Anne Müsch

Organization of vesicular trafficking in epithelia

Experiments using mammalian epithelial cell lines have elucidated biosynthetic and recycling pathways for apical and basolateral plasma-membrane proteins, and have identified components that guide apical and basolateral proteins along these pathways. These components include apical and basolateral sorting signals, adaptors for basolateral signals, and docking and fusion proteins for vesicular trafficking. Recent live-cell-imaging studies provide a real-time view of sorting processes in epithelial cells, including key roles for actin, microtubules and motors in the organization of post-Golgi trafficking.

Microtubule organization and function in epithelial cells.

Microtubules are essential for many aspects of polarity in multicellular organisms, ranging from the asymmetric distribution of cell‐fate determinants in the one‐cell embryo to the transient polarity generated in migrating fibroblasts. Epithelial cells exhibit permanent cell polarity characterized by apical and basolateral surface domains of distinct protein and lipid composition that are segregated by tight junctions. They are also endowed with a microtubule network that reflects the asymmetry of their cell surface: microtubule minus‐ends face the apical‐ and microtubule plus‐ends the basal domain. Strikingly, the formation of distinct surface domains during epithelial differentiation is accompanied by the re‐organization of microtubules from a uniform array focused at the centrosome to the noncentrosomal network that aligns along the apico‐basolateral polarity axis. The significance of this coincidence for epithelial morphogenesis and the signaling mechanisms that drive microtubule repolymerization in developing epithelia remain major unresolved questions that we are only beginning to address. Studies in cultured polarized epithelial cells have established that microtubules serve as tracks that facilitate targeted vesicular transport. Novel findings suggest, moreover, that microtubule‐based transport promotes protein sorting, and even the generation of transport carriers in the endo‐ and exocytic pathways.

cdc42 regulates the exit of apical and basolateral proteins from the trans-Golgi network

It is well established that Rho‐GTPases regulate vesicle fusion and fission events at the plasma membrane through their modulatory role on the cortical actin cytoskeleton. In contrast, their effects on intracellular transport processes and actin pools are less clear. It was recently shown that cdc42 associates with the Golgi apparatus in an ARF‐dependent manner, similarly to coat proteins involved in vesicle formation and to several actin‐binding proteins. We report here that mutants of cdc42 inhibited the exit of basolateral proteins from the trans‐Golgi network (TGN), while stimulating the exit of an apical marker, in two different transport assays. This regulation may result from modulation of the actin cytoskeleton, as GTPase‐deficient cdc42 depleted a perinuclear actin pool that rapidly exchanges with exogenous fluorescent actin.

Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton

Epithelial differentiation involves the generation of luminal surfaces and of a noncentrosomal microtubule (MT) network aligned along the polarity axis. Columnar epithelia (e.g., kidney, intestine, and Madin-Darby canine kidney [MDCK] cells) generate apical lumina and orient MT vertically, whereas liver epithelial cells (hepatocytes and WIFB9 cells) generate lumina at cell–cell contact sites (bile canaliculi) and orient MTs horizontally. We report that knockdown or inhibition of the mammalian orthologue of Caenorhabditis elegans Par-1 (EMK1 and MARK2) during polarization of cultured MDCK and WIFB9 cells prevented development of their characteristic lumen and nonradial MT networks. Conversely, EMK1 overexpression induced the appearance of intercellular lumina and horizontal MT arrays in MDCK cells, making EMK1 the first known candidate to regulate the developmental branching decision between hepatic and columnar epithelial cells. Our experiments suggest that EMK1 primarily promotes reorganization of the MT network, consistent with the MT-regulating role of this gene product in other systems, which in turn controls lumen formation and position.

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.

Selective control of basolateral membrane protein polarity by cdc42.

The rho GTPase cdc42 is implicated in several aspects of cell polarity. A recent study (Kroschewski R, Hall A, Mellman I. Nat Cell Biol 1999;1:8–13) demonstrated that a dominant negative mutant of cdc42 abolishes the polarity of basolateral membrane proteins in MDCK cells, but did not elucidate whether this effect was selective for basolateral proteins or nonselective for all secreted proteins. To answer this question, we analyzed the polarity of newly synthesized membrane and soluble proteins in MDCK cell lines previously induced to overexpress mutant forms of cdc42. GTPase‐deficient and dominant negative cdc42 did not affect the apical targeting of a newly synthesized apical membrane protein, but reversed to apical the distribution of two exogenous basolateral membrane proteins. In striking contrast, GTPase‐deficient cdc42 did not affect polarized exocytosis of endogenous soluble proteins, either apical or basolateral. The exquisitely selective regulation of polarized protein targeting by cdc42 may allow cells to fine‐tune their membrane composition in response to extracellular signals during development, migration and in response to injury.

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

Par1b regulates cell–ECM signaling and dictates epithelial lumenal organization by targeting IRSp53, a Rho GTPase adaptor and scaffolding protein.

Apical surface formation in MDCK cells: regulation by the serine/threonine kinase EMK1.

It has recently become evident that basic mechanisms for the establishment of cell polarity are conserved between epithelial and nonepithelial systems. The vast catalogue of known gene products involved in various aspects of invertebrate and yeast cell polarity provides a repertoire of candidate proteins that can be tested for their roles in the organization of mammalian epithelia. Here, we describe cell biological approaches to study the development and maintenance of cell polarity in Mardin-Darby canine kidney (MDCK) cells, an established mammalian model cell line for simple epithelia. The assays allowed us to characterize the Caenorhabditis elegans PAR-1 homologue EMK1 as a novel regulator of apical surface formation in epithelial cells.

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

Positioning of the mitotic spindle relative to the luminal domain in epithelial cells is determined by Par1b in a RhoA-mediated signaling pathway that involves the astral microtubule anchoring complex LGN–NuMA.

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

Proliferating hepatocytes in the liver show an atypical, asymmetric mode of cell division, which is coordinated by Par1b and LGN and may explain the unique tissue architecture of the liver.