Live-cell Imaging of Biosynthetic Protein Transport in Hepatocytes.

Here, we describe a strategy to analyze the exit of apical and basolateral cargo from the trans-Golgi network in primary hepatocytes. The method is based on recombinant adenovirus-mediated infection combined with a pulse-chase regimen and live-cell imaging analysis of fluorescent protein-tagged dipeptidyl peptidase IV (DPPIV) and vesicular stomatitis virus G (VSVG) cargo proteins, coexpressed and accumulated in the endoplasmic reticulum via DPPIV aggregation through an engineered conditional aggregation domain and VSVG by exploiting the aggregation of the ts045 mutant at its non-permissive temperature of 40 °C.

Low Rho activity in hepatocytes prevents apical from basolateral cargo separation during trans-Golgi network to surface transport.

Hepatocytes, the main epithelial cells of the liver, organize their polarized membrane domains differently from ductal epithelia. They also differ in their biosynthetic delivery of single-membrane-spanning and glycophosphatidylinositol-anchored proteins to the apical domain. While ductal epithelia target apical proteins to varying degrees from the trans-Golgi network (TGN) to the apical surface directly, hepatocytes target them first to the basolateral domain, from where they undergo basolateral-to-apical transcytosis. How TGN-to-surface transport differs in both scenarios is unknown. Here, we report that the basolateral detour of a hepatocyte apical protein is due, in part, to low RhoA activity at the TGN, which prevents its segregation from basolateral transport carriers. Activating Rho in hepatocytic cells, which switches their polarity from hepatocytic to ductal, also led to apical-basolateral cargo segregation at the TGN as is typical for ductal cells, affirming a central role for Rho-signaling in different aspects of the hepatocytic polarity phenotype. Nevertheless, Rho-induced cargo segregation was not sufficient to target the apical protein directly; thus, failure to recruit apical targeting machinery also contributes to its indirect itinerary.

Inhibition of polarity-regulating kinase PAR1b contributes to Helicobacter pylori inflicted DNA Double Strand Breaks in gastric cells.

The serine/threonine kinase Par1 is a core component of the machinery that sets up polarity in the embryo and regulates cell fate decisions but its role in the homeostasis of adult tissues is poorly understood. Inhibition of Par1 by the bacterium Helicobacter pylori (H. pylori) represents the only established pathology that affects Par1 function in an adult epithelium. Thus, during chronic H. pylori infection of the gastric mucosa Par1 is one of the targets of the non-obligate H.pylori cytotoxic protein and oncogene CagA, which stimulates inflammation and triggers morphological changes, both believed to contribute to the gastric cancer risk imposed by H. pylori infection. Based on Par1's role in cell polarity, it has been speculated that Par1 inhibition affects epithelial polarity. Here we report the unexpected finding that CagA-mediated Par1-inhibition promotes the generation of DNA Double Strand Breaks in primary gastric epithelial cells, which likely contributes to the reported accumulation of mutations in chronically infected mucosal cells. Abbreviations: AGS: human gastric adenocarcinoma cell line; CM: CagA Multimerization (and Par1 binding) domain; H. pylori: Helicobacter pylori; DSB: Double Strand Break; HGECs: human (primary) gastric epithelial cells; IB: immunoblot; IF: immunofluorescence; MOI: Multiplicity of Infection; ROS: reactive oxygen species; Par1: Partitioning Defective 1 kinase; WT: wild type.

From a common progenitor to distinct liver epithelial phenotypes.

The vertebrate liver presents a fascinating case study for how cell form is optimized for function. To execute its duties the liver assembles two distinct lumen-forming epithelial phenotypes: Firstly, cords with a branched, capillary-like luminal network formed between hepatocytes (bile canaliculi); and secondly, tubular ducts formed by biliary epithelial cells arranged around a central cavity and connected to the bile canaliculi. How these remarkably different epithelial polarity phenotypes are generated and joined into a contiguous luminal network are major unresolved questions. Recent studies have characterized the divergence of the two epithelial lineages from common progenitors, described the coordination of bile canaliculi formation with bile duct branching during biliary tree morphogenesis and implicated RhoA-dependent E-cadherin adhesion in the decision to polarize with hepatocytic or biliary phenotype.

Cell-cell adhesion accounts for the different orientation of columnar and hepatocytic cell divisions.

Mitotic spindle alignment with the basal or substrate-contacting domain ensures that dividing epithelial cells remain in the plane of the monolayer. Spindle orientation with respect to the substratum is established in metaphase coincident with maximal cell rounding, which enables unobstructed spindle rotation. Misaligned metaphase spindles are believed to result in divisions in which one daughter loses contact with the basal lamina. Here we describe a rescue mechanism that drives substrate-parallel spindle alignment of quasi-diagonal metaphase spindles in anaphase. It requires a Rho- and E-cadherin adhesion-dependent, substrate-parallel contractile actin belt at the apex that governs anaphase cell flattening. In contrast to monolayered Madin-Darby canine kidney cells, hepatocytic epithelial cells, which typically feature tilted metaphase spindles, lack this anaphase flattening mechanism and as a consequence maintain their spindle tilt through cytokinesis. This results in out-of-monolayer divisions, which we propose contribute to the stratified organization of hepatocyte cords in vivo.

Partitioning-Defective 1a/b Depletion Impairs Glomerular and Proximal Tubule Development.

The kidney is a highly polarized epithelial organ that develops from undifferentiated mesenchyme, although the mechanisms that regulate the development of renal epithelial polarity are incompletely understood. Partitioning-defective 1 (Par1) proteins have been implicated in cell polarity and epithelial morphogenesis; however, the role of these proteins in the developing kidney has not been established. Therefore, we studied the contribution of Par1a/b to renal epithelial development. We examined the renal phenotype of newborn compound mutant mice carrying only one allele of Par1a or Par1b. Loss of three out of four Par1a/b alleles resulted in severe renal hypoplasia, associated with impaired ureteric bud branching. Compared with kidneys of newborn control littermates, kidneys of newborn mutant mice exhibited dilated proximal tubules and immature glomeruli, and the renal proximal tubular epithelia lacked proper localization of adhesion complexes. Furthermore, Par1a/b mutants expressed low levels of renal Notch ligand Jag1, activated Notch2, and Notch effecter Hes1. Together, these data demonstrate that Par1a/b has a key role in glomerular and proximal tubule development, likely via modulation of Notch signaling.

Iterative sorting of apical and basolateral cargo in Madin-Darby canine kidney cells.

For several decades, the trans-Golgi network (TGN) was considered the most distal stop and hence the ultimate protein-sorting station for distinct apical and basolateral transport carriers that reach their respective surface domains in the direct trafficking pathway. However, recent reports of apical and basolateral cargoes traversing post-Golgi compartments accessible to endocytic ligands before their arrival at the cell surface and the post-TGN breakup of large pleomorphic membrane fragments that exit the Golgi region toward the surface raised the possibility that compartments distal to the TGN mediate or contribute to biosynthetic sorting. Here we describe the development of a novel assay that quantitatively distinguishes different cargo pairs by their degree of colocalization at the TGN and by the evolution of colocalization during their TGN-to-surface transport. Keys to the high resolution of our approach are 1) conversion of perinuclear organelle clustering into a two-dimensional microsomal spread and 2) identification of TGN and post-TGN cargo without the need for a TGN marker that universally cosegregates with all cargo. Using our assay, we provide the first evidence that apical NTRp75 and basolateral VSVG in Madin-Darby canine kidney cells still undergo progressive sorting after they exit the TGN toward the cell surface.

Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum.

All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow ß angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell's height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule-mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning.

The unique polarity phenotype of hepatocytes.

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.

The special case of hepatocytes: unique tissue architecture calls for a distinct mode of cell division.

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 kinase Par1b determines both the epithelial lumen polarity and cell division phenotype via cell adhesion signaling that converges on the small GTPase RhoA.

KIFC3 promotes mitotic progression and integrity of the central spindle in cytokinesis.

Kinesin-14 motor proteins play a variety of roles during metaphase and anaphase. However, it is not known whether members of this family of motors also participate in the dramatic changes in mitotic spindle organization during the transition from telophase to cytokinesis. We have identified the minus-end-directed motor, KIFC3, as an important contributor to central bridge morphology at this stage. KIFC3's unique motor-dependent localization at the central bridge allows it to congress microtubules, promoting efficient progress through cytokinesis. Conversely, when KIFC3 function is perturbed, abscission is delayed, and the central bridge is both widened and extended. Examination of KIFC3 on growing microtubules in interphase indicates that it caps microtubules released from the centrosome, both in the region of the centrosome and in the cell periphery. In line with other kinesin-14 family members, KIFC3 may guide free microtubules to their destination at the bridge and/or may slide and crosslink central bridge microtubules in order to stage the cells for abscission.

Par1b induces asymmetric inheritance of plasma membrane domains via LGN-dependent mitotic spindle orientation in proliferating hepatocytes.

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 hepatocytes thus display an asymmetric inheritance of their apical domains via a mechanism that involves Par1b and LGN, which we postulate serves the unique tissue architecture of the developing liver parenchyma.

Par1b links lumen polarity with LGN-NuMA positioning for distinct epithelial cell division phenotypes.

Columnar epithelia establish their luminal domains and their mitotic spindles parallel to the basal surface and undergo symmetric cell divisions in which the cleavage furrow bisects the apical domain. Hepatocyte lumina interrupt the lateral domain of neighboring cells perpendicular to two basal domains and their cleavage furrow rarely bifurcates the luminal domains. We determine that the serine/threonine kinase Par1b defines lumen position in concert with the position of the astral microtubule anchoring complex LGN-NuMA to yield the distinct epithelial division phenotypes. Par1b signaling via the extracellular matrix (ECM) in polarizing cells determined RhoA/Rho-kinase activity at cell-cell contact sites. Columnar MDCK and Par1b-depleted hepatocytic HepG2 cells featured high RhoA activity that correlated with robust LGN-NuMA recruitment to the metaphase cortex, spindle alignment with the substratum, and columnar organization. Reduced RhoA activity at the metaphase cortex in HepG2 cells and Par1b-overexpressing MDCK cells correlated with a single or no LGN-NuMA crescent, tilted spindles, and the development of lateral lumen polarity.

A model for the self-organization of vesicular flux and protein distributions in the Golgi apparatus.

The generation of two non-identical membrane compartments via exchange of vesicles is considered to require two types of vesicles specified by distinct cytosolic coats that selectively recruit cargo, and two membrane-bound SNARE pairs that specify fusion and differ in their affinities for each type of vesicles. The mammalian Golgi complex is composed of 6-8 non-identical cisternae that undergo gradual maturation and replacement yet features only two SNARE pairs. We present a model that explains how distinct composition of Golgi cisternae can be generated with two and even a single SNARE pair and one vesicle coat. A decay of active SNARE concentration in aging cisternae provides the seed for a cis[Formula: see text]trans SNARE gradient that generates the predominantly retrograde vesicle flux which further enhances the gradient. This flux in turn yields the observed inhomogeneous steady-state distribution of Golgi enzymes, which compete with each other and with the SNAREs for incorporation into transport vesicles. We show analytically that the steady state SNARE concentration decays exponentially with the cisterna number. Numerical solutions of rate equations reproduce the experimentally observed SNARE gradients, overlapping enzyme peaks in cis, medial and trans and the reported change in vesicle nature across the Golgi: Vesicles originating from younger cisternae mostly contain Golgi enzymes and SNAREs enriched in these cisternae and extensively recycle through the Endoplasmic Reticulum (ER), while the other subpopulation of vesicles contains Golgi proteins prevalent in older cisternae and hardly reaches the ER.

Hepatocyte polarity.

Hepatocytes, like other epithelia, are situated at the interface between the organism's exterior and the underlying internal milieu and organize the vectorial exchange of macromolecules between these two spaces. To mediate this function, epithelial cells, including hepatocytes, are polarized with distinct luminal domains that are separated by tight junctions from lateral domains engaged in cell-cell adhesion and from basal domains that interact with the underlying extracellular matrix. Despite these universal principles, hepatocytes distinguish themselves from other nonstriated epithelia by their multipolar organization. Each hepatocyte participates in multiple, narrow lumina, the bile canaliculi, and has multiple basal surfaces that face the endothelial lining. Hepatocytes also differ in the mechanism of luminal protein trafficking from other epithelia studied. They lack polarized protein secretion to the luminal domain and target single-spanning and glycosylphosphatidylinositol-anchored bile canalicular membrane proteins via transcytosis from the basolateral domain. We compare this unique hepatic polarity phenotype with that of the more common columnar epithelial organization and review our current knowledge of the signaling mechanisms and the organization of polarized protein trafficking that govern the establishment and maintenance of hepatic polarity. The serine/threonine kinase LKB1, which is activated by the bile acid taurocholate and, in turn, activates adenosine monophosphate kinase-related kinases including AMPK1/2 and Par1 paralogues has emerged as a key determinant of hepatic polarity. We propose that the absence of a hepatocyte basal lamina and differences in cell-cell adhesion signaling that determine the positioning of tight junctions are two crucial determinants for the distinct hepatic and columnar polarity phenotypes.

The serine/threonine kinase Par1b regulates epithelial lumen polarity via IRSp53-mediated cell-ECM signaling.

The serine/threonine kinase Par1b promotes cell-cell adhesion and determines the polarity of the luminal domain in epithelial cells. In this study, we demonstrate that Par1b also regulates cell-extracellular matrix (ECM) signaling in kidney-derived Madin-Darby canine kidney (MDCK) cells and identified the rho-guanosine triphosphatase adaptor and scaffolding protein IRSp53 as a Par1b substrate involved in this pathway. Par1b overexpression inhibits basal lamina formation, cell spreading, focal adhesion, stress fiber formation, and compaction, whereas Par1b depletion has the opposite effect. IRSp53 depletion mimics Par1b overexpression on cell-ECM signaling and lumen polarity but had no effect on adherens junction formation. Par1b directly phosphorylates IRSp53 on S366 in cell lysates and stimulates phosphorylation on S453/3/5 via an indirect mechanism. A Par1b phosphorylation-deficient IRSp53 mutant but not the wild-type protein efficiently rescues both the cell spreading and the lumen polarity defects in Par1b MDCK cells. Our data suggest a model in which Par1b phosphorylation prevents recruitment of IRSp53 effector proteins to its Src homology domain 3 by promoting 14-3-3 binding in the vicinity of that domain.

Analysis of detergent-resistant membranes of Helicobacter pylori infected gastric adenocarcinoma cells reveals a role for MARK2/Par1b in CagA-mediated disruption of cellular polarity.

Detergent-resistant membranes of eukaryotic cells are enriched in many important cellular signalling molecules and frequently targeted by bacterial pathogens. To learn more about pathogenic mechanisms of Helicobacter pylori and to elucidate novel effects on host epithelial cells, we investigated how bacterial co-cultivation changes the protein composition of detergent-resistant membranes of gastric adenocarcinoma (AGS) tissue culture cells. Using iTRAQ (isobaric tags for relative and absolute quantification) analysis we identified several cellular proteins, which are potentially related to H. pylori virulence. One of the proteins, which showed a significant infection-dependent increase in detergent resistance, was the polarity-associated serine/threonine kinase MARK2 (EMK1/Par-1b). We demonstrate that H. pylori causes the recruitment of MARK2 from the cytosol to the plasma membrane, where it colocalizes with the bacteria and interacts with CagA. Using Mardin Darby Canine Kidney (MDCK) monolayers and a three-dimensional MDCK tissue culture model we showed that association of CagA with MARK2 not only causes disruption of apical junctions, but also inhibition of tubulogenesis and cell differentiation.

Par1b promotes hepatic-type lumen polarity in Madin Darby canine kidney cells via myosin II- and E-cadherin-dependent signaling.

Kidney-derived Madin Darby canine kidney (MDCK) cells form lumina at their apices, and target luminal proteins to an intracellular vacuolar apical compartment (VAC) when prevented from polarizing. Hepatocytes, by contrast, organize their luminal surfaces (the bile canaliculi; BC) between their lateral membranes, and, when nonpolarized, they display an intracellular luminal compartment that is distinct from the VACs of MDCK cells. Overexpression of the serine/threonine kinase Par1b/EMK1/MARK2 induces BC-like lateral lumina and a hepatic-type intracellular luminal compartment in MDCK cells, suggesting a role for Par1b in the branching decision between kidney- and hepatic-type epithelial phenotypes. Here, we report that Par1b promotes lateral lumen polarity in MDCK cells independently of Ca(2+)-mediated cell-cell adhesion by inhibiting myosin II in a rho kinase-dependent manner. Polarization was inhibited by E-cadherin depletion but promoted by an adhesion-defective E-cadherin mutant. By contrast, apical surface formation in control MDCK cells required Ca(2+)-dependent cell-cell adhesion, but it occurred in the absence of E-cadherin. We propose that E-cadherin, when in an adhesion-incompetent state at the lateral domain, serves as targeting patch for the establishment of lateral luminal surfaces. E-cadherin depletion also reverted the hepatic-type intracellular luminal compartment in Par1b-MDCK cells to VACs characteristic of control MDCK cells, indicating a novel link between E-cadherin and luminal protein targeting.

PAR1b promotes cell-cell adhesion and inhibits dishevelled-mediated transformation of Madin-Darby canine kidney cells.

Mammalian Par1 is a family of serine/threonine kinases comprised of four homologous isoforms that have been associated with tumor suppression and differentiation of epithelial and neuronal cells, yet little is known about their cellular functions. In polarizing kidney epithelial (Madin-Darby canine kidney [MDCK]) cells, the Par1 isoform Par1b/MARK2/EMK1 promotes the E-cadherin-dependent compaction, columnarization, and cytoskeletal organization characteristic of differentiated columnar epithelia. Here, we identify two functions of Par1b that likely contribute to its role as a tumor suppressor in epithelial cells. 1) The kinase promotes cell-cell adhesion and resistance of E-cadherin to extraction by nonionic detergents, a measure for the association of the E-cadherin cytoplasmic domain with the actin cytoskeleton, which is critical for E-cadherin function. 2) Par1b attenuates the effect of Dishevelled (Dvl) expression, an inducer of wnt signaling that causes transformation of epithelial cells. Although Dvl is a known Par1 substrate in vitro, we determined, after mapping the PAR1b-phosphorylation sites in Dvl, that PAR1b did not antagonize Dvl signaling by phosphorylating the wnt-signaling molecule. Instead, our data suggest that both proteins function antagonistically to regulate the assembly of functional E-cadherin-dependent adhesion complexes.

Protein sorting in the Golgi complex: shifting paradigms.

The paradigms for transport along the biosynthetic route have changed dramatically over the past 15 years. Unlike the situation 15 years ago, the current paradigm involves sorting signals practically at every step of the pathway. In particular, at the exit from the Golgi complex, apical, basolateral and lysosomal targeting signals result in the generation of a variety of routes. Furthermore, it is now quite clear that not all sorting in the biosynthetic route occurs in the Golgi complex or the Trans Golgi Network (TGN). Sorting may occur distally to the Golgi, in recycling endosomes or in budded tubulosaccular structures, or it may occur proximally to the Golgi complex, at the exit from the ER. Several adaptors are candidates to sort apical and basolateral proteins but only AP1B and AP4 are currently involved. Progress is fast and future work should elucidate many of the open questions.