Several tissues contain cells with multiple motile cilia that generate a fluid or particle flow to support development and organ functions; defective motility causes human disease. Developmental cues ...orient motile cilia, but how cilia are locked into their final position to maintain a directional flow is not understood. Here we find that the actin cytoskeleton is highly dynamic during early development of multiciliated cells (MCCs). While apical actin bundles become increasingly more static, subapical actin filaments are nucleated from the distal tip of ciliary rootlets. Anchorage of these subapical actin filaments requires the presence of microridge-like structures formed during MCC development, and the activity of Nonmuscle Myosin II. Optogenetic manipulation of Ezrin, a core component of the microridge actin-anchoring complex, or inhibition of Myosin Light Chain Kinase interfere with rootlet anchorage and orientation. These observations identify microridge-like structures as an essential component of basal body rootlet anchoring in MCCs.
Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central ...role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.
Src homology 2-containing phosphotyrosine phosphatase (Shp2) functions as a positive effector in receptor tyrosine kinase (RTK) signaling immediately proximal to activated receptors. However, neither ...its physiological substrate(s) nor its mechanism of action in RTK signaling has been defined. In this study, we demonstrate that Sprouty (Spry) is a possible target of Shp2. Spry acts as a conserved inhibitor of RTK signaling, and tyrosine phosphorylation of Spry is indispensable for its inhibitory activity. Shp2 was able to dephosphorylate fibroblast growth factor receptor-induced phosphotyrosines on Spry both in vivo and in vitro. Shp2-mediated dephosphorylation of Spry resulted in dissociation of Spry from Grb2. Furthermore, Shp2 could reverse the inhibitory effect of Spry on FGF-induced neurite outgrowth and MAP kinase activation. These findings suggest that Shp2 acts as a positive regulator in RTK signaling by dephosphorylating and inactivating Spry.
Sprouty (Spry) inhibits signalling by receptor tyrosine kinases; however, the molecular mechanism underlying this function has not been defined. Here we show that after stimulation by growth factors ...Spry1 and Spry2 translocate to the plasma membrane and become phosphorylated on a conserved tyrosine. Next, they bind to the adaptor protein Grb2 and inhibit the recruitment of the Grb2-Sos complex either to the fibroblast growth factor receptor (FGFR) docking adaptor protein FRS2 or to Shp2. Membrane translocation of Spry is necessary for its phosphorylation, which is essential for its inhibitor activity. A tyrosine-phosphorylated octapeptide derived from mouse Spry2 inhibits Grb2 from binding FRS2, Shp2 or mouse Spry2 in vitro and blocks activation of the extracellular-signal-regulated kinase (ERK) in cells stimulated by growth factor. A non-phosphorylated Spry mutant cannot bind Grb2 and acts as a dominant negative, inducing prolonged activation of ERK in response to FGF and promoting the FGF-induced outgrowth of neurites in PC12 cells. Our findings suggest that Spry functions in a negative feedback mechanism in which its inhibitor activity is controlled rapidly and reversibly by post-translational mechanisms.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Nephronophthisis (NPH) is a heterogenetic autosomal recessive disorder associated with kidney cysts and multiple extrarenal manifestations. The disease-associated gene products (NPHPs) typically ...contain domains involved in protein–protein interactions, and appear to exert their tissue-specific functions in large protein complexes. Most NPHPs localize to the cilium and/or basal body; however, their precise molecular functions remain largely unknown. We have recently identified the SAM-domain containing protein Anks3 as a potential ANKS6/NPHP16-interacting protein, and report now that Anks3 interacts with several NPHPs as well as with Bicc1 and the oxygen-sensitive asparaginyl hydroxylase HIF1AN. Knockdown of anks3 in zebrafish embryos was associated with NPH-typical manifestations, including ciliary abnormalities, cyst formation, and laterality defects. In multi-ciliated epidermal cells, GFP-tagged Anks3 localizes to the cilium, but forms large aggregates in the absence of NPHP1, indicating that the negatively charged NPHP1 curtails the polymerization of Anks3. Collectively, these findings suggest that Anks3 is a cilia-associated molecule that partners with the ANKS6- and via NPHP1 to the NPHP1-4-8 module. Thus, developmental defects associated with Anks3 depletion in zebrafish suggest that ANKS3 mutations may cause NPH or NPH-like disease in humans.
► Diversin is localized to the basal body of multi-ciliated skin cells. ► Overexpression of Diversin causes the disruption of basal body polarization. ► Diversin depletion leads to absent or ...nonfunctional cilia in multi-ciliated cells. ► Diversin depletion interferes with ciliogenesis in the gastrocoel roof plate.
The centrosome is essential for the formation of the cilia and has been implicated in cell polarization and signaling during early embryonic development. A number of Wnt pathway components were found to localize at the centrosome, but how this localization relates to their signaling functions is unclear. In this study, we assessed a role for Diversin, a putative Wnt pathway mediator, in developmental processes that involve cilia. We find that Diversin is specifically localized to the basal body compartment near the base of the cilium in
Xenopus multi-ciliated skin cells. Overexpression of Diversin RNA disrupted basal body polarization in these cells, suggesting that tightly regulated control of Diversin levels is crucial for this process. In cells depleted of endogenous Diversin, basal body structure appeared abnormal and this was accompanied by disrupted polarity, shortened or absent cilia and defective ciliary flow. These results are consistent with the involvement of Diversin in processes that are related to the acquisition of cell polarity and require ciliary functions.
Cilia are microtubule-based organelles that are present on most cells and are required for normal tissue development and function. Defective cilia cause complex syndromes with multiple organ ...manifestations termed ciliopathies. A crucial step during ciliogenesis in multiciliated cells (MCCs) is the association of future basal bodies with the apical plasma membrane, followed by their correct spacing and planar orientation. Here, we report a novel role for ELMO-DOCK1, which is a bipartite guanine nucleotide exchange factor complex for the small GTPase Rac1, and for the membrane-cytoskeletal linker Ezrin, in regulating centriole/basal body migration, docking and spacing. Downregulation of each component results in ciliopathy-related phenotypes in zebrafish and disrupted ciliogenesis in Xenopus epidermal MCCs. Subcellular analysis revealed a striking impairment of basal body docking and spacing, which is likely to account for the observed phenotypes. These results are substantiated by showing a genetic interaction between elmo1 and ezrin b. Finally, we provide biochemical evidence that the ELMO-DOCK1-Rac1 complex influences Ezrin phosphorylation and thereby probably serves as an important molecular switch. Collectively, we demonstrate that the ELMO-Ezrin complex orchestrates ciliary basal body migration, docking and positioning in vivo.
It has been suggested that ILK (integrin‐linked kinase) participates in integrin‐ and growth factor‐mediated signaling pathways and also functions as a scaffold protein at cell–extracellular matrix ...(ECM) adhesion sites. As the recently reported ILK knockout mice were found to die at the peri‐implantation stage, the stage specific to mammals, little is known about the function of ILK in early developmental processes common to every vertebrate. To address this, we isolated a Xenopus ortholog of ILK (XeILK) and characterized its role in early Xenopus embryogenesis. XeILK was expressed constitutively and ubiquitously throughout the early embryogenesis. Depletion of XeILK with morpholino oligonucleotides (XeILK MO) caused severe defects in blastopore closure and axis elongation without affecting the mesodermal specification. Furthermore, XeILK MO was found to interfere with cell–cell and cell–ECM adhesions in dorsal marginal zone explants and to result in a significant loss of cell–ECM adhesions in activin‐treated dissociated animal cap cells. These results thus indicate that XeILK plays an essential role in morphogenetic movements during gastrulation.
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