Mesenchymal cells regulate enteric neural crest cell migration via RET-GFRA1b trans-signaling.

During early development, the enteric nervous system forms from the migration of enteric neural crest cells (ENCCs) from the foregut to the hindgut, where they undergo proliferation and differentiation facilitated by interactions with enteric mesenchymal cells (EMCs). This study investigates the impact on ENCC migration of EMC-ENCC communication mediated by GFRA1b expressed in EMCs. GFRA1-expressing cells in day 11-12 (E11-12) mouse embryos differentiated into smooth muscle cells from E12 onwards. Observations at E12-13.5 revealed high levels of GFRA1 expression on the anti-mesenteric side of the hindgut, correlating with enhanced ENCC migration. This indicates that GFRA1 in EMCs plays a role in ENCC migration during development. Examining GFRA1 isoforms, we found high levels of GFRA1b, which lacks amino acids 140-144, in EMCs. To assess the impact of GFRA1 isoforms on EMC-ENCC communication, we conducted neurosphere drop assays. This revealed that GFRA1b-expressing cells promoted GDNF-dependent extension and increased neurite density in ENCC neurospheres. Co-culture of ENCC mimetic cells expressing RET and GFRA1a with EMC mimetic cells expressing GFRA1a, GFRA1b, or vector alone showed that only GFRA1b-expressing co-cultured cells sustained RET phosphorylation in ENCC-mimetic cells for over 120 min upon GDNF stimulation. Our study provides evidence that GFRA1b-mediated cell-to-cell communication plays a critical role in ENCC motility in enteric nervous system development. These findings contribute to understanding the cellular interactions and signaling mechanisms that underlie enteric nervous system formation and highlight potential therapeutic targets for gastrointestinal motility disorders.

Recipient colon preoperative treatment with type I collagenase and fibronectin promotes the growth of transplanted enteric neural crest cells into Auerbach's plexus.

PURPOSE: Cell-based therapy is a potential treatment option for neurointestinal diseases by serving as a source of neural progenitor cells to replace missing or abnormal enteric neurons. Using an ex vivo transplantation model, we recently demonstrated that treatment with collagenase and fibronectin promotes infiltration of transplanted enteric neural crest cells (ENCCs) toward the colon lumen. The aim of this study was to determine whether this new method also promotes colonization of transplanted ENCCs in vivo. METHODS: Collagenase was applied locally on the anti-mesenteric area of the recipient colon using filter paper, followed by fibronectin. Neurospheres were generated from ENCCs isolated from fetal mouse intestines and transplanted into the collagenase and fibronectin-treated colon. Engraftment of neurospheres was confirmed by immunofluorescence. RESULTS: Neurospheres transplanted onto PBS- or fibronectin-treated colons were not observed to infiltrate to the muscle layer. However, when used in combination with type I collagenase and fibronectin in the recipient colon, transplanted neurospheres reached Auerbach's plexus. CONCLUSION: We demonstrated that transplanted neurospheres grow into Auerbach's plexus in the recipient colon pretreated with collagenase and fibronectin.

Transplanted neural crest cells migrate toward Auerbach's plexus layer instead of the colon surface in recipient colon pretreated with collagenase and fibronectin.

The enteric nervous system (ENS) regulates gastrointestinal motility, secretion, and absorption. Developmental ENS dysplasia causes intestinal ganglion dysfunction, including Hirschsprung's disease. Given their potential ability to replenish insufficient neurons, transplantation of enteric neural cells provides the prospect of a cure. In this study, we used an ex vivo mouse colon transplant model to demonstrate that treatment with collagenase and fibronectin altered the migration of transplanted cells from the direction of the colon surface toward the lumen. Collagenase-treated colons exhibited enhanced expression of type III and VI collagens, which inhibited fibronectin-induced enteric neural crest cell (ENCC) migration. Invasion of neurospheres into colon was dependent on preoperative treatment of recipient colon with collagenase and fibronectin, which enhanced neurosphere motility towards the direction of colon lumen. Infiltration of transplanted ENCCs into the colon increased proportionally to the degree of dedifferentiation of surrounding smooth muscle cells, which was induced in a neurosphere-dependent manner in collagenase-treated colon. Furthermore, induction of GDNF expression, a Ret ligand that promotes enteric neural cell migration, was observed in treated colons. Our results suggest that the environment provided by the extracellular matrix of the colon surface affects the direction of transplanted ENCC migration. Moreover, these findings demonstrating that ENCCs can be accepted by the recipient colon will help to refine current strategies for cell therapy.

Collagen VI suppresses fibronectin-induced enteric neural crest cell migration by downregulation of focal adhesion proteins.

The enteric nervous system (ENS) is a network of neurons and glia that are derived from enteric neural crest cells (ENCCs) and essential for regulating peristaltic activity of the colon. ENCCs migrate along the gastrointestinal tract to form the ENS, and disruption of ENCC motility leads to ENS disorders, such as Hirschsprung's disease. Previous ENCC-transplant experiments show that ENCCs can invade into isolated mouse intestines by age E13.5, but not after E15.5. We hypothesized that altered age-specific micro-environments in the intestine are responsible for ENCC invasion/migration. Here, we compared gene expression in the intestine between at E11.5 and E15.5 and identified 1355 differentially expressed transcripts. Among these, we found that genes encoding extracellular matrix (ECM) proteins were enriched. Notably, collagen VI (ColVI) family members were upregulated in the E15.5 mouse intestine at the mRNA and protein levels, whereas fibronectin (FN) was downregulated; however, both proteins showed colocalization at E15.5. To understand the mechanisms of ColVI and FN in ENCC migration, we examined neurosphere or individual ENCC-adherence capabilities toward the ECM. ColVI suppressed FN-induced ENCC spreading/migration, whereas ColVI induced morphologically narrow ENCC spreading and weak stress-fiber formation as compared with those with FN. Additionally, in ENCCs cultured on plates containing ColVI, the expression and phosphorylation of p130Cas, a members of focal adhesion complexes, was reduced. These data indicated an inhibitory role of ColVI in ENCC migration and suggested that ColVI suppression in the intestine might represent a novel therapeutic strategy for aganglionic colonic diseases.

Upregulation of multiple signaling pathways by Dock5 deletion in epithelial cells.

Purpose: Rupture of lens cataract (RLC) is a hereditary mouse model that shows spontaneous rupture of the lens at the posterior pole at 45-100 days of age. The responsible gene for this phenotype was identified as Dock5, a guanine nucleotide exchange factor for small GTPase Rac1. This study was performed to elucidate the pathway initiating this phenotype. Methods: We examined the RNA expression by microarray in lens epithelial cells (LECs) from wild-type and RLC mice at the pre-rupture age of 21 days. We applied the list of altered genes to an Ingenuity Pathway Analysis (IPA) to predict the pathways that are altered upon dedicator of cytokinesis-5 (Dock5) protein loss. The activation status of the predicted pathways was examined by western blotting in the cultured epithelial cells treated with a Dock5 inhibitor. Results: The highest-scored network was "Antimicrobial Response, Inflammatory Response, Dermatological Diseases and Conditions." In that network, it is predicted that extracellular signal-regulated kinase (Erk) is activated in LECs from RLC mice. Our test confirmed that Erk was more phosphorylated in the LECs at the equator in both Dock5-knockout mice and RLC mice. In an in vitro experiment of the cultured epithelial cells, the inhibition of Dock5 activity significantly induced Erk activation. It was also confirmed that Akt (cellular homolog of murine thymoma virus akt8 oncogene, also called protein kinase B) and nuclear factor-kappa B (NFκB), predicted to be the key molecules in two other high-scoring networks by IPA, were activated upon Dock5 inhibition in the cultured epithelial cells. Conclusions: Dock5 participates in epithelial cell maintenance by regulating gene expression.

Comparative lipid analysis in the normal and cancerous organoids of MDCK cells.

Epithelial organs are made of a well-polarized monolayer of epithelial cells, and their morphology is maintained strictly for their proper functioning. The roles of lipids are not only to generate the membrane, but also to provide the specific domains for signal transduction, or to transmit signals as second messengers. By using a liquid chromatography-electrospray ionization mass spectrometry (LC-MS)/MS method, we here analyzed sphingolipids in MDCK cysts under various conditions. Our result showed that, compared to the three-dimensional cyst, the two-dimensional MDCK sheet is relatively enriched in sphingolipids. During cystogenesis, the contents of sphingomyelin (SM) and lactocylceramide (LacCer)-but, none those of ceramide, hexocylceramide, or GM3-are altered depending on their acyl chains. While the total SM is decreased more efficiently by SMS-1 knockdown than by SMS-2 knockdown, depletion of SMS-2, but not SMS-1, inhibits cyst growth. Finally upon the switching on of activated K-Ras expression which induces luminal cell filling, ceramide and LacCer are increased. Our parallel examinations of the microarray data for mRNA of sphingolipid metabolic enzymes failed to fully explain the remodelling of the sphingolipids of MDCK cysts. However, these results should be useful to investigate the cell-type- and structure-specific lipid metabolism.

Regulation of Ripply1 expression in MDCK organoids.

Epithelial organs are made of a well-polarized monolayer of epithelial cells, and their morphology must be maintained for their proper function. To examine the genes that are specifically expressed in the late stages of cystogenesis and are involved in maintaining the morphology of the mature cysts, we performed a microarray analysis comparing the mRNA expression between the early and late stages of Madin-Darby Canine Kidney (MDCK) cystogenesis. We found that one of the gene candidates, Ripply1, was expressed higher in the late stages, and its expression was also transiently much higher in the early stages. Although the protein expression showed similar kinetics, depletion of Ripply1 had only a slight effect on organoid growth. Unexpectedly, we found that the Ripply1 protein is degraded by the proteasome system. Mutant analysis suggests that Ripply1 is not ubiquitinated directly, but rather is degraded only after binding to Transducin-like Enhancer of Split (TLE)1, a transcriptional repressor. Ripply1 is degraded in the nucleus, and this degradation is inhibited during the mitosis. These data indicate for the first time that Ripply1 expression is regulated at the protein level.

Large-scale analysis of the evolutionary histories of phosphorylation motifs in the human genome.

BACKGROUND: Protein phosphorylation is a post-translational modification that is essential for a wide range of eukaryotic physiological processes, such as transcription, cytoskeletal regulation, cell metabolism, and signal transduction. Although more than 200,000 phosphorylation sites have been reported in the human genome, the physiological roles of most remain unknown. In this study, we provide some useful datasets for the assessment of functional phosphorylation signaling using a comparative genome analysis of phosphorylation motifs. FINDINGS: We described the evolutionary patterns of conservation of these and comparative genomic data for 93,101 phosphosites and 1,003,756 potential phosphosites in human phosphomotifs, using 178 phosphomotifs identified in a previous study that occupied 69% of known phosphosites in public databases. Comparative genomic analyses were performed using genomes from nine species from yeast to humans. Here we provide an overview of the evolutionary patterns of phosphomotif acquisition and indicate the dependence on motif structures. Using the data from our previous study, we describe the interaction networks of phosphoproteins, identify the kinase substrates associated with phosphoproteins, and perform gene ontology enrichment analyses. In addition, we show how this dataset can help to elucidate the function of phosphomotifs. CONCLUSIONS: Our characterizations of motif structures and assessments of evolutionary conservation of phosphosites reveal physiological roles of unreported phosphosites. Thus, interactions between protein groups that share motifs are likely to be helpful for inferring kinase-substrate interaction networks. Our computational methods can be used to elucidate the relationships between phosphorylation signaling and cellular functions.

Elucidation of the evolutionary expansion of phosphorylation signaling networks using comparative phosphomotif analysis.

BACKGROUND: Protein phosphorylation is catalyzed by kinases and is involved in the regulation of a wide range of processes. The phosphosites in protein sequence motifs determine the types of kinases involved. The development of phosphoproteomics has allowed the identification of huge numbers of phosphosites, some of which are not involved in physiological functions. RESULTS: We developed a method for extracting phosphosites with important roles in cellular functions and determined 178 phosphomotifs based on the analysis of 34,366 phosphosites. We compared the conservation of serine/threonine/tyrosine residues observed in humans and seven other species. Consequently, we identified 16 phosphomotifs, where the level of conservation increased among species. The highly conserved phosphomotifs in humans and the worm were kinase regulatory sites. The motifs present in the fly were novel phosphomotifs, including zinc finger motifs involved in the regulation of gene expression. Subsequently, we found that this zinc finger motif contributed to subcellular protein localization. The motifs identified in fish allowed us to detect the expansion of phosphorylation signals related to alternative splicing. We also showed that the motifs present in specific species functioned in an additional network that interacted directly with the core signaling network conserved from yeast to humans. CONCLUSIONS: Our method may facilitate the efficient extraction of novel phosphomotifs with physiological functions, thereby contributing greatly to the analysis of complex phosphorylation signaling cascades. Our study suggests that the phosphorylation networks acquired during evolution have added signaling network modules to the core signaling networks.

Rapid turnover rate of phosphoinositides at the front of migrating MDCK cells.

Phosphoinositides (PtdInss) play key roles in cell polarization and motility. With a series of biosensors based on Förster resonance energy transfer, we examined the distribution and metabolism of PtdInss and diacylglycerol (DAG) in stochastically migrating Madin-Darby canine kidney (MDCK) cells. The concentrations of phosphatidylinositol (4,5)-bisphosphate, phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), phosphatidylinositol (3,4)-bisphosphate, and DAG were higher at the plasma membrane in the front of the cell than at the plasma membrane of the rear of the cell. The difference in the concentrations of PtdInss was estimated to be less than twofold between the front and rear of the migrating MDCK cells. To decode the spatial activities of PtdIns metabolic enzymes from the obtained concentration maps of PtdInss, we developed a one-dimensional reaction diffusion model of PtdIns metabolism. In this model, the activities of phosphatidylinositol monophosphate 5-kinase, phosphatidylinositol 3-kinase, phospholipase C, and PIP(3) 5-phosphatases were higher at the plasma membrane of the front than at the plasma membrane of the rear of the cell. This result suggests that, although the difference in the steady-state level of PtdInss is less than twofold, PtdInss were more rapidly turned over at the front than the rear of the migrating MDCK cells.

Akt-PDK1 complex mediates epidermal growth factor-induced membrane protrusion through Ral activation.

We studied the spatiotemporal regulation of Akt (also called protein kinase B), phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] by using probes based on the principle of fluorescence resonance energy transfer. On epidermal growth factor (EGF) stimulation, the amount of PtdIns(3,4,5)P3 was increased diffusely in the plasma membrane, whereas that of PtdIns(3,4)P2 was increased more in the nascent lamellipodia than in the plasma membrane of the central region. The distribution and time course of Akt activation were similar to that of increased PtdIns(3,4)P2 levels, which were most prominent in the nascent lamellipodia. Moreover, we found that upon EGF stimulation 3-phosphoinositide-dependent protein kinase-1 (PDK1) was also recruited to nascent lamellipodia in an Akt-dependent manner. Because PDK1 is known to activate Ral GTPase and because Ral is required for EGF-induced lamellipodial protrusion, we speculated that the PDK1-Akt complex may be indispensable for the induction of lamellipodia. In agreement with this idea, EGF-induced lamellipodia formation was promoted by the overexpression of Akt and inhibited by an Akt inhibitor or a Ral-binding domain of Sec5. These results identified the Akt-PDK1 complex as an upstream positive regulator of Ral GTPase in the induction of lamellipodial protrusion.

STAT3 noncell-autonomously controls planar cell polarity during zebrafish convergence and extension.

Zebrafish signal transducer and activator of transcription 3 (STAT3) controls the cell movements during gastrulation. Here, we show that noncell-autonomous activity of STAT3 signaling in gastrula organizer cells controls the polarity of neighboring cells through Dishevelled-RhoA signaling in the Wnt-planar cell polarity (Wnt-PCP) pathway. In STAT3-depleted embryos, although all the known molecules in the Wnt-PCP pathway were expressed normally, the RhoA activity in lateral mesendodermal cells was down-regulated, resulting in severe cell polarization defects in convergence and extension movements identical to Strabismus-depleted embryos. Cell-autonomous activation of Wnt-PCP signaling by DeltaN-dishevelled rescued the defect in cell elongation, but not the orientation of lateral mesendodermal cells in STAT3-depleted embryos. The defect in the orientation could be rescued by transplantation of shield cells having noncell-autonomous activity of STAT3 signaling. These results suggest that the cells undergoing convergence and extension movement may sense the gradient of signaling molecules, which are expressed in gastrula organizer by STAT3 and noncell-autonomously activate PCP signaling in neighboring cells during zebrafish gastrulation.

Cell type-specific regulation of RhoA activity during cytokinesis.

Rho family GTPases play pivotal roles in cytokinesis. By using probes based on the principle of fluorescence resonance energy transfer (FRET), we have shown that in HeLa cells RhoA activity increases with the progression of cytokinesis. Here we show that in Rat1A cells RhoA activity remained suppressed during most of the cytokinesis. Consistent with this observation, the expression of C3 toxin inhibited cytokinesis in HeLa cells but not in Rat1A cells. Furthermore, the expression of a dominant negative mutant of Ect2, a Rho GEF, or Y-27632, an inhibitor of the Rho-dependent kinase ROCK, inhibited cytokinesis in HeLa cells but not in Rat1A cells. In contrast to the activity of RhoA, the activity of Rac1 was suppressed during cytokinesis and started increasing at the plasma membrane of polar sides before the abscission of the daughter cells in both HeLa and Rat1A cells. This type of Rac1 suppression was shown to be essential for cytokinesis because a constitutively active mutant of Rac1 induced a multinucleated phenotype in both HeLa and Rat1A cells. Moreover, the involvement of MgcRacGAP/CYK-4 in this suppression of Rac1 during cytokinesis was shown by the use of a dominant negative mutant. Because ML-7, an inhibitor of myosin light chain kinase, delayed the cytokinesis of Rat1A cells and because Pak, a Rac1 effector, is known to suppress myosin light chain kinase, the suppression of the Rac1-Pak pathway by MgcRacGAP may play a pivotal role in the cytokinesis of Rat1A cells.

Coactivation of Rac1 and Cdc42 at lamellipodia and membrane ruffles induced by epidermal growth factor.

A major function of Rho-family GTPases is to regulate the organization of the actin cytoskeleton; filopodia, lamellipodia, and stress fiber are regarded as typical phenotypes of the activated Cdc42, Rac, and Rho, respectively. Using probes based on fluorescent resonance energy transfer, we report on the spatiotemporal regulation of Rac1 and Cdc42 at lamellipodia and membrane ruffles. In epidermal growth factor (EGF)-stimulated Cos1 and A431 cells, both Rac1 and Cdc42 were activated diffusely at the plasma membrane, followed by lamellipodial protrusion and membrane ruffling. Although Rac1 activity subsided rapidly, Cdc42 activity was sustained at lamellipodia. A critical role of Cdc42 in these EGF-induced morphological changes was demonstrated as follows. First, phorbol 12-myristate 13-acetate, which activated Rac1 but not Cdc42, could not induce full-grown lamellipodia in Cos1 cells. Second, a GTPase-activating protein for Cdc42, KIAA1204/CdGAP, inhibited lamellipodial protrusion and membrane ruffling without interfering with Rac1 activation. Third, expression of the Cdc42-binding domain of N-WASP inhibited the EGF-induced morphological changes. Therefore, Rac1 and Cdc42 seem to synergistically induce lamellipodia and membrane ruffles in EGF-stimulated Cos1 cells and A431 cells.

Activity of Rho-family GTPases during cell division as visualized with FRET-based probes.

Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)-based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582-6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.

Activation of rac and cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells.

Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42- and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPs, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.