Retromer is a protein assembly that plays a central role in orchestrating export of transmembrane-spanning cargo proteins from endosomes into retrieval pathways destined for the Golgi apparatus and ...the plasma membrane 1. Recently, a specific mutation in the retromer component VPS35, VPS35(D620N), has linked retromer dysfunction to familial autosomal dominant and sporadic Parkinson disease 2, 3. However, the effect of this mutation on retromer function remains poorly characterized. Here we established that in cells expressing VPS35(D620N) there is a perturbation in endosome-to-TGN transport but not endosome-to-plasma membrane recycling, which we confirm in patient cells harboring the VPS35(D620N) mutation. Through comparative stable isotope labeling by amino acids in cell culture (SILAC)-based analysis of wild-type VPS35 versus the VPS35(D620N) mutant interactomes, we establish that the major defect of the D620N mutation lies in the association to the actin-nucleating Wiskott-Aldrich syndrome and SCAR homolog (WASH) complex. Moreover, using isothermal calorimetry, we establish that the primary defect of the VPS35(D620N) mutant is a 2.2 ± 0.5-fold decrease in affinity for the WASH complex component FAM21. These data define the primary molecular defect in retromer assembly that arises from the VPS35(D620N) mutation and, by revealing functional effects on retromer-mediated endosome-to-TGN transport, provide new insight into retromer deregulation in Parkinson disease.
•VPS35(D620N) mutation leads to a defect in endosome-to-TGN transport•Mutant has a perturbed association with the actin-polymerizing WASH complex•Primary defect is a reduced affinity for binding WASH component FAM21•Provides new molecular insight into retromer deregulation in Parkinson disease
Using quantitative proteomics and functional analysis in patient-derived fibroblasts, McGough et al. define the primary molecular defect associated with the Parkinson disease-linked retromer VPS35(D620N) mutation and, by revealing effects on retromer-mediated endosome-to-TGN transport, provide new insight into retromer deregulation in this disease.
Recent Advances in Retromer Biology McGough, Ian J.; Cullen, Peter J.
Traffic (Copenhagen, Denmark),
August 2011, Letnik:
12, Številka:
8
Journal Article
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The endosomal network is an organized array of intracellular, membranous compartments that function as sorting sites for endosomal and biosynthetic cargo. The fate of endocytic cargo is reliant upon ...interactions with a number of molecularly distinct sorting complexes, which tightly control the relationship between sorting of their respective cargo and the physical process of membrane re‐scuplturing required for the formation of transport carries. One such complex, retromer, mediates retrograde transport from endosomes to the trans‐Golgi network (TGN). Disregulation of retromer has been implicated in a host of disease states including late‐onset Alzheimer's. Rather than give a broad overview of retromer biology, here we aim to outline the recent advances in understanding this complex, focussing on the involvement of both clathrin and the cytoskeleton in retromer function.
Retromer is a protein assembly that orchestrates the sorting of transmembrane cargo proteins into endosome-to-Golgi and endosome-to-plasma-membrane transport pathways. Here, we have employed ...quantitative proteomics to define the interactome of human VPS35, the core retromer component. This has identified a number of new interacting proteins, including ankyrin-repeat domain 50 (ANKRD50), seriologically defined colon cancer antigen 3 (SDCCAG3) and VPS9-ankyrin-repeat protein (VARP, also known as ANKRD27). Depletion of these proteins resulted in trafficking defects of retromer-dependent cargo, but differential and cargo-specific effects suggested a surprising degree of functional heterogeneity in retromer-mediated endosome-to-plasma-membrane sorting. Extending this, suppression of the retromer-associated WASH complex did not uniformly affect retromer cargo, thereby confirming cargo-specific functions for retromer-interacting proteins. Further analysis of the retromer-VARP interaction identified a role for retromer in endosome-to-melanosome transport. Suppression of VPS35 led to mistrafficking of the melanogenic enzymes, tyrosinase and tryrosine-related protein 1 (Tyrp1), establishing that retromer acts in concert with VARP in this trafficking pathway. Overall, these data reveal hidden complexities in retromer-mediated sorting and open up new directions in our molecular understanding of this essential sorting complex.
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.
This one-step method generates, for any locus, a conditional knockout allele in Drosophila. The allele carries a bright fluorescent marker for easy screening and an attP landing site for subsequent ...genetic manipulations. After removing the selectable marker with Cre, the attP site can be used to insert DNA fragments expressing tagged or mutant alleles to determine protein localization and function in a tissue- and stage-specific manner. Only a single round of CRISPR-Cas9-mediated editing is required.
For complete details on the use and execution of this protocol, please refer to the DWnt4cKO example in Yu et al. (2020).
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•One-step CRISPR-Cas9 protocol to generate conditional knockout alleles in Drosophila•Facile screening with a removeable fluorescent marker•Integrase site included to enable subsequent “plug&play” manipulations
This one-step method generates, for any locus, a conditional knockout allele in Drosophila. The allele carries a bright fluorescent marker for easy screening and an attP landing site for subsequent genetic manipulations. After removing the selectable marker with Cre, the attP site can be used to insert DNA fragments expressing tagged or mutant alleles to determine protein localization and function in a tissue- and stage-specific manner. Only a single round of CRISPR-Cas9-mediated editing is required.
A relatively small number of proteins have been suggested to act as morphogens-signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during ...development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity
. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem
. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.
Planar cell polarity (PCP) organizes the orientation of cellular protrusions and migratory activity within the tissue plane. PCP establishment involves the subcellular polarization of core PCP ...components. It has been suggested that Wnt gradients could provide a global cue that coordinates local PCP with tissue axes. Here, we dissect the role of Wnt ligands in the orientation of hairs of Drosophila wings, an established system for the study of PCP. We found that PCP was normal in quintuple mutant wings that rely solely on the membrane-tethered Wingless for Wnt signaling, suggesting that a Wnt gradient is not required. We then used a nanobody-based approach to trap Wntless in the endoplasmic reticulum, and hence prevent all Wnt secretion, specifically during the period of PCP establishment. PCP was still established. We conclude that, even though Wnt ligands could contribute to PCP, they are not essential, and another global cue must exist for tissue-wide polarization.
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•Reporter knockin alleles identify all the Wnt genes expressed in the Drosophila wing•A quintuple mutant removes all diffusible DWnts from the wing but leaves PCP unperturbed•The secretion and release of Wnts is dispensable for Drosophila wing PCP establishment•The wing margin does not provide a global cue for PCP
Planar cell polarity (PCP) requires Frizzled, which transduces Wnt signaling. Yu et al show that the secretion and release of Wnts is dispensable for proper PCP establishment in the Drosophila wing. The global cue organizing PCP in this tissue remains unknown but does not originate from the wing margin.
A key step in the canonical Wnt signalling pathway is the inhibition of GSK3β, which results in the accumulation of nuclear β-catenin (also known as CTNNB1), and hence regulation of target genes. ...Evidence suggests that endocytosis is required for signalling, yet its role and the molecular understanding remains unclear. A recent and controversial model suggests that endocytosis contributes to Wnt signalling by causing the sequestration of the ligand-receptor complex, including LRP6 and GSK3 to multivesicular bodies (MVBs), thus preventing GSK3β from accessing β-catenin. Here, we use specific inhibitors (Dynasore and Dyngo-4a) to confirm the essential role of endocytosis in Wnt/Wingless signalling in human and Drosophila cells. However, we find no evidence that, in Drosophila cells or wing imaginal discs, LRP6/Arrow traffics to MVBs or that MVBs are required for Wnt/Wingless signalling. Moreover, we show that activation of signalling through chemical blockade of GSK3β is prevented by endocytosis inhibitors, suggesting that endocytosis impacts on Wnt/Wingless signalling downstream of the ligand-receptor complex. We propose that, through an unknown mechanism, endocytosis boosts the resting pool of β-catenin upon which GSK3β normally acts.
Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless ...(Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. Here, we report that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1-SNX2 and SNX5-SNX6. We have found that the unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion and show that SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo.
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