

Exp Cell Res

Gastroenterology

Methods Cell Biol

Cell

FEBS Lett

Gastroenterology

J Biol Chem

J Biol Chem

J Biol Chem

Curr Opin Cell Biol

Gastroenterology

Gastroenterology

Gastroenterology

Endocytosis and molecular sorting

Annu Rev Cell Dev Biol

The biogenesis of lysosomes

Annu Rev Cell Biol

Effects of cholestasis on membrane flow and surface polarity in hepatocytes

J Hepatol

Endocytosis in filter-grown Madin-Darby canine kidney cells

J Cell Biol

Meeting of the apical and basolateral endocytic pathways of the Madin-Darby canine kidney cell in late endosomes

J Cell Biol

Convergence of apical and basolateral endocytic pathways for beta(2)-microglobulin in LLC-PK1 cells

Am J Physiol

Endocytic pathways in polarized Caco-2 cells: identification of an endosomal compartment accessible from both apical and basolateral surfaces

J Cell Biol

Convergence of apical and basolateral endocytic pathways at apical late endosomes in absorptive cells of suckling rat ileum in vivo

J Cell Sci

Transepithelial transport of immunoglobulins

Annu Rev Immunol

Membrane protein trafficking through the common apical endosome compartment of polarized Caco-2 cells

Mol Biol Cell

Apical and basolateral endosomes of MDCK cells are interconnected and contain polarized sorting mechanisms

J Cell Biol

Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes

J Cell Biol

Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome

J Cell Biol

Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells

J Cell Biol

Rab17 regulates membrane trafficking through apical recycling endosomes in polarized epithelial cells

J Cell Biol

Endocytosis

Physiol Rev

Liver, Pancreas, and Biliary Tract
Apical endocytosis in rat hepatocytes in situ involves clathrin, traverses a subapical compartment, and leads to lysosomes☆,☆☆,★