The Adenomatous polyposis coli tumour suppressor is essential for Axin complex assembly and function and opposes Axin's interaction with Dishevelled

Most cases of colorectal cancer are linked to mutational inactivation of the Adenomatous polyposis coli (APC) tumour suppressor. APC downregulates Wnt signalling by enabling Axin to promote the degradation of the Wnt signalling effector β-catenin (Armadillo in flies). This depends on Axin's DIX domain whose polymerization allows it to form dynamic protein assemblies (‘degradasomes’). Axin is inactivated upon Wnt signalling, by heteropolymerization with the DIX domain of Dishevelled, which recruits it into membrane-associated ‘signalosomes’. How APC promotes Axin's function is unclear, especially as it has been reported that APC's function can be bypassed by overexpression of Axin. Examining apc null mutant Drosophila tissues, we discovered that APC is required for Axin degradasome assembly, itself essential for Armadillo downregulation. Degradasome assembly is also attenuated in APC mutant cancer cells. Notably, Axin becomes prone to Dishevelled-dependent plasma membrane recruitment in the absence of APC, indicating a crucial role of APC in opposing the interaction of Axin with Dishevelled. Indeed, co-expression experiments reveal that APC displaces Dishevelled from Axin assemblies, promoting degradasome over signalosome formation in the absence of Wnts. APC thus empowers Axin to function in two ways—by enabling its DIX-dependent self-assembly, and by opposing its DIX-dependent copolymerization with Dishevelled and consequent inactivation

DLGS97/SAP97 is developmentally upregulated and is required for complex adult behaviors and synapse morphology and function

The synaptic membrane-associated guanylate kinase (MAGUK) scaffolding protein family is thought to play key roles in synapse assembly and synaptic plasticity. Evidence supporting these roles in vivo is scarce, as a consequence of gene redundancy in mammals. The genome of Drosophila contains only one MAGUK gene, discs large (dlg), from which two major proteins originate: DLGA [PSD95 (postsynaptic density 95)-like] and DLGS97 [SAP97 (synapse-associated protein)-like]. These differ only by the inclusion in DLGS97 of an L27 domain, important for the formation of supramolecular assemblies. Known dlg mutations affect both forms and are lethal at larval stages attributable to tumoral overgrowth of epithelia. We generated independent null mutations for each, dlgA and dlgS97. These allowed unveiling of a shift in expression during the development of the nervous system: predominant expression of DLGA in the embryo, balanced expression of both during larval stages, and almost exclusive DLGS97 expression in the adult brain. Loss of embryonic DLGS97 does not alter the development of the nervous system. At larval stages, DLGA and DLGS97 fulfill both unique and partially redundant functions in the neuromuscular junction. Contrary to dlg and dlgA mutants, dlgS97 mutants are viable to adulthood, but they exhibit marked alterations in complex behaviors such as phototaxis, circadian activity, and courtship, whereas simpler behaviors like locomotion and odor and light perception are spared. We propose that the increased repertoire of associations of a synaptic scaffold protein given by an additional domain of protein-protein interaction underlies its ability to integrate molecular networks required for complex functions in adult synapses

Proper Splicing Contributes to Visual Function in The Aging Drosophila Eye

Changes in splicing patterns are a characteristic of the aging transcriptome; however, it is unclear whether these age‐related changes in splicing facilitate the progressive functional decline that defines aging. In Drosophila, visual behavior declines with age and correlates with altered gene expression in photoreceptors, including downregulation of genes encoding splicing factors. Here, we characterized the significance of these age‐regulated splicing‐associated genes in both splicing and visual function. To do this, we identified differential splicing events in either the entire eye or photoreceptors of young and old flies. Intriguingly, aging photoreceptors show differential splicing of a large number of visual function genes. In addition, as shown previously for aging photoreceptors, aging eyes showed increased accumulation of circular RNAs, which result from noncanonical splicing events. To test whether proper splicing was necessary for visual behavior, we knocked down age‐regulated splicing factors in photoreceptors in young flies and examined phototaxis. Notably, many of the age‐regulated splicing factors tested were necessary for proper visual behavior. In addition, knockdown of individual splicing factors resulted in changes in both alternative splicing at age‐spliced genes and increased accumulation of circular RNAs. Together, these data suggest that cumulative decreases in splicing factor expression could contribute to the differential splicing, circular RNA accumulation, and defective visual behavior observed in aging photoreceptors

Caveolae protect endothelial cells from membrane rupture during increased cardiac output.

Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae in endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under the mechanical forces experienced by endothelial cells in vivo is unclear. In this study we have sought to determine whether endothelial caveolae disassemble under increased hemodynamic forces, and whether caveolae help prevent acute rupture of the plasma membrane under these conditions. Experiments in cultured cells established biochemical assays for disassembly of caveolar protein complexes, and assays for acute loss of plasma membrane integrity. In vivo, we demonstrate that caveolae in endothelial cells of the lung and cardiac muscle disassemble in response to acute increases in cardiac output. Electron microscopy and two-photon imaging reveal that the plasma membrane of microvascular endothelial cells in caveolin 1(-/-) mice is much more susceptible to acute rupture when cardiac output is increased. These data imply that mechanoprotection through disassembly of caveolae is important for endothelial function in vivo

MAGI-1 Modulates AMPA Receptor Synaptic Localization and Behavioral Plasticity in Response to Prior Experience

It is well established that the efficacy of synaptic connections can be rapidly modified by neural activity, yet how the environment and prior experience modulate such synaptic and behavioral plasticity is only beginning to be understood. Here we show in C. elegans that the broadly conserved scaffolding molecule MAGI-1 is required for the plasticity observed in a glutamatergic circuit. This mechanosensory circuit mediates reversals in locomotion in response to touch stimulation, and the AMPA-type receptor (AMPAR) subunits GLR-1 and GLR-2, which are required for reversal behavior, are localized to ventral cord synapses in this circuit. We find that animals modulate GLR-1 and GLR-2 localization in response to prior mechanosensory stimulation; a specific isoform of MAGI-1 (MAGI-1L) is critical for this modulation. We show that MAGI-1L interacts with AMPARs through the intracellular domain of the GLR-2 subunit, which is required for the modulation of AMPAR synaptic localization by mechanical stimulation. In addition, mutations that prevent the ubiquitination of GLR-1 prevent the decrease in AMPAR localization observed in previously stimulated magi-1 mutants. Finally, we find that previously-stimulated animals later habituate to subsequent mechanostimulation more rapidly compared to animals initially reared without mechanical stimulation; MAGI-1L, GLR-1, and GLR-2 are required for this change in habituation kinetics. Our findings demonstrate that prior experience can cause long-term alterations in both behavioral plasticity and AMPAR localization at synapses in an intact animal, and indicate a new, direct role for MAGI/S-SCAM proteins in modulating AMPAR localization and function in the wake of variable sensory experience

BioID identifies proteins involved in the cell biology of caveolae.

The mechanisms controlling the abundance and sub-cellular distribution of caveolae are not well described. A first step towards determining such mechanisms would be identification of relevant proteins that interact with known components of caveolae. Here, we applied proximity biotinylation (BioID) to identify a list of proteins that may interact with the caveolar protein cavin1. Screening of these candidates using siRNA to reduce their expression revealed that one of them, CSDE1, regulates the levels of mRNAs and protein expression for multiple components of caveolae. A second candidate, CD2AP, co-precipitated with cavin1. Caveolar proteins were observed in characteristic and previously un-described linear arrays adjacent to cell-cell junctions in both MDCK cells, and in HeLa cells overexpressing an active form of the small GTPase Rac1. CD2AP was required for the recruitment of caveolar proteins to these linear arrays. We conclude that BioID will be useful in identification of new proteins involved in the cell biology of caveolae, and that interaction between CD2AP and cavin1 may have an important role in regulating the sub-cellular distribution of caveolae

Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating β-catenin

Wnt/β-catenin signaling controls numerous steps in normal animal development and can also cause cancer if inappropriately activated. In the absence of Wnt, β-catenin is targeted continuously for proteasomal degradation by the Axin destruction complex, whose activity is blocked upon Wnt stimulation by Dishevelled, which recruits Axin to the plasma membrane and assembles it into a signalosome. This key event during Wnt signal transduction depends on dynamic head-to-tail polymerization by the DIX domain of Dishevelled. Here, we use rescue assays in Drosophila tissues and functional assays in human cells to show that polymerization-blocking mutations in the DIX domain of Axin disable its effector function in down-regulating Armadillo/β-catenin and its response to Dishevelled during Wnt signaling. Intriguingly, NMR spectroscopy revealed that the purified DIX domains of the two proteins interact with each other directly through their polymerization interfaces, whereby the same residues mediate both homo- and heterotypic interactions. This result implies that Dishevelled has the potential to act as a “natural” dominant-negative, binding to the polymerization interface of Axin's DIX domain to interfere with its self-assembly, thereby blocking its effector function