Research ArticleCalmodulin modulates hepatic membrane polarity by protein kinase C-sensitive steps in the basolateral endocytic pathway
Introduction
Polarized cells have distinct plasma membrane (PM) domains, which are separated by tight junctions. Such cells exhibit cell-surface polarity with a basolateral PM domain, which faces the blood circulation and adjacent cells, and an apical domain, which is in contact with the luminal environment, such as the bile canaliculus (BC) in hepatocytes. These membrane domains differ in both lipid and protein composition. To generate and maintain this polarized membrane composition, specific vectorial flow of membrane carriers must be ensured. This implies that apically and basolaterally destined lipids and proteins must be segregated and sorted along the biosynthetic pathway. In addition, there must be appropriate targeting of endocytosed material that is selectively returned to either PM domain, emphasizing a role of endocytosis in the maintenance of cell surface polarity [1]. There is good evidence to suggest that the subapical compartment, SAC (also known as the common endosome [CE]), where endocytic pathways from both surfaces merge, functions as a polarized recycling system, thus playing a crucial role in the maintenance/biogenesis of cell surface polarity [2]. Indeed, disrupting trafficking via SAC, as occurs upon actin depolymerization [3], [4] or brefeldin-A treatment [5], leads to missorting of basolateral proteins and thereby depolarization. SAC also plays a crucial role in lipid sorting. In polarized hepatocytes, sphingomyelin (SM) and galactosylceramide (GalCer) are transcytosed from the apical membrane via SAC to the basolateral membrane, whereas glucosylceramide (GlcCer) primarily recycles between SAC and the apical membrane [6], [7]. Both SM and GalCer adopt an SAC–apical membrane directed pathway under conditions that promote apical membrane biogenesis, as occurs upon exogenous or intrinsic activation of PKA activity [8], an event in which distinct cytokines appear to be instrumental as well [9].
Although some insight has been obtained in the regulation of lipid trafficking along the reverse endocytic pathway in HepG2 cells, it is far less clear how trafficking along the basolateral endocytic pathway connects with transport to the apical membrane and how this trafficking governs maintenance and biogenesis of the apical membrane. The relevance of the basolateral endocytic pathway for apical membrane homeostasis is inferred from observations that perturbing actin filament organization frustrates transport from early endosomes to CE, which causes a diminishment in the available pool of CE and protein missorting, thus resulting in membrane depolarization [4].
To monitor basolateral membrane derived flow, a fluorescent sphingolipid analog (C6-NBD-SM) was inserted into the basolateral PM domain of polarized HepG2 cells, and its (polarized) transport was investigated. The effects of calmodulin antagonists, known to interfere with various aspects of endocytic and transcytotic trafficking of ligand/receptor complexes [10], [11], [12], [13], on apical membrane directed flow were subsequently investigated. Our data revealed that calmodulin antagonists cause a drastic depolarization of HepG2 cells. The loss of polarity does not reflect a decrease of sphingomyelin internalization, but instead the lipid was found to strongly accumulate in dilated/vacuolar early sorting endosomes. These observations were related to previous work [14] in which we observed that PKC activation inhibited apical membrane biogenesis, which was similarly reflected by an inhibition of transport of lipid analogues along the basolateral endocytic pathway. Indeed, since the effects of the calmodulin antagonists are effectively abolished by treatment with PKC inhibitors, our data suggest a correlation between calmodulin and PKC activity in regulating basolateral endocytic trafficking that connects to apical membrane biogenesis.
Section snippets
Cell culture
HepG2 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) with 4.5 g/l glucose and supplemented with 10% fetal calf serum and antibiotics. Cells were trypsinized and splitted twice a week, and media were changed every other day. For experiments, cells were seeded at 2 × 104 cells per cm2, in six-well plates for quantitative experiments and in dishes onto ethanol-sterilized coverslips for microscopy experiments. In all experiments, cells were grown for 3 days after plating. At this
Calmodulin antagonists induce depolarization of HepG2 cells
To examine the overall effect of calmodulin on polarity development, the number of BC/100 cells as a measure of polarity development was counted as previously described (see Materials and methods) in cells treated or not with calmodulin antagonists (Fig. 1). In control 3-day-old HepG2 cells, polarity development in terms of BC/100 cells reached a value of 17.0 ± 1.6% (mean ± SD from 6 independent experiments with 3 measurements each), indicating that ∼35% of the cells were polarized since at
Discussion
In this work, we have investigated how calmodulin antagonists, in particular CPM, interfered with transcytotic membrane flow in polarized HepG2 cells, and the potential consequence of this interference for apical membrane biogenesis. Previously, we observed [14] that activation of PKC inhibited basolateral-to-apical membrane transport of sphingolipid analogues, which had been inserted into the basolateral plasma membrane of HepG2 cells. Whereas PKC activation did not affect endocytosis per se,
Acknowledgments
D.T. was supported (in part) by a grant from the Fonds Spécial de Recherches de l'UCL and S.C.D.vIJ. by a fellowship from the Royal Dutch Acedemy of Sciences (KNAW). This work was supported by grants from the Belgian Fonds National de la Recherche Scientifique (FNRS), the Région Wallonne, the Actions de Recherches Concertées and the Inter-University Attraction Poles.
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