The cell adhesion molecule Fasciclin2 regulates brush border length and organization in Drosophila renal tubules

Multicellular organisms rely on cell adhesion molecules to coordinate cell–cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most abundantly expressed in the adult renal (Malpighian) tubule rather than in neuronal tissues. The role Fas2 serves in this epithelium is unknown. Here we show that Fas2 is essential to brush border maintenance in renal tubules of Drosophila. Fas2 is dynamically expressed during tubule morphogenesis, localizing to the brush border whenever the tissue is transport competent. Genetic manipulations of Fas2 expression levels impact on both microvilli length and organization, which in turn dramatically affect stimulated rates of fluid secretion by the tissue. Consequently, we demonstrate a radically different role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border

Primary cilia and signaling pathways in mammalian development, health and disease

Although first described 1898 and long considered a vestigial organelle of little functional importance, the primary cilium has become one of the hottest research topics in modern cell biology and physiology. Primary cilia are non-motile sensory organelles present in a single copy on the surface of most growth-arrested or differentiated mammalian cells, and defects in their assembly or function are tightly coupled to many developmental defects, diseases and disorders. In normal tissues the primary cilium coordinates a series of signal transduction pathways, including Hedgehog, Wnt, PDGFRα and integrin signaling. In the kidney the primary cilium may function as a mechano-, chemo- and osmosensing unit that probes the extracellular environment and transmits signals to the cell via e.g. polycystins, which depend on ciliary localization for appropriate function. Indeed, hypomorphic mutations in the mouse ift88 (previously called Tg737) gene, which encodes a ciliogenic intraflagellar transport (IFT) protein, result in malformation of primary cilia, and in the collecting ducts of kidney tubules this is accompanied by development of autosomal recessive polycystic kidney disease (PKD; (1)). While PKD was one of the first diseases to be linked to dysfunctional primary cilia, defects in this organelle have subsequently been associated with many other phenotypes, including cancer, obesity, diabetes as well as a number of developmental defects. Collectively, these disorders of the cilium are now referred to as the ciliopathies. In this review we provide a brief overview of the structure and function of primary cilia and some of their roles in coordinating signal transduction pathways in mammalian development, health and disease. This review was written in conjunction with the Takis Anagnostopoulos Symposium on Renal and Epithelial Physiology and Physiopathology at Faculté de Médecine Necker in Paris, June 26-27, 2008

Directional Cell Migration and Chemotaxis in Wound Healing Response to PDGF-AA are Coordinated by the Primary Cilium in Fibroblasts

Cell motility and migration play pivotal roles in numerous physiological and pathophysiological processes including development and tissue repair. Cell migration is regulated through external stimuli such as platelet-derived growth factor-AA (PDGF-AA), a key regulator in directional cell migration during embryonic development and a chemoattractant during postnatal migratory responses including wound healing. We previously showed that PDGFRα signaling is coordinated by the primary cilium in quiescent cells. However, little is known about the function of the primary cilium in cell migration. Here we used micropipette analysis to show that a normal chemosensory response to PDGF-AA in fibroblasts requires the primary cilium. In vitro and in vivo wound healing assays revealed that in ORPK mouse (IFT88Tg737Rpw) fibroblasts, where ciliary assembly is defective, chemotaxis towards PDGF-AA is absent, leading to unregulated high speed and uncontrolled directional cell displacement during wound closure, with subsequent defects in wound healing. These data suggest that in coordination with cytoskeletal reorganization, the fibroblast primary cilium functions via ciliary PDGFRα signaling to monitor directional movement during wound healing

KIF13B establishes a CAV1-enriched microdomain at the ciliary transition zone to promote Sonic hedgehog signalling

Ciliary membrane composition is controlled by transition zone (TZ) proteins such as RPGRIP1, RPGRIPL and NPHP4, which are vital for balanced coordination of diverse signalling systems like the Sonic hedgehog (Shh) pathway. Activation of this pathway involves Shh-induced ciliary accumulation of Smoothened (SMO), which is disrupted by disease-causing mutations in TZ components. Here we identify kinesin-3 motor protein KIF13B as a novel member of the RPGRIP1N-C2 domain-containing protein family and show that KIF13B regulates TZ membrane composition and ciliary SMO accumulation. KIF13B is upregulated during ciliogenesis and is recruited to the ciliary base by NPHP4, which binds to two distinct sites in the KIF13B tail region, including an RPGRIP1N-C2 domain. KIF13B and NPHP4 are both essential for establishment of a CAV1 membrane microdomain at the TZ, which in turn is required for Shh-induced ciliary SMO accumulation. Thus KIF13B is a novel regulator of ciliary TZ configuration, membrane composition and Shh signalling

KIF13B establishes a CAV1-enriched microdomain at the ciliary transition zone to promote Sonic hedgehog signalling

Ciliary membrane composition is controlled by transition zone (TZ) proteins such as RPGRIP1, RPGRIPL and NPHP4, which are vital for balanced coordination of diverse signalling systems like the Sonic hedgehog (Shh) pathway. Activation of this pathway involves Shh-induced ciliary accumulation of Smoothened (SMO), which is disrupted by disease-causing mutations in TZ components. Here we identify kinesin-3 motor protein KIF13B as a novel member of the RPGRIP1N-C2 domain-containing protein family and show that KIF13B regulates TZ membrane composition and ciliary SMO accumulation. KIF13B is upregulated during ciliogenesis and is recruited to the ciliary base by NPHP4, which binds to two distinct sites in the KIF13B tail region, including an RPGRIP1N-C2 domain. KIF13B and NPHP4 are both essential for establishment of a CAV1 membrane microdomain at the TZ, which in turn is required for Shh-induced ciliary SMO accumulation. Thus KIF13B is a novel regulator of ciliary TZ configuration, membrane composition and Shh signalling

Inversin/Nephrocystin-2 Is Required for Fibroblast Polarity and Directional Cell Migration

<div><p>Inversin is a ciliary protein that critically regulates developmental processes and tissue homeostasis in vertebrates, partly through the degradation of Dishevelled (Dvl) proteins to coordinate Wnt signaling in planar cell polarity (PCP). Here, we investigated the role of Inversin in coordinating cell migration, which highly depends on polarity processes at the single-cell level, including the spatial and temporal organization of the cytoskeleton as well as expression and cellular localization of proteins in leading edge formation of migrating cells. Using cultures of mouse embryonic fibroblasts (MEFs) derived from <i>inv^−/−</i> and <i>inv^+/+</i> animals, we confirmed that both <i>inv^−/−</i> and <i>inv^+/+</i> MEFs form primary cilia, and that Inversin localizes to the primary cilium in <i>inv^+/+</i> MEFs. In wound healing assays, <i>inv^−/−</i> MEFs were severely compromised in their migratory ability and exhibited cytoskeletal rearrangements, including distorted lamellipodia formation and cilia orientation. Transcriptome analysis revealed dysregulation of Wnt signaling and of pathways regulating actin organization and focal adhesions in <i>inv^−/−</i> MEFs as compared to <i>inv^+/+</i> MEFs. Further, Dvl-1 and Dvl-3 localized to MEF primary cilia, and β-catenin/Wnt signaling was elevated in <i>inv^−/−</i> MEFs, which moreover showed reduced ciliary localization of Dvl-3. Finally, <i>inv^−/−</i> MEFs displayed dramatically altered activity and localization of RhoA, Rac1, and Cdc42 GTPases, and aberrant expression and targeting of the Na⁺/H⁺ exchanger NHE1 and ezrin/radixin/moesin (ERM) proteins to the edge of cells facing the wound. Phosphorylation of β-catenin at the ciliary base and formation of well-defined lamellipodia with localization and activation of ERM to the leading edge of migrating cells were restored in <i>inv^−/−</i> MEFs expressing Inv-GFP. Collectively, our findings point to the significance of Inversin in controlling cell migration processes, at least in part through transcriptional regulation of genes involved in Wnt signaling and pathways that control cytoskeletal organization and ion transport.</p> </div

Inversin affects expression, regulation and localization of ERM proteins.

<p>(<b>A</b>) WB analysis of growth arrested <i>inv^+/+</i> and <i>inv^−/−</i> MEFs with antibodies against total ezrin/radixin/moesin (ERM), phosphorylated ERM (T⁵⁶⁷ of ezrin, T⁵⁶⁴ of Radixin, T⁵⁵⁸ of moesin, p-ERM), ezrin, radixin and moesin, and α-tubulin as control, with indications of the 80 (1) and 75 (2) kDa bands. (<b>B</b>) Quantification of WB from (A); Histograms represent mean ± S.E.M. (n≥3). (<b>C,E</b>) IFM analysis of growth arrested <i>inv^+/+</i> and <i>inv^−/−</i> MEFs in wound healing assays, with phalloidin staining of the actin cytoskeleton (F-actin, <i>red</i>) and nuclei with DAPI (<i>blue</i>). Open arrows indicate direction of migration, and arrowhead indicate leading edge staining of ezrin (C, <i>green</i>), Radixin (D; upper panel, <i>green</i>), moesin (D; lower panel, <i>green</i>), and p-ERM (E, <i>green</i>). (<b>F,G</b>) DIC and IFM analysis on lamellipodium formation and localization of ERM (F, <i>red</i>) and p-ERM (G, <i>red</i>) to the leading edge of migrating cells in wound healing assays in Inv-GFP (<i>green</i>) transfected <i>inv^−/−</i> MEFs. The actin cytoskeleton is stained with phalloidin (F-actin, <i>blue</i>). Open arrows indicate direction of migration.</p