Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads (original) (raw)
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Hepatocyte apical bulkheads provide a mechanical means to oppose bile pressure
Journal of Cell Biology
Hepatocytes grow their apical surfaces anisotropically to generate a 3D network of bile canaliculi (BC). BC elongation is ensured by apical bulkheads, membrane extensions that traverse the lumen and connect juxtaposed hepatocytes. We hypothesize that apical bulkheads are mechanical elements that shape the BC lumen in liver development but also counteract elevated biliary pressure. Here, by resolving their structure using STED microscopy, we found that they are sealed by tight junction loops, connected by adherens junctions, and contain contractile actomyosin, characteristics of mechanical function. Apical bulkheads persist at high pressure upon microinjection of fluid into the BC lumen, and laser ablation demonstrated that they are under tension. A mechanical model based on ablation results revealed that apical bulkheads double the pressure BC can hold. Apical bulkhead frequency anticorrelates with BC connectivity during mouse liver development, consistent with predicted changes in ...
Bioarchitecture
Columnar epithelia (e.g., kidney, intestine) and hepatocytes embody the two major organizational phenotypes of non-stratified epithelial cells. Columnar epithelia establish their apical and basal domains at opposing poles and organize in monolayered cysts and tubules, in which their apical surfaces form a single continuous lumen whereas hepatocytes establish their apical domains in the midst of their basolateral domains and organize a highly branched capillary luminal network, the bile canaliculi, in which a single hepatocyte can engage in lumen formation with multiple neighbors. To maintain their distinct tissue architectures, columnar epithelial cells bisect their luminal domains during symmetric cell divisions, while the cleavage furrow in dividing hepatocytes avoids bisecting the bile canalicular domains. We discuss recently discovered molecular mechanisms that underlie the different cell division phenotypes in columnar and hepatocytic model cell lines. The serine/threonine kina...
Cytokinesis defines a spatial landmark for hepatocyte polarization and apical lumen formation
Journal of Cell Science, 2014
By definition, all epithelial cells have apical-basal polarity, but it is unclear how epithelial polarity is acquired and how polarized cells engage in tube formation. Here, we show that hepatocyte polarization is linked to cytokinesis using the rat hepatocyte cell line Can 10. Before abscission, polarity markers are delivered to the site of cell division in a strict spatiotemporal order. Immediately after abscission, daughter cells remain attached through a unique disc-shaped structure, which becomes the site for targeted exocytosis, resulting in the formation of a primitive bile canaliculus. Subsequently, oriented cell division and asymmetric cytokinesis occur at the bile canaliculus midpoint, resulting in its equal partitioning into daughter cells. Finally, successive cycles of oriented cell division and asymmetric cytokinesis lead to the formation of a tubular bile canaliculus, which is shared by two rows of hepatocytes. These findings define a novel mechanism for cytokinesis-linked polarization and tube formation, which appears to be broadly conserved in diverse cell types.
Rab35 controls formation of luminal projections required for bile canalicular morphogenesis
Journal of Cell Biology, 2021
Hepatocytes display a unique biaxial polarity with shared apical luminal connections between adjacent hepatocytes that merge into a network of bile canaliculi. Belicova et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202103003) discovered that hepatocyte apical membranes generate Rab35-dependent extensions that traverse the lumen and are essential for bile canalicular formation and maintenance.
The polarized architecture of hepatocytes
2014
The development and maintenance of polarized epithelial tissue requires a tightly controlled orientation of mitotic cell division relative to the apical polarity axis. Hepatocytes display a unique polarized architecture. We demonstrate that mitotic hepatocytes asymmetrically segregate their apical plasma membrane domain to the nascent daughter cells. The non-polarized nascent daughter cell can form a de novo apical domain with its new neighbor. This asymmetric segregation of apical domains is facilitated by a geometrically distinct “apicolateral” subdomain of the lateral surface present in hepatocytes. The polarity protein partitioning-defective 1/microtubule-affinity regulating kinase 2 (Par1b/MARK2) translates this positional landmark to cortical polarity by promoting the apicolateral accumulation of Leu-Gly-Asn repeat-enriched protein (LGN) and the capture of nuclear mitotic apparatus protein (NuMA)–positive astral microtubules to orientate the mitotic spindle. Proliferating hepa...
Molecular Biology of the Cell, 2006
The molecular mechanisms that regulate multicellular architecture and the development of extended apical bile canalicular lumens in hepatocytes are poorly understood. Here, we show that hepatic HepG2 cells cultured on glass coverslips first develop intercellular apical lumens typically formed by a pair of cells. Prolonged cell culture results in extensive organizational changes, including cell clustering, multilayering, and apical lumen morphogenesis. The latter includes the development of large acinar structures and subsequent elongated canalicular lumens that span multiple cells. These morphological changes closely resemble the early organizational pattern during development, regeneration, and neopla-
Journal of Visualized Experiments
Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function. Video Link The video component of this article can be found at https://www.jove.com/video/60507/ 8. This suggests a critical role of keratin intermediate filaments in supporting of the canalicular structure. In vitro studies utilizing 3D hepatocyte collagen sandwiches have also shown the importance of the AMP-activated protein kinase and its upstream activator LKB1 in bile canalicular network formation 9. These findings were then further confirmed by subsequent in vivo studies 10,11. Thus, it has become clear that in vitro studies are necessary to further the understanding of signaling processes involved in the establishment of hepatocyte polarization, proper canalicular network formation, and bile secretion.
HAL (Le Centre pour la Communication Scientifique Directe), 2021
In biological and medical literature, alternative hypotheses for initial bile duct lumen formation during embryogenesis exist, which so far remained largely untested. Guided by the quantification of morphological features and expression of genes in developing bile ducts from embryonic mouse liver, these hypotheses were sharpened and data collected that permitted to develop a high resolution individual-based computational model to test the alternative hypotheses in silico. Simulations with this model suggest that successful bile duct lumen formation primarily requires the simultaneous contribution of directed cell division of cholangiocytes, local osmotic effects generated by salt excretion in the lumen, and temporally-controlled differentiation of hepatoblasts to cholangiocytes, with apical constriction of cholangiocytes only moderately affecting luminal size.
Molecular Biology of The Cell, 2003
Epithelial cells form monolayers of polarized cells with apical and basolateral surfaces. Madin-Darby canine kidney epithelial cells transiently lose their apico-basolateral polarity and become motile by treatment with hepatocyte growth factor (HGF), which causes the monolayer to remodel into tubules. HGF induces cells to produce basolateral extensions. Cells then migrate out of the monolayer to produce chains of cells, which go on to form tubules. Herein, we have analyzed the molecular mechanisms underlying the production of extensions and chains. We find that cells switch from an apico-basolateral polarization in the extension stage to a migratory cell polarization when in chains. Extension formation requires phosphatidyl-inositol 3-kinase activity, whereas Rho kinase controls their number and length. Microtubule dynamics and cell division are required for the formation of chains, but not for extension formation. Cells in the monolayer divide with their spindle axis parallel to the monolayer. HGF causes the spindle axis to undergo a variable "seesaw" motion, so that a daughter cells can apparently leave the monolayer to initiate a chain. Our results demonstrate the power of direct observation in investigating how individual cell behaviors, such as polarization, movement, and division are coordinated in the very complex process of producing multicellular structures.
The endodermal epithelium in the mammalian liver consists of hepatocytes and biliary epithelial cells. Both cell types originate from hepatoblasts, which may be hepatic stem cells in fetal stages. When hepatoblasts are located around the portal veins, they give rise to biliary epithelial cells under an inductive action by portal connective tissues. Nonperiportal hepatoblasts differentiate into mature hepa-tocytes. Recent studies on naturally mutated or genetically engineered animals have highlighted several new aspects of the hepatoblast differentiation. Mosaic analysis using random inactivation of either paternal or maternal X-chromosome carrying a spf ash mutation in the heterozygous females has demonstrated that hepatoblasts may be bipotential for their differentiation potency into hepatocytes and biliary epithelial cells. This idea is strongly supported by the observation that hepatoblasts in C/EBP-knockout mouse livers abundantly develop pseudoglandular structures, which resemble precursor structures for intrahepatic bile ducts (pearl-like structures or ductal plates). The data have also shown that the suppression of C/EBP expression in periportal hepatoblasts is a key for biliary cell differentiation, which leads to elevation of HNF6 and HNF1 expression. In this review article, molecular mechanisms for bile duct formation are discussed, including roles of the suppression of C/EBP expression and Jagged1/Notch2 or activin/TGF signaling. For hepatocyte maturation and growth, cell-cell interactions such as hepatoblast-stellate or endothelial cell interactions are also required. Our present understanding of their molecular mechanisms is summarized with cell lineages and roles of nonparenchymal cells in liver development.