Retromer- and WASH-dependent sorting of nutrient transporters requires a multivalent interaction network with ANKRD50

Retromer and the associated actin-polymerizing WASH complex are essential for the endocytic recycling of a wide range of integral membrane proteins. A hereditary Parkinson's-disease-causing point mutation (D620N) in the retromer subunit VPS35 perturbs retromer's association with the WASH complex and also with the uncharacterized protein ankyrin-repeat-domain-containing protein 50 (ANKRD50). Here, we firmly establish ANKRD50 as a new and essential component of the SNX27– retromer–WASH super complex. Depletion of ANKRD50 in HeLa or U2OS cells phenocopied the loss of endosome-to-cell-surface recycling of multiple transmembrane proteins seen upon suppression of SNX27, retromer or WASH- complex components. Mass-spectrometry-based quantification of the cell surface proteome of ANKRD50-depleted cells identified amino acid transporters of the SLC1A family, among them SLC1A4, as additional cargo molecules that depend on ANKRD50 and retromer for their endocytic recycling. Mechanistically, we show that ANKRD50 simultaneously engages multiple parts of the SNX27–retromer–WASH complex machinery in a direct and co-operative interaction network that is needed to efficiently recycle the nutrient transporters GLUT1 (also known as SLC2A1) and SLC1A4, and potentially many other surface proteins

Retromer/WASH dependent sorting of nutrient transporters requires a multivalent interaction network with ANKRD50

Retromer and the associated actin-polymerizing WASH complex are essential for the endocytic recycling of a wide range of integral membrane proteins. A hereditary Parkinson's-disease-causing point mutation (D620N) in the retromer subunit VPS35 perturbs retromer's association with the WASH complex and also with the uncharacterized protein ankyrin-repeat-domain-containing protein 50 (ANKRD50). Here, we firmly establish ANKRD50 as a new and essential component of the SNX27–retromer–WASH super complex. Depletion of ANKRD50 in HeLa or U2OS cells phenocopied the loss of endosome-to-cell-surface recycling of multiple transmembrane proteins seen upon suppression of SNX27, retromer or WASH-complex components. Mass-spectrometry-based quantification of the cell surface proteome of ANKRD50-depleted cells identified amino acid transporters of the SLC1A family, among them SLC1A4, as additional cargo molecules that depend on ANKRD50 and retromer for their endocytic recycling. Mechanistically, we show that ANKRD50 simultaneously engages multiple parts of the SNX27–retromer–WASH complex machinery in a direct and co-operative interaction network that is needed to efficiently recycle the nutrient transporters GLUT1 (also known as SLC2A1) and SLC1A4, and potentially many other surface proteins

Retromer and TBC1D5 maintain late endosomal RAB7 domains to enable amino acid–induced mTORC1 signaling

Retromer is an evolutionarily conserved multiprotein complex that orchestrates the endocytic recycling of integral membrane proteins. Here, we demonstrate that retromer is also required to maintain lysosomal amino acid signaling through mTORC1 across species. Without retromer, amino acids no longer stimulate mTORC1 translocation to the lysosomal membrane, which leads to a loss of mTORC1 activity and increased induction of autophagy. Mechanistically, we show that its effect on mTORC1 activity is not linked to retromer’s role in the recycling of transmembrane proteins. Instead, retromer cooperates with the RAB7-GAP TBC1D5 to restrict late endosomal RAB7 into microdomains that are spatially separated from the amino acid– sensing domains. Upon loss of retromer, RAB7 expands into the ragulator-decorated amino acid–sensing domains and interferes with RAG-GTPase and mTORC1 recruitment. Depletion of retromer in Caenorhabditis elegans reduces mTORC1 signaling and extends the lifespan of the worms, confirming an evolutionarily conserved and unexpected role for retromer in the regulation of mTORC1 activity and longevity

Reciprocal integrin/integrin antagonism through kindlin-2 and Rho GTPases regulates cell cohesion and collective migration

Collective cell behaviour during embryogenesis and tissue repair requires the coordination of intercellular junctions, cytoskeleton-dependent shape changes controlled by Rho GTPases, and integrin-dependent cell-matrix adhesion. Many different integrins are simultaneously expressed during wound healing, embryonic development, and sprouting angiogenesis, suggesting that there is extensive integrin/integrin cross-talk to regulate cell behaviour. Here, we show that fibronectin-binding β1 and β3 integrins do not act synergistically, but rather antagonize each other during collective cell processes in neuro-epithelial cells, placental trophoblasts, and endothelial cells. Reciprocal β1/β3 antagonism controls RhoA activity in a kindlin-2-dependent manner, balancing cell spreading, contractility, and intercellular adhesion. In this way, reciprocal β1/β3 antagonism controls cell cohesion and cellular plasticity to switch between extreme and opposing states, including epithelial versus mesenchymal-like phenotypes and collective versus individual cell migration. We propose that integrin/integrin antagonism is a universal mechanism to effectuate social cellular interactions, important for tissue morphogenesis, endothelial barrier function, trophoblast invasion, and sprouting angiogenesis

Suicidal Autointegration of <i>Sleeping Beauty</i> and <i>piggyBac</i> Transposons in Eukaryotic Cells

<div><p>Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. ‘Cut and paste’ DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, <i>Sleeping Beauty</i> (<i>SB</i>) and <i>piggyBac (PB)</i> that are widely used for genome manipulation in vertebrate species. <i>SB</i> originates from fish and <i>PB</i> from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither <i>SB</i> nor <i>PB</i> is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1), a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the <i>SB</i> transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both <i>SB</i> and <i>PB</i> were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, <i>SB</i> is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for <i>PB</i>.</p></div

Bimolecular transposition events generated by <i>PB</i>.

<p>Transposition assay was performed by using ‘solo’ transposon substrates, either alone or mixed in equimolar ratios, in the present of the mPB transposase. The statistical significance of differences is shown by asterisk above the bars, *P<0.05. Molecular analysis identified no transposase-mediated integration events in the resistant colonies using <i>PBΔright</i> (background). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen-1004103-t001" target="_blank">Table 1</a>.</p

The cellular factor of BANF1 interferes with autointegration.

<p>A. Relative autointegration frequencies of <i>SB</i> (<i>SB7K</i>, left panel) and PB (<i>PB7K</i>, right panel) in HeLa cells, where BANF1 was either knocked-down or overexpressed. Knocking down of BANF1 stimulated, whereas overexpressing of BANF1 inhibited autointegration of both <i>SB</i> and <i>PB</i> transposons (n = 3). The statistical significance of differences is shown by asterisk above the bars *P<0.05. B. A SILAC pull-down experiment using anti-HA resin to investigate interaction partners of HMGXB4 in the presence/absence of <i>SB10</i> transposase in transiently transfected HEK293T cells. Schematic representation of the SILAC/pull-down experimental approach in which stable isotope labeled amino acids [Light (L) or Medium heavy (M)] are added in the form of medium supplement to culture HEK293T cells. Detection of interaction partners is performed by mass spectrometry. Scatter plot displays the normalized log2 SILAC ratio M/L values (X-axis) versus log2 intensity (Y-axis) of proteins detected in the interactome around HMGXB4⁻ in presence of the SB transposase. Each dot represents an individual protein, while their position indicates their abundance in the complex pulled down by the bait of HMGXB4. Proteins with a positive log2 SILAC M/L ratio, including BANF1 and <i>SB</i> are enriched in the protein complex around HMGXB4. C. Co-immunoprecipitation assay to investigate the interaction partners of HMGXB4, a physical interaction partner of the <i>Sleeping Beauty</i> transposase, SB10 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen.1004103-Walisko2" target="_blank">[13]</a>. <i>SB10</i> and HA-tagged HMGXB4 were transiently transfected into HEK293T cells (see Methods). In comparison to negative control, BANF1 and <i>SB</i> are enriched in the pull-down by HMGXB4-HA.</p

Comparing autointegration profile to the predicted, close-to-random target site distribution of <i>SB</i> transposition.

<p>A. Distribution of 53 <i>de novo</i> autointegration events (triangles) detected by the assay shown in (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen-1004103-g001" target="_blank">Figure 1B</a>). Autointegration products were isolated from individual bacterial clones, sequenced and mapped to the <i>SBrescue</i> construct. The thin arrow indicates the location of the sequencing primer on the left IR. B. Comparison of the predicted and experimental insertion events. The <i>SBrescue</i> construct is shown in a linear mode. The <i>SB Vstep</i> scores and experimental insertion events were shown below. Un, undetectable.</p

Autointegration properties of <i>PiggyBac</i> transposition.

<p>A. Frequency of <i>PB</i> autointegration events in HeLa cells using the PB2K construct. PBase, m<i>PB</i> transposase <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen.1004103-Cadinanos1" target="_blank">[50]</a>. B. The structure of the <i>PB2K</i> construct. For explanation, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen-1004103-g001" target="_blank">Figure 1A</a>. Distribution of <i>de novo PB</i> insertions indicated by black triangles (n = 22) on the <i>PB2K</i> construct. C. Sequence of three (3/22) representative single-ended transposition events mapped to the B, C and rpsL regions of <i>PB2K</i>. Sequences flanking the right inverted repeat of the <i>PB</i> transposon in <i>PB2K</i>. Original sequences (bold); <i>de novo</i> integration events (normal); target site of PB transposition, TTAA (italic). D. Distribution of six single-ended transposition events on the <i>PBsingle</i> construct. <i>Kan</i>: kanamycin resistant gene (<i>Kan</i>). Dark bars indicate the control experiment with only transposon vector; light bars indicate the experiment with both transposon vector and transposase expressing vector. E. Sequence of the six individual single-ended transposition shown on <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen-1004103-g003" target="_blank">Figure 3D</a>. The <i>PB</i> transposon is shown as a two-headed arrow, representing the IRs (black). Frequencies are shown in parentheses.</p

Transposase-mediated integration events of ‘solo’ substrates.

<p>HeLa cells were co-transfected with the ‘solo’ transposon constructs in the present of either mPB or SB100X transposases, while a catalytically inactive <i>SB</i> transposase, D3 was used as a control. Frequency of substrate integration was calculated as a ratio of colony numbers in the presence vs absence of transposases. Colonies were picked and analysed for transposase-mediated integration events. Transposase-mediated integration is defined when the IR of the transposon is integrated into a respective target site in the genome (see also Supporting <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen.1004103.s006" target="_blank">Text S1</a>). ND: not detected.</p