No abstract

SUMMARY Canonical Wnt signaling requires inhibition of Glycogen Synthase Kinase 3 (GSK3) activity, but the molecular mechanism by which this is achieved remains unclear. Here we report that Wnt signaling triggers the sequestration of GSK3 from the cytosol into multivesicular bodies (MVBs), so that this enzyme becomes separated from its many cytosolic substrates. Endocytosed Wnt co-localized with GSK3 in acidic vesicles positive for endosomal markers. After Wnt addition, endogenous GSK3 activity decreased in the cytosol, and GSK3 became protected from protease treatment inside membrane-bounded organelles. Cryoimmuno electron microscopy showed that these corresponded to multivesicular bodies. Two proteins essential for MVB formation, HRS/Vps27 and Vps4, were required for Wnt signaling. The sequestration of GSK3 extended the half-life of many other proteins in addition to β-Catenin, including an artificial Wnt-regulated reporter protein containing GSK3 phosphorylation sites. We conclude that multivesicular endosomes are essential components of the Wnt signal transduction pathway.

Oculodentodigital dysplasia (ODDD) is a dominant negatively inherited disorder with variable but characteristic anomalies of the fingers and toes, eyes, face and teeth, which are caused by mutations in the connexin 43 (Cx43) gene. All mutations analyzed so far have a negative influence on the conductance through gap junctional channels and hemichannels, as well as trafficking of Cx43 protein in transfected cells. In this study, we inserted the human Cx43G138R point mutation into the mouse Cx43 gene and generated mice conditionally expressing this mutation. All ODDD phenotypic manifestations observed in humans, including syndactyly and enamel hypoplasia as well as craniofacial, bone and heart anomalies, were also observed with significant penetrance in Cx43G138R mice. When this mutation was specifically expressed in cardiomyocytes, characteristic alterations in the electrocardiogram and spontaneous arrhythmias were recorded. In vitro studies with Cx43G138R-expressing cells revealed loss of the Cx43 P2 phosphorylation state, which was also absent in the mutated hearts. This loss has previously been associated with gap junctional dysfunction and increased cellular ATP release. The Cx43G138R mutated mice show significantly increased arrhythmogeneity ex vivo in Langendorff experiments with explanted hearts and in vivo in particular under hypoxic conditions. Our results suggest that the increased activity of ATP-releasing channels in Cx43G138R mutated cardiomyocytes may further reduce the already decreased gap junctional communication and thus aggravate arrhythmogenesis in the mouse mutant.

Signal transduction and endocytosis are intertwined processes. The internalization of ligandactivated receptors by endocytosis has classically been thought to attenuate signals by targeting receptors for degradation in lysosomes, but it can also maintain signals in early signalling endosomes. In both cases, localization to multivesicular endosomes en route to lysosomes is thought to terminate signalling. However, during WNT signal transduction, sequestration of the enzyme glycogen synthase kinase 3 (GSK3) inside multivesicular endosomes results in the stabilization of many cytosolic proteins. Thus, the role of endocytosis during signal transduction may be more diverse than anticipated, and multivesicular endosomes may constitute a crucial signalling organelle.The role of endocytosis in signal transduction has been known and debated for many years. After endocytosis, signalling receptors and their factors are targeted to endosomes and multivesicular bodies (MVBs) and eventually fuse to lysosomes to be degraded. Thus, the established view has been that the internalization of most growth factor receptors bound to their ligands constitutes a way to downregulate activated receptors and attenuate the signal 1,2 .In this model, internalized vesicles mature into MVBs, which then fuse with lysosomes to allow degradation of their content. This was first shown in early work by Cohen 3 , who observed that epidermal growth factor (EGF) coupled to ferritin was rapidly internalized upon binding to EGF receptor (EGFR) and found inside MVBs after only 15 minutes exposure of cells to ligand. MVBs form as endosomes mature, through invagination of small intraluminal vesicles (ILVs) of about 50 nm in diameter (BOX 1), which then pinch off. This requires the help of the ESCRT (endosomal sorting complex required for transport) machinery 2,4 , the components of which were first identified in budding yeast as Vps (vacuolar protein sorting) mutants 1 and regulate membrane scission during ILV formation. Biogenesis and functions of multivesicular endosomesMultivesicular endosomes are characterized by the internalization of small intraluminal vesicles (ILVs) of about 50 nm in diameter. This requires the orderly recruitment of components of the ESCRT (endosomal sorting complex required for transport) machinery 4,70 . In addition, ILV formation requires the endosome-specific lipid phosphatidylinositol-3-phosphate (PtdIns3P), and the AAA-ATPase vacuolar protein sorting-associated 4 (VPS4) to pinch-off the vesicles 71,72 . The matrix of endosomes is gradually acidified by vacuolar ATPases (v-ATPases) as they undergo maturation, enlarge and convert the early RAB5-positive compartments into RAB7-positive late endosomes 73,74 . The lumen of early recycling endosomes has a pH of 6.5-6.4 (compared with pH 7.2 in the cytosol), that of late multivesicular endosomes has a pH of 6.0-5.0 and, after fusing with lysosomes, a pH of 5.0-4.5 is reached 75,76 . Lysosomal hydrolases degrade proteins and lipids at acid pH.The diverse functions of multiv...