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Scribble was originally identified as a Drosophila protein that regulates epithelial polarity and formation of the basolateral surface. The mammalian orthologue, Scrib, is evolutionarily conserved, but does not appear to be necessary for apical-basolateral epithelial polarity. Instead, it is implicated in the regulation of cell survival, protein trafficking, adhesion and migration. A key issue is to understand the molecular pathway by which Scrib participates in these processes. Here, we investigate Scrib using a 3D epithelial cell culture system. We show a novel association between the LRR domain of Scrib and the co-chaperone Sgt1 and demonstrate that these proteins are necessary for epithelial morphogenesis and tubulogenesis following HGF stimulation. The molecular chaperone HSP90 is also required for Sgt1 association with Scrib, and both Sgt1 and HSP90 are needed to ensure proper Scrib protein levels. Furthermore, reduced Scrib stability, following inhibition of Sgt1-HSP90, lowers the cellular abundance of the Scrib- βPix-PAK complex. Inhibition of any member of this complex, Scrib, βPix or PAK, is sufficient to block HGF-mediated epithelial morphogenesis. The identification of Scrib as an Sgt1-HSP90 client protein required for 3D cell migration suggests that chaperonemediated regulation of polarity protein stability and homeostasis is an unappreciated mechanism underlying dynamic rearrangements during morphogenesis. © 2012.

Cell-cell adhesion by adherens junctions controls proliferation and cell polarization and is crucial to maintain epithelial architecture and homeostasis. Downregulation of two of the main components of adherens junctions, E-cadherin and p120, is an often recurring hallmark of carcinomas, causing loss of polarity and increased proliferation, survival and invasion of epithelial cells. On the other hand, tumor-promoting effects of both E-cadherin and p120 have been reported, substantiated by sustained, or even elevated expression of these molecules in many cancers. In this review, we will discuss how expression regulation by EMT, E-cadherin cleavage or p120 isoform expression can contribute to either tumor-supressing or tumor-promoting processes. Furthermore, we will focus on the contradictory functions of E-cadherin and p120 in the different phases of tumor progression, from carcinoma in situ up to the formation of distant metastasis. Finally, we will discuss the possibilities and challenges when using either protein as a biomarker., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Tumor metastasis is the endpoint of tumor progression and depends on the ability of tumor cells to locally invade tissue, transit through the bloodstream and ultimately to colonize secondary organs at distant sites. P120 catenin (P120) has been implicated as an important regulator of metastatic dissemination because of its roles in cell-cell junctional stability, cytoskeletal dynamics, growth and survival. However, conflicting roles for P120 in different tumor models and steps of metastasis have been reported, and the understanding of P120 functions is confounded by the differential expression of P120 isoforms, which differ in N-terminal length, tissue localization and, likely, function. Here, we used in silico exon expression analyses, in vitro invasion assays and both RT-PCR and immunofluorescence of human tumors. We show that alternative exon usage favors expression of short isoform P120-3 in 1098 breast tumors and correlates with poor prognosis. P120-3 is upregulated at the invasive front of breast cancer cells migrating as collective groups in vitro. Furthermore, we demonstrate in histological sections of 54 human breast cancer patients that P120-3 expression is maintained throughout the metastatic cascade, whereas P120-1 is differentially expressed and diminished during invasion and in metastases. These data suggest specific regulation and functions of P120-3 in breast cancer invasion and metastasis.

L1 cell adhesion molecule (L1CAM) is a glycoprotein involved in cancer development and is associated with metastases and poor prognosis. Cellular processing of L1CAM results in expression of either full-length or cleaved forms of the protein. The different forms of L1CAM may localize at the plasma membrane as a transmembrane protein, or in the intra- or extracellular environment as cleaved or exosomal forms. Here, we systematically analyze available literature that directly relates to L1CAM domains and associated signaling pathways in cancer. Specifically, we chart its domain-specific functions in relation to cancer progression, and outline pre-clinical assays used to assess L1CAM. It is found that full-length L1CAM has both intracellular and extracellular targets, including interactions with integrins, and linkage with ezrin. Cellular processing leading to proteolytic cleavage and/or exosome formation results in extracellular soluble forms of L1CAM that may act through similar mechanisms as compared to full-length L1CAM, such as integrin-dependent signals, but also through distinct mechanisms. We provide an algorithm to guide a step-wise analysis on L1CAM in clinical samples, to promote interpretation of domain-specific expression. This systematic review infers that L1CAM has an important role in cancer progression that can be attributed to domain-specific forms. Most studies focus on the full-length plasma membrane L1CAM, yet knowledge on the domain-specific forms is a prerequisite for selective targeting treatment., Competing Interests: The authors declare no conflict of interest.

P120 catenin (p120) is a non-redundant master regulatory protein of cadherin-based cell-cell junctions, intracellular signaling, and tissue homeostasis and repair. Alternative splicing can generate p120 isoforms 1 and 3 (p120-1 and p120-3), which are implicated in non-overlapping functions by differential expression regulation and unique interactions in different cell types, with often predominant expression of p120-1 in mesenchymal cells, and p120-3 generally prevalent in epithelial cells. However, the lack of specific p120-3 protein detection has precluded analysis of their relative abundance in tissues. Here, we have developed a p120-3 isoform-specific antibody and analyzed the p120-3 localization relative to p120-1 in human tissues. p120-3 but not p120-1 is highly expressed in cell-cell junctions of simple gastrointestinal epithelia such as colon and stomach, and the acini of salivary glands and the pancreas. Conversely, the basal layer of the epidermis and hair follicles expressed p120-1 with reduced p120-3, whereas most other epithelia co-expressed p120-3 and p120-1, including bronchial epithelia and mammary luminal epithelial cells. These data provide an inventory of tissue-specific p120 isoform expression and suggest a link between p120 isoform expression and epithelial differentiation.

Collective cell migration is paramount to morphogenesis and contributes to the pathogenesis of cancer. To migrate directionally and reach their site of destination, migrating cells must distinguish a front and a rear. In addition to polarizing individually, cell-cell interactions in collectively migrating cells give rise to a higher order of polarity, which allows them to move as a supracellular unit. Rather than just conferring adhesion, emerging evidence indicates that cadherin-based adherens junctions intrinsically polarize the cluster and relay mechanical signals to establish both intracellular and supracellular polarity. In this review, we discuss the various functions of adherens junctions in polarity of migrating cohorts., (Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.)

The transformation of polarized epithelial cells into cells with mesenchymal characteristics by the morphogenetic process of epithelial-mesenchymal transition (EMT) is a well-characterized process essential for embryonic development and associated with cancer progression. EMT is a program driven by changes in gene expression induced by several EMT-specific transcription factors, which inhibit the expression of cell-cell adhesion proteins and other epithelial markers, causing a characteristic loss of cell-cell adhesion, a switch to mesenchymal cell morphology, and increased migratory capabilities. Recently, it has become apparent that in addition to these transcriptionally regulated changes, EMT may also be regulated posttranscriptionally, that is, by alternative splicing. Specifically, the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) have been described as epithelial-specific splicing master regulators specifically involved in EMT-associated alternative splicing. Here, we discuss the regulation of ESRP activity, as well as the evidence supporting a causal role of ESRPs in EMT., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Collective cell migration results from the establishment and maintenance of collective polarization, mechanocoupling, and cytoskeletal kinetics. The guidance of collective cell migration depends on a reciprocal process between cell-intrinsic multicellular organization with leader-follower cell behavior and results in mechanosensory integration of extracellular guidance cues. Important guidance mechanisms include chemotaxis, haptotaxis, durotaxis, and strain-induced mechanosensing to move cell groups along interfaces and paths of least resistance. Additional guidance mechanisms steering cell groups during specialized conditions comprise electrotaxis and passive drift. To form higher-order cell and tissue structures during morphogenesis and cancer invasion, these guidance principles act in parallel and are integrated for collective adaptation to and shaping of varying tissue environments. We review mechanochemical and electrical inputs and multiparameter signal integration underlying collective guidance, decision making, and outcome., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A recent article by Tsujita et al. (2015) in Nature Cell Biology provides insight into how cells sense and translate plasma membrane tension toward polarized actin polymerization and migration. They identify FBP17 as a multifunctional adaptor that senses membrane curvature and delivers feedback to actin dynamics and directed cell migration., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Building the complex architecture of tubular organs is a highly dynamic process that involves cell migration, polarization, shape changes, adhesion to neighboring cells and the extracellular matrix, physicochemical characteristics of the extracellular matrix and reciprocal signaling with the mesenchyme. Understanding these processes in vivo has been challenging as they take place over extended time periods deep within the developing organism. Here, I will discuss 3D in vitro models that have been crucial to understand many of the molecular and cellular mechanisms and key concepts underlying branching morphogenesis in vivo., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Collective cell migration depends on multicellular mechanocoupling between leader and follower cells to coordinate traction force and position change. Co-registration of Rho GTPase activity and forces in migrating epithelial cell sheets now shows how RhoA controls leader-follower cell hierarchy, multicellular cytoskeletal contractility and mechanocoupling, to prevent ectopic leading edges and to move the cell sheet forward.

The family of Rho GTPases are intracellular signal transducers that link cell surface signals to multiple intracellular responses. They are best known for their role in regulating actin dynamics required for cell migration, but in addition control cell-cell adhesion, polarization, vesicle trafficking, and the cell cycle. The roles of Rho GTPases in single mesenchymal cell migration are well established and rely on Cdc42- and Rac-dependent cell protrusion of a leading edge, coupled to Rho-dependent contractility required to move the cell body forward. In cells migrating collectively, cell-cell junctions are maintained, and migrating leader cells are mechanically coupled to, and coordinate, migration with follower cells. Recent evidence suggests that Rho GTPases provide multifunctional input to collective cell polarization, cell-cell interaction, and migration. Here, we discuss the role of Rho GTPases in initiating and maintaining front-rear, apical-basal cell polarization, mechanotransduction, and cell-cell junction stability between leader and follower cells, and how these roles are integrated in collective migration. Thereby, spatiotemporal fine-tuning of Rho GTPases within the same cell and among cells in the cell group are crucial in controlling potentially conflicting, divergent cell adhesion and cytoskeletal functions to achieve supracellular coordination and mechanocoupling.

Scribble was originally identified as a Drosophila protein that regulates epithelial polarity and formation of the basolateral surface. The mammalian orthologue, Scrib, is evolutionarily conserved, but does not appear to be necessary for apical-basolateral epithelial polarity. Instead, it is implicated in the regulation of cell survival, protein trafficking, adhesion and migration. A key issue is to understand the molecular pathway by which Scrib participates in these processes. We have investigated Scrib using a three-dimensional epithelial cell culture system. We show a novel association between the leucine-rich repeat domain of Scrib and the co-chaperone Sgt1 and demonstrate that these proteins are necessary for epithelial morphogenesis and tubulogenesis following hepatocyte growth factor (HGF) stimulation. The molecular chaperone HSP90 is also required for Sgt1 association with Scrib, and both Sgt1 and HSP90 are needed to ensure proper Scrib protein levels. Furthermore, reduced Scrib stability, following inhibition of Sgt1-HSP90, lowers the cellular abundance of the Scrib-βPix-PAK complex. Inhibition of any member of this complex, Scrib, βPix or PAK, is sufficient to block HGF-mediated epithelial morphogenesis. The identification of Scrib as an Sgt1-HSP90 client protein required for three-dimensional cell migration suggests that chaperone-mediated regulation of polarity protein stability and homeostasis is an unappreciated mechanism underlying dynamic rearrangements during morphogenesis.

The development of the basic architecture of branching tubules enclosing a central lumen that characterizes most epithelial organs crucially depends on the apico-basolateral polarization of epithelial cells. Signals from the extracellular matrix control the orientation of the apical surface, so that it faces the lumen interior, opposite to cell-matrix adhesion sites. This orientation of the apical surface is thought to be intrinsically linked to the formation of single lumens. We previously demonstrated in three-dimensional cyst cultures of Madin-Darby canine kidney (MDCK) cells that signaling by β1 integrins regulates the orientation of the apical surface, via a mechanism that depends on the activity of the small GTPase Rac1. Here, we investigated whether the Rac1 effector Pak1 is a downstream effector in this pathway. Expression of constitutive active Pak1 phenocopies the effect of β1 integrin inhibition in that it misorients the apical surface and induces a multilumen phenotype. The misorientation of apical surfaces depends on the interaction of active Pak1 with PIX proteins and is linked to defects in basement membrane assembly. In contrast, the multilumen phenotype was independent of PIX and the basement membrane. Therefore, Pak1 likely regulates apical polarization and lumen formation by two distinct pathways.

Classic cadherins are important regulators of tissue morphogenesis. The predominant cadherin in epithelial cells, E-cadherin, has been extensively studied because of its critical role in normal epithelial development and carcinogenesis. Epithelial cells may also coexpress other cadherins, but their roles are less clear. The Madin Darby canine kidney (MDCK) cell line has been a popular mammalian model to investigate the role of E-cadherin in epithelial polarization and tubulogenesis. However, MDCK cells also express relatively high levels of cadherin-6, and it is unclear whether the functions of this cadherin are redundant to those of E-cadherin. We investigate the specific roles of both cadherins using a knockdown approach. Although we find that both cadherins are able to form adherens junctions at the basolateral surface, we show that they have specific and mutually exclusive roles in epithelial morphogenesis. Specifically, we find that cadherin-6 functions as an inhibitor of tubulogenesis, whereas E-cadherin is required for lumen formation. Ablation of cadherin-6 leads to the spontaneous formation of tubules, which depends on increased phosphoinositide 3-kinase (PI3K) activity. In contrast, loss of E-cadherin inhibits lumen formation by a mechanism independent of PI3K.

To generate and maintain epithelial cell polarity, specific sorting of proteins into vesicles destined for the apical and basolateral domain is required. Syntaxin 3 and 4 are apical and basolateral SNARE proteins important for the specificity of vesicle fusion at the apical and basolateral plasma membrane domains, respectively, but how these proteins are specifically targeted to these domains themselves is unclear. Munc18/SM proteins are potential regulators of this process. Like syntaxins, they are crucial for exocytosis and vesicle fusion. However, how munc18c and syntaxin 4 regulate the function of each other is unclear. Here, we investigated the requirement of syntaxin 4 in the delivery of basolateral membrane and secretory proteins, the basolateral targeting of syntaxin 4, and the role of munc18c in this targeting. Depletion of syntaxin 4 resulted in significant reduction of basolateral targeting, suggesting no compensation by other syntaxin forms. Mutational analysis identified amino acids Leu-25 and to a lesser extent Val-26 as essential for correct localization of syntaxin 4. Recently, it was shown that the N-terminal peptide of syntaxin 4 is involved in binding to munc18c. A mutation in this region that affects munc18c binding shows that munc18c binding is required for stabilization of syntaxin 4 at the plasma membrane but not for its correct targeting. We conclude that the N terminus serves two functions in membrane targeting. First, it harbors the sorting motif, which targets syntaxin 4 basolaterally in a munc18c-independent manner and second, it allows for munc18c binding, which stabilizes the protein in a munc18c-dependent manner.

Branching morphogenesis is a fundamental process in the development of the kidney. This process gives rise to a network of ducts, which form the collecting system. Defective branching can lead to a multitude of kidney disorders including agenesis and reduced nephron number. The formation of branching tubules involves changes in cell shape, cell motility, and reorganization of the cytoskeleton. However, the exact intracellular mechanisms involved are far from understood. We have used the three-dimensional (3D) Madin-Darby canine kidney (MDCK) cell culture system to study how p21-activated kinase 1 (Pak1), which is an important regulator of the cytoskeleton, modulates branching. Our data reveal that Pak1 plays a crucial role in regulating branching morphogenesis. Expression of a dominant-negative Pak1 mutant (DN-Pak1) in MDCK cysts resulted in the spontaneous formation of extensions and branching tubules. Cellular contractility and levels of phosphorylated myosin light chain (pMLC) were increased in DN-Pak1 cells in collagen. Expression of a DN-Pak1 mutant that does not bind to PIX (DN-Pak1-DeltaPIX) failed to form extensions in collagen and did not have increased contractility. This shows that the DN-Pak1 mutant requires PIX binding to generate extensions and increased contractility in 3D culture. Furthermore, a beta1-integrin function-blocking antibody (AIIB2) inhibited the formation of branches and blocked the increased contractility in DN-Pak1 cysts. Taken together, our work shows that DN-Pak1-induced branching morphogenesis requires PIX binding and beta1-integrin signaling.

It is crucial for organ homeostasis that epithelia have effective mechanisms to restrict motility and cell proliferation in order to maintain tissue architecture. On the other hand, epithelial cells need to rapidly and transiently acquire a more mesenchymal phenotype, with high levels of cell motility and proliferation, in order to repair epithelia upon injury. Cross talk between cell-cell and cell-matrix signaling is crucial for regulating these transitions. The Pak1-betaPIX-GIT complex is an effector complex downstream of the small GTPase Rac1. We previously showed that translocation of this complex from cell-matrix to cell-cell adhesion sites was required for the establishment of contact inhibition of proliferation. In this study, we provide evidence that this translocation depends on cadherin function. Cadherins do not recruit the complex by direct interaction. Rather, we found that inhibition of the normal function of cadherin or Pak1 leads to defects in focal adhesion turnover and to increased signaling by phosphatidylinositol 3-kinase. We propose that cadherins are involved in regulation of contact inhibition by controlling the function of the Pak1-betaPIX-GIT complex at focal contacts.

In multicellular epithelial tissues, the orientation of polarity of each cell must be coordinated. Previously, we reported that for Madin-Darby canine kidney cells in three-dimensional collagen gel culture, blockade of beta1-integrin by the AIIB2 antibody or expression of dominant-negative Rac1N17 led to an inversion of polarity, such that the apical surfaces of the cells were misorientated towards the extracellular matrix. Here, we show that this process results from the activation of RhoA. Knockdown of RhoA by short hairpin RNA reverses the inverted orientation of polarity, resulting in normal cysts. Inhibition of RhoA downstream effectors, Rho kinase (ROCK I) and myosin II, has similar effects. We conclude that the RhoA-ROCK I-myosin II pathway controls the inversion of orientation of epithelial polarity caused by AIIB2 or Rac1N17. These results might be relevant to the hyperactivation of RhoA and disruption of normal polarity frequently observed in human epithelial cancers.

Rho GTPases are critical regulators of epithelial morphogenesis. A powerful means to investigate their function is three-dimensional (3D) cell culture, which mimics the architecture of epithelia in vivo. However, the nature of 3D culture requires specialized techniques for morphological and biochemical analyses. Here, we describe protocols for 3D culture studies with Madin-Darby Canine Kidney (MDCK) epithelial cells: establishing cultures, immunostaining, and expressing, detecting, and assaying Rho proteins. These protocols enable the regulation of epithelial morphogenesis to be explored at a detailed molecular level.

Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures. We show that interaction of collagen I with apical beta1-integrins after collagen overlay of a polarized MDCK monolayer induces activation of Rac1, which is required for collagen overlay-induced tubulocyst formation. Cysts, comprised of a monolayer enclosing a central lumen, form after embedding single cells in collagen. In those cultures, addition of a beta1-integrin function-blocking antibody to the collagen matrix gives rise to cysts that have defects in the organization of laminin into the basement membrane and have inverted polarity. Normal polarity is restored by either expression of activated Rac1, or the inclusion of excess laminin-1 (LN-1). Together, our results suggest a signaling pathway in which the activation of beta1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane.

Wound healing in epithelia requires coordinated cell migration and proliferation regulated by signaling mechanisms that are poorly understood. Here we show that epithelial cells expressing constitutively active or kinase-dead mutants of the Rac/Cdc42 effector Pak1 fail to undergo growth arrest upon wound closure. Strikingly, this phenotype is only observed when the Pak1 kinase mutants are expressed in cells possessing a free lateral surface, i.e. one that is not engaged in contact with neighboring cells. The Pak1 kinase mutants perturb contact inhibition by a mechanism that depends on the Pak-interacting Rac-GEF PIX. In control cells, endogenous activated Pak and PIX translocate from focal complexes to cell-cell contacts during wound closure. This process is abrogated in cells expressing Pak1 kinase mutants. In contrast, Pak1 mutants rendered defective in PIX binding do not impede translocation of activated Pak and PIX, and exhibit normal wound healing. Thus, recruitment of activated Pak and PIX to cell-cell contacts is pivotal to transduction of growth-inhibitory signals from neighboring cells in epithelial wound healing.

The most fundamental type of organization of cells in metazoa is that of epithelia, which comprise sheets of adherent cells that divide the organism into topologically and physiologically distinct spaces. Some epithelial cells cover the outside of the organism; these often form multiple layers, such as in skin. Other epithelial cells form monolayers that line internal organs, and yet others form tubes that infiltrate the whole organism, carrying liquids and gases containing nutrients, waste and other materials. These tubes can form elaborate networks in the lung, kidney, reproductive passages and vasculature tree, as well as the many glands branching from the digestive system such as the liver, pancreas and salivary glands. In vitro systems can be used to study tube formation and might help to define common principles underlying the formation of diverse types of tubular organ.

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.

How do individual cells organize into multicellular tissues? Here, we propose that the morphogenetic behaviour of epithelial cells is guided by two distinct elements: an intrinsic differentiation programme that drives formation of a lumen-enclosing monolayer, and a growth factor-induced, transient de-differentiation that allows this monolayer to be remodelled.

Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and Raf exhibit cell multilayering and alterations in the actin cytoskeleton. The changes in the actin cytoskeleton comprise a loss of actin stress fibers and enhanced cortical actin. Using MDCK cells expressing a conditionally active form of Raf, we have explored the molecular mechanisms that underlie these observations. Raf activation elicited a robust increase in Rac1 activity consistent with the observed increase in cortical actin. Loss of actin stress fibers is indicative of attenuated Rho function, but no change in Rho-GTP levels was detected following Raf activation. However, the loss of actin stress fibers in Raf-transformed cells was preceded by the induced expression of Rnd3, an endogenous inhibitor of Rho protein function. Expression of Rnd3 alone at levels equivalent to those observed following Raf transformation led to a substantial loss of actin stress fibers. Moreover, cells expressing activated RhoA failed to multilayer in response to Raf. Pharmacological inhibition of MEK activation prevented all of the biological and biochemical changes described above. Consequently, the data are consistent with a role for induced Rnd3 expression downstream of the Raf-MEK-extracellular signal-regulated kinase pathway in epithelial oncogenesis.

Epithelial cells express plasma-membrane polarity in order to meet functional requirements that are imposed by their interaction with different extracellular environments. Thus apical and basolateral membrane domains are distinguished that are separated by tight junctions in order to maintain the specific lipid and protein composition of each domain. In hepatic cells, the plasma membrane is also polarized, containing a sinusoidal (basolateral) and a bile canalicular (apical)-membrane domain. Relevant to the biogenesis of these domains are issues concerning sorting, (co-)transport and regulation of transport of domain-specific membrane components. In epithelial cells, specific proteins and lipids, destined for the apical membrane, are sorted in the trans-Golgi network (TGN), which involves their sequestration into cholesterol/sphingolipid 'rafts', followed by 'direct' transport to the apical membrane. In hepatic cells, a direct apical transport pathway also exists, as revealed by transport of sphingolipids from TGN to the apical membrane. This is remarkable, since in these cells numerous apical membrane proteins are 'indirectly' sorted, i.e. they are first transferred to the basolateral membrane prior to their subsequent transcytosis to the apical membrane. This raises intriguing questions as to the existence of specific lipid rafts in hepatocytes. As demonstrated in studies with HepG2 cells, it has become evident that, in hepatic cells, apical transport pathways can be regulated by protein kinase activity, which in turn modulates cell polarity. Finally, an important physiological function of hepatic cells is their involvement in intracellular transport and secretion of bile-specific lipids. Mechanisms of these transport processes, including the role of multidrug-resistant proteins in lipid translocation, will be discussed in the context of intracellular vesicular transport. Taken together, hepatic cell systems provide an important asset to studies aimed at elucidating mechanisms of sorting and trafficking of lipids (and proteins) in polarized cells in general.

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and sphingomyelin, whereas the other involves basolateral to apical transcytosis of both sphingolipids. We show that these distinct routes display a different sensitivity toward nocodazole and cytochalasin D, implying a specific transport dependence on either microtubules or actin filaments, respectively. Thus, nocodazole strongly inhibited the direct route, whereas sphingolipid transport by transcytosis was hardly affected. Moreover, nocodazole blocked "hyperpolarization," i.e., the enlargement of the apical membrane surface, which is induced by treating cells with dibutyryl-cAMP. By contrast, the transcytotic route but not the direct route was inhibited by cytochalasin D. The actin-dependent step during transcytotic lipid transport probably occurs at an early endocytic event at the basolateral plasma membrane, because total lipid uptake and fluid phase endocytosis of horseradish peroxidase from this membrane were inhibited by cytochalasin D as well. In summary, the results show that the two sphingolipid transport pathways to the apical membrane must have a different requirement for cytoskeletal elements.

A photoreactive sphingolipid precursor was used to investigate the potential involvement of protein-lipid interactions that may convey specificity to sphingolipid transport in the human hepatoma cell line, HepG2. A 125I-labeled, photoreactive ceramide, 125I-N3-Cer, was incubated with the cells and became incorporated into two sphingolipid products. The major product was photoreactive sphingomyelin (125I-N3-SM) (25% of total radioactivity), while only minor amounts of photoreactive glucosylceramide (125I-N3-GlcCer) were formed (< 2%). After photoactivation, a restricted number of proteins was labeled. Given the absolute amounts of the newly synthesized, photoreactive lipids and their precursor present in the cells, labeling of the proteins can be assumed to be derived from interaction with either ceramide (Cer) or sphingomyelin (SM), or both. To discriminate between these possibilities, photoactivation and protein analysis was performed in cells treated with D-threo-1-phenyl-2-decanoyl amino-3-morpholino-1-propanol (PDMP), an inhibitor of sphingolipid biosynthesis. In treated cells, the radioactive SM pool was reduced by approximately 80%. Concomitantly, labeling of a 60-kd protein, seen in control cells, decreased. Furthermore, the 60-kd protein is membrane-associated and insoluble in detergent at low temperature. Moreover, when cells containing photoreactive sphingolipids after a preincubation with the photoreactive Cer were photoactivated and subsequently incubated with fluorescent sphingolipid analogs, transport of the latter to the bile canalicular membrane, as observed in control cells, was inhibited. Taken together, the data suggest that distinct proteins, among them a 60-kd protein, may play a specific and functional role in sphingolipid transport to the bile canalicular membrane.

Photoactivatable derivatives of ceramide, glucosylceramide (GlcCer) and sphingomyelin {3-(p-azido-m-[125I]iodophenyl)propionylceramide, 3-(p-azido-m-[125I]iodophenyl)propionyl-GlcCer and 3-(p-azido-m-[125I]iodophenyl)propionylsphingomyelin} were synthesized in an attempt to identify compartment-specific proteins involved in sphingolipid sorting or metabolism. In HT29 and BHK cells the ceramide analogue entered the cell by monomeric diffusion, as evidenced by the probe's efficient internalization at low temperature (4 degrees C). In contrast, the photoactivatable GlcCer was internalized only at elevated temperatures (37 degrees C), presumably reflecting an endocytic mechanism of uptake. The photoactivatable ceramide was mainly metabolized to the corresponding sphingomyelin analogue, but small amounts of GlcCer and galactosylceramide were also synthesized. The newly synthesized photoreactive sphingomyelin was subsequently transported to the cell surface, a process that was effectively inhibited by the presence of brefeldin A. The incubation of cells with photoactivatable analogues at 4 degrees C, followed by illumination, led to the association of sphingolipid with a specific subset of proteins. The protein labelling pattern of ceramide differed from that of glucosylceramide. A further shift in labelling pattern was apparent when the cells were incubated with the lipid analogues at 37 degrees C. Moreover, most of the proteins labelled by photoreactive sphingomyelin seemed to be detergent-insoluble, which is indicative of a location in sphingolipid-rich microdomains at the plasma membrane. The potential of applying photoactivatable sphingolipids to further define and identify the role of distinct proteins in sphingolipid biosynthesis, transport and sorting, is discussed.

The regulation of sphingolipid transport to the bile canalicular apical membrane in the well differentiated HepG2 hepatoma cells was studied. By employing fluorescent lipid analogs, trafficking in a transcytosis-dependent pathway and a transcytosis-independent ('direct') route between the trans-Golgi network and the apical membrane were examined. The two lipid transport routes were shown to operate independently, and both were regulated by kinase activity. The kinase inhibitor staurosporine inhibited the direct lipid transport route but slightly stimulated the transcytosis-dependent route. The protein kinase C (PKC) activator phorbol-12 myristate-13 acetate (PMA) inhibited apical lipid transport via both transport routes, while a specific inhibitor of this kinase stimulated apical lipid transport. Activation of protein kinase A (PKA) had opposing effects, in that a stimulation of apical lipid transport via both transport routes was seen. Interestingly, the regulatory effects of either kinase activity in sphingolipid transport correlated with changes in cell polarity. Stimulation of PKC activity resulted in a disappearance of the bile canalicular structures, as evidenced by the redistribution of several apical markers upon PMA treatment, which was accompanied by an inhibition of apical sphingolipid transport. By contrast, activation of PKA resulted in an increase in the number and size of bile canaliculi and a concomitant enhancement of apical sphingolipid transport. Taken together, our data indicate that apical membrane-directed sphingolipid transport in HepG2 cells is regulated by kinases, which could play a role in the biogenesis of the apical plasma membrane domain.

HepG2 cells are highly differentiated hepatoma cells that have retained an apical, bile canalicular (BC) plasma membrane polarity. We investigated the dynamics of two BC-associated sphingolipids, glucosylceramide (GlcCer) and sphingomyelin (SM). For this, the cells were labeled with fluorescent acyl chain-labeled 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]hexanoic acid (C6-NBD) derivatives of either GlcCer (C6-NBD-GlcCer) or SM (C6-NBD-SM). The pool of the fluorescent lipid analogues present in the basolateral plasma membrane domain was subsequently depleted and the apically located C6-NBD-lipid was chased at 37 degrees C. By using fluorescence microscopical analysis and a new assay that allows an accurate estimation of the fluorescent lipid pool in the apical membrane, qualitative and quantitative insight was obtained concerning kinetics, extent and (intra)cellular sites of the redistribution of apically located C6-NBD-GlcCer and C6-NBD-SM. It is demonstrated that both lipids display a preferential localization, C6-NBD-GlcCer in the apical and C6-NBD-SM in the basolateral area. Such a preference is expressed during transcytosis of both sphingolipids from the apical to the basolateral plasma membrane domain, a novel lipid trafficking route in HepG2 cells. Whereas the vast majority of the apically derived C6-NBD-SM was rapidly transcytosed to the basolateral surface, most of the apically internalized C6-NBD-GlcCer was efficiently redirected to the BC. The redirection of C6-NBD-GlcCer did not involve trafficking via the Golgi apparatus. Evidence is provided which suggests the involvement of vesicular compartments, located subjacent to the apical plasma membrane. Interestingly, the observed difference in preferential localization of C6-NBD-GlcCer and C6-NBD-SM was perturbed by treatment of the cells with dibutyryl cAMP, a stable cAMP analogue. While the preferential apical localization of C6-NBD-GlcCer was amplified, dibutyryl cAMP-treatment caused apically retrieved C6-NBD-SM to be processed via a similar pathway as that of C6-NBD-GlcCer. The data unambiguously demonstrate that segregation of GlcCer and SM occurs in the reverse transcytotic route, i.e., during apical to basolateral transport, which results in the preferential localization of GlcCer and SM in the apical and basolateral region of the cells, respectively. A role for non-Golgi-related, sub-apical vesicular compartments in the sorting of GlcCer and SM is proposed.