Glia detect and mount a protective response to loss of dendrite substructure integrity in C. elegans.

Neurons have elaborate structures that determine their connectivity and functions. Changes in neuronal structure accompany learning and memory formation and are hallmarks of neurological disease. Here we show that glia monitor dendrite structure and respond to dendrite perturbation. In C. elegans mutants with defective sensory-organ dendrite cilia, adjacent glia accumulate extracellular matrix-laden vesicles, secrete excess matrix around cilia, alter gene expression, and change their secreted protein repertoire. Inducible cilia disruption reveals that this response is acute. DGS-1, a 7-transmembrane domain neuronal protein, and FIG-1, a multifunctional thrombospondin-domain glial protein, are required for glial detection of cilia integrity, and exhibit mutually-dependent localization to and around cilia, respectively. While inhibiting glial secretion disrupts dendritic cilia properties, hyperactivating the glial response protects against dendrite damage. Our studies uncover a homeostatic protective dendrite-glia interaction and suggest that similar signaling occurs at other sensory structures and at synapses, which resemble sensory organs in architecture and molecules.

SNX3-retromer requires an evolutionary conserved MON2:DOPEY2:ATP9A complex to mediate Wntless sorting and Wnt secretion.

Wntless transports Wnt morphogens to the cell surface and is required for Wnt secretion and morphogenic gradients formation. Recycling of endocytosed Wntless requires the sorting nexin-3 (SNX3)-retromer-dependent endosome-to-Golgi transport pathway. Here we demonstrate the essential role of SNX3-retromer assembly for Wntless transport and report that SNX3 associates with an evolutionary conserved endosome-associated membrane re-modelling complex composed of MON2, DOPEY2 and the putative aminophospholipid translocase, ATP9A. In vivo suppression of Ce-mon-2, Ce-pad-1 or Ce-tat-5 (respective MON2, DOPEY2 and ATP9A orthologues) phenocopy a loss of SNX3-retromer function, leading to enhanced lysosomal degradation of Wntless and a Wnt phenotype. Perturbed Wnt signalling is also observed upon overexpression of an ATPase-inhibited TAT-5(E246Q) mutant, suggesting a role for phospholipid flippase activity during SNX3-retromer-mediated Wntless sorting. Together, these findings provide in vitro and in vivo mechanistic details to describe SNX3-retromer-mediated transport during Wnt secretion and the formation of Wnt-morphogenic gradients.

Retromer Endosome Exit Domains Serve Multiple Trafficking Destinations and Regulate Local G Protein Activation by GPCRs.

Retromer mediates sequence-directed cargo exit from endosomes to support both endosome-to-Golgi (retrograde transport) and endosome-to-plasma membrane (recycling) itineraries. It is not known whether these trafficking functions require cargos to exit endosomes separately via distinct transport intermediates or whether the same retromer-coated carriers can support both itineraries. We addressed this question by comparing human Wntless (Wls) and ß2 adrenergic receptor (ß2AR), which require retromer physiologically for retrograde transport and recycling, respectively. We show here by direct visualization in living cells that both cargos transit primarily the same endosomes and exit via shared transport vesicles generated from a retromer-coated endosome domain. While both Wls and ß2AR clearly localize to the same retromer-coated endosome domains, Wls is consistently enriched more strongly. This enrichment difference is determined by distinct motifs present in the cytoplasmic tail of each cargo, with Wls using tandem Φ-X-[L/M] motifs and ß2AR using a PDZ motif. Exchanging these determinants reverses the enrichment phenotype of each cargo but does not change cargo itinerary, verifying the multifunctional nature of retromer and implying that additional sorting must occur downstream. Quantitative differences in the degree of cargo enrichment instead underlie a form of kinetic sorting that impacts the rate of cargo delivery via both itineraries and determines the ability of ß2AR to activate its cognate G protein transducer locally from endosomes. We propose that mammalian retromer forms a multifunctional membrane coat that supports shared cargo exit for divergent trafficking itineraries and regulates signaling from endosomes.

Mena associates with Rac1 and modulates connexin 43 remodeling in cardiomyocytes.

Mena, a member of the Ena/VASP family of actin regulatory proteins, modulates microfilaments and interacts with cytoskeletal proteins associated with heart failure. Mena is localized at the intercalated disc (ICD) of adult cardiac myocytes, colocalizing with numerous cytoskeletal proteins. Mena's role in the maintainence of mechanical myocardial stability at the cardiomyocyte ICD remains unknown. We hypothesized that Mena may modulate signals from the sarcolemma to the actin cytoskeleton at the ICD to regulate the expression and localization of connexin 43 (Cx43). The small GTPase Rac1 plays a pivotal role in the regulation of actin cytoskeletal reorganization and mediating morphological and transcriptional changes in cardiomyocytes. We found that Mena is associated with active Rac1 in cardiomyocytes and that RNAi knockdown of Mena increased Rac1 activity significantly. Furthermore, Mena knockdown increased Cx43 expression and altered Cx43 localization and trafficking at the ICD, concomitant with faster intercellular communication, as assessed by dye transfer between cardiomyocyte pairs. In mice overexpressing constitutively active Rac1, left ventricular Mena expression was increased significantly, concomitant with lateral redistribution of Cx43. These results suggest that Mena is a critical regulator of the ICD and is required for normal localization of Cx43 in part via regulation of Rac1.