Rab4A-directed endosome traffic shapes pro-inflammatory mitochondrial metabolism in T cells via mitophagy, CD98 expression, and kynurenine-sensitive mTOR activation.

Activation of the mechanistic target of rapamycin (mTOR) is a key metabolic checkpoint of pro-inflammatory T-cell development that contributes to the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), however, the underlying mechanisms remain poorly understood. Here, we identify a functional role for Rab4A-directed endosome traffic in CD98 receptor recycling, mTOR activation, and accumulation of mitochondria that connect metabolic pathways with immune cell lineage development and lupus pathogenesis. Based on integrated analyses of gene expression, receptor traffic, and stable isotope tracing of metabolic pathways, constitutively active Rab4AQ72L exerts cell type-specific control over metabolic networks, dominantly impacting CD98-dependent kynurenine production, mTOR activation, mitochondrial electron transport and flux through the tricarboxylic acid cycle and thus expands CD4+ and CD3+CD4-CD8- double-negative T cells over CD8+ T cells, enhancing B cell activation, plasma cell development, antinuclear and antiphospholipid autoantibody production, and glomerulonephritis in lupus-prone mice. Rab4A deletion in T cells and pharmacological mTOR blockade restrain CD98 expression, mitochondrial metabolism and lineage skewing and attenuate glomerulonephritis. This study identifies Rab4A-directed endosome traffic as a multilevel regulator of T cell lineage specification during lupus pathogenesis.

Receptor-mediated internalization promotes increased endosome size and number in a RAB4- and RAB5-dependent manner.

Despite their significance in receptor-mediated internalization and continued signal transduction in cells, early/sorting endosomes (EE/SE) remain incompletely characterized, with many outstanding questions that surround the dynamics of their size and number. While several studies have reported increases in EE/SE size and number resulting from endocytic events, few studies have addressed such dynamics in a methodological and quantitative manner. Herein we apply quantitative fluorescence microscopy to measure the size and number of EE/SE upon internalization of two different ligands: transferrin and epidermal growth factor. Additionally, we used siRNA knock-down to determine the involvement of 5 different endosomal RAB proteins (RAB4, RAB5, RAB8A, RAB10 and RAB11A) in EE/SE dynamics. Our study provides new information on the dynamics of endosomes during endocytosis, an important reference for researchers studying receptor-mediated internalization and endocytic events.

Proximity labelling identifies pro-migratory endocytic recycling cargo and machinery of the Rab4 and Rab11 families.

Endocytic recycling controls the return of internalised cargoes to the plasma membrane to coordinate their positioning, availability and downstream signalling. The Rab4 and Rab11 small GTPase families regulate distinct recycling routes, broadly classified as fast recycling from early endosomes (Rab4) and slow recycling from perinuclear recycling endosomes (Rab11), and both routes handle a broad range of overlapping cargoes to regulate cell behaviour. We adopted a proximity labelling approach, BioID, to identify and compare the protein complexes recruited by Rab4a, Rab11a and Rab25 (a Rab11 family member implicated in cancer aggressiveness), revealing statistically robust protein-protein interaction networks of both new and well-characterised cargoes and trafficking machinery in migratory cancer cells. Gene ontological analysis of these interconnected networks revealed that these endocytic recycling pathways are intrinsically connected to cell motility and cell adhesion. Using a knock-sideways relocalisation approach, we were further able to confirm novel links between Rab11, Rab25 and the ESCPE-1 and retromer multiprotein sorting complexes, and identify new endocytic recycling machinery associated with Rab4, Rab11 and Rab25 that regulates cancer cell migration in the 3D matrix.

Exploring the role of the multiple sclerosis susceptibility gene CLEC16A in T cells.

C-type lectin-like domain family 16 member A (CLEC16A) is associated with autoimmune disorders, including multiple sclerosis (MS), but its functional relevance is not completely understood. CLEC16A is expressed in several immune cells, where it affects autophagic processes and receptor expression. Recently, we reported that the risk genotype of an MS-associated single nucleotide polymorphism in CLEC16A intron 19 is associated with higher expression of CLEC16A in CD4+ T cells. Here, we show that CLEC16A expression is induced in CD4+ T cells upon T cell activation. By the use of imaging flow cytometry and confocal microscopy, we demonstrate that CLEC16A is located in Rab4a-positive recycling endosomes in Jurkat TAg T cells. CLEC16A knock-down in Jurkat cells resulted in lower cell surface expression of the T cell receptor, however, this did not have a major impact on T cell activation response in vitro in Jurkat nor in human, primary CD4+ T cells.

Intracellular trafficking of Notch orchestrates temporal dynamics of Notch activity in the fly brain.

While Delta non-autonomously activates Notch in neighboring cells, it autonomously inactivates Notch through cis-inhibition, the molecular mechanism and biological roles of which remain elusive. The wave of differentiation in the Drosophila brain, the 'proneural wave', is an excellent model for studying Notch signaling in vivo. Here, we show that strong nonlinearity in cis-inhibition reproduces the second peak of Notch activity behind the proneural wave in silico. Based on this, we demonstrate that Delta expression induces a quick degradation of Notch in late endosomes and the formation of the twin peaks of Notch activity in vivo. Indeed, the amount of Notch is upregulated and the twin peaks are fused forming a single peak when the function of Delta or late endosomes is compromised. Additionally, we show that the second Notch peak behind the wavefront controls neurogenesis. Thus, intracellular trafficking of Notch orchestrates the temporal dynamics of Notch activity and the temporal patterning of neurogenesis.

A conserved haplotype in Wagyu cattle contains RAB4A whose encoded protein regulates glucose trafficking in muscle and fat cells.

The Wagyu breed of taurine cattle possess favourable genetics for intramuscular fat (IMF) but genomic loci associated with the trait remain under characterised. Here, we report the identification of a previously unidentified genomic region possessing a particular haplotype structure in Wagyu. Through deployment of a genome-wide haplotype detection analysis that captures regions conserved in a target population but not other populations we screened 100 individual Wagyu and contrasted them with 100 individuals from two independent comparison breeds, Charolais and Angus, using high-density SNPs. An extreme level of Wagyu conservation was assigned to a single genomic window (spanning genomic coordinates BTA28:41 088-300 265 bp). In fact, a five-SNP region spanning 27 096 bp is almost perfectly conserved among the 100 Wagyu individuals assayed and partially overlaps RAB4A. Focussing in, two consecutive SNPs (genomic coordinates 236 949 and 239 950) are apparently fixed within the Wagyu (BB and AA respectively), but at mixed frequencies in the other two breeds. These SNPs are located in the two introns straddling exon 7. In a separate analysis using the 1000 Bulls database, we found that, coincident with exon 7 of RAB4A first allele frequencies were highest in the high IMF Japanese Native (Wagyu) breeds (0.78) and lowest in the low IMF indicine breeds (Nelore and Brahman), with intermediate marbling breeds (Angus and Charolais) assigned intermediate rankings (0.42). RAB4A is known to encode a protein that regulates intracellular trafficking of the insulin-regulated glucose transporter GLUT4. RAB4A can be considered an attractive new positional candidate for IMF development.

ß-Adrenergic control of sarcolemmal CaV1.2 abundance by small GTPase Rab proteins.

The number and activity of Cav1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. ß-Adrenergic receptor (ßAR) activation stimulates sarcolemmal insertion of CaV1.2. This supplements the preexisting sarcolemmal CaV1.2 population, forming large "superclusters" wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca2+ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized CaV1.2 channels. ßAR-activation decreased CaV1.2/endosome colocalization in ventricular myocytes, as it triggered "emptying" of endosomal CaV1.2 cargo into the t-tubule sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that CaV1.2 are often inserted into the sarcolemma as preformed, multichannel clusters. Similarly, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by coexpression of constitutively active Rab4a, halved by coexpression of dominant-negative Rab11a, and abolished by coexpression of dominant-negative mutant Rab4a. In ventricular myocytes, ßAR-stimulated recycling of CaV1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented ßAR-activated Ca2+ current augmentation. Moreover, ßAR-regulation of CaV1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that ßAR-stimulation triggers an on-demand boost in sarcolemmal CaV1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for ßAR-regulation of cardiac CaV1.2.

A feed-forward loop between SorLA and HER3 determines heregulin response and neratinib resistance.

Current evidence indicates that resistance to the tyrosine kinase-type cell surface receptor (HER2)-targeted therapies is frequently associated with HER3 and active signaling via HER2-HER3 dimers, particularly in the context of breast cancer. Thus, understanding the response to HER2-HER3 signaling and the regulation of the dimer is essential to decipher therapy relapse mechanisms. Here, we investigate a bidirectional relationship between HER2-HER3 signaling and a type-1 transmembrane sorting receptor, sortilin-related receptor (SorLA; SORL1). We demonstrate that heregulin-mediated signaling supports SorLA transcription downstream of the mitogen-activated protein kinase pathway. In addition, we demonstrate that SorLA interacts directly with HER3, forming a trimeric complex with HER2 and HER3 to attenuate lysosomal degradation of the dimer in a Ras-related protein Rab4-dependent manner. In line with a role for SorLA in supporting the stability of the HER2 and HER3 receptors, loss of SorLA compromised heregulin-induced cell proliferation and sensitized metastatic anti-HER2 therapy-resistant breast cancer cells to neratinib in cancer spheroids in vitro and in vivo in a zebrafish brain xenograft model.

Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling.

The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein-coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule-binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.

UNC0321 inhibits high glucose induced apoptosis in HUVEC by targeting Rab4.

The vascular complications in heart, brain, kidney and retina are the most common chronic complications of diabetes mellitus (DM). At present, it has become a research hotspot to regulate the abnormal apoptosis of vascular endothelial cells for DM treatment. UNC0321 is a high affinity GPCRs inhibitor, and has potential practical value in chromatin remodeling. In this study, we treated HUVEC with UNC0321 in vitro, and found that UNC0321 inhibit the level of Cleaved-Caspase3 and Bax, thus inhibiting the apoptosis caused by high glucose. In addition, UNC0321 also promoted cell proliferation and migration by activating Akt / mTOR pathway. The transcriptome changes of HUVEC cells cultured with high glucose with or without the treatment of UNC0321 were analysis using sequencing. It was found that Rab4 expression was significantly inhibited after UNC0321 treatment. Subsequently, we overexpressed Rab4 in HUVEC cells cultured with high glucose, and found that overexpression of Rab4 promoted the apoptosis, and inhibited cell proliferation and migration. At the same time, after overexpression of Rab4 in HUVEC cells treated with UNC0321, the number of apoptosis was significantly increased, cell proliferation and migration were inhibited, and the activity of Akt / mTOR pathway decreased. These data suggested that overexpression of Rab4 effectively blocked the inhibition of apoptosis and the increase of cell proliferation induced by UNC0321. In conclusion, we found that UNC0321 inhibits the apoptosis of HUVEC cells caused by high glucose through inhibiting Rab4 expression, providing new potential drugs and targets for the treatment of diabetic vascular complications.

Excess Rab4 rescues synaptic and behavioral dysfunction caused by defective HTT-Rab4 axonal transport in Huntington's disease.

Huntington's disease (HD) is characterized by protein inclusions and loss of striatal neurons which result from expanded CAG repeats in the poly-glutamine (polyQ) region of the huntingtin (HTT) gene. Both polyQ expansion and loss of HTT have been shown to cause axonal transport defects. While studies show that HTT is important for vesicular transport within axons, the cargo that HTT transports to/from synapses remain elusive. Here, we show that HTT is present with a class of Rab4-containing vesicles within axons in vivo. Reduction of HTT perturbs the bi-directional motility of Rab4, causing axonal and synaptic accumulations. In-vivo dual-color imaging reveal that HTT and Rab4 move together on a unique putative vesicle that may also contain synaptotagmin, synaptobrevin, and Rab11. The moving HTT-Rab4 vesicle uses kinesin-1 and dynein motors for its bi-directional movement within axons, as well as the accessory protein HIP1 (HTT-interacting protein 1). Pathogenic HTT disrupts the motility of HTT-Rab4 and results in larval locomotion defects, aberrant synaptic morphology, and decreased lifespan, which are rescued by excess Rab4. Consistent with these observations, Rab4 motility is perturbed in iNeurons derived from human Huntington's Disease (HD) patients, likely due to disrupted associations between the polyQ-HTT-Rab4 vesicle complex, accessory proteins, and molecular motors. Together, our observations suggest the existence of a putative moving HTT-Rab4 vesicle, and that the axonal motility of this vesicle is disrupted in HD causing synaptic and behavioral dysfunction. These data highlight Rab4 as a potential novel therapeutic target that could be explored for early intervention prior to neuronal loss and behavioral defects observed in HD.

Distinct Roles for RAB10 and RAB29 in Pathogenic LRRK2-Mediated Endolysosomal Trafficking Alterations.

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease, and sequence variations are associated with the sporadic form of the disease. LRRK2 phosphorylates a subset of RAB proteins implicated in secretory and recycling trafficking pathways, including RAB8A and RAB10. Another RAB protein, RAB29, has been reported to recruit LRRK2 to the Golgi, where it stimulates its kinase activity. Our previous studies revealed that G2019S LRRK2 expression or knockdown of RAB8A deregulate epidermal growth factor receptor (EGFR) trafficking, with a concomitant accumulation of the receptor in a RAB4-positive recycling compartment. Here, we show that the G2019S LRRK2-mediated EGFR deficits are mimicked by knockdown of RAB10 and rescued by expression of active RAB10. By contrast, RAB29 knockdown is without effect, but expression of RAB29 also rescues the pathogenic LRRK2-mediated trafficking deficits independently of Golgi integrity. Our data suggest that G2019S LRRK2 deregulates endolysosomal trafficking by impairing the function of RAB8A and RAB10, while RAB29 positively modulates non-Golgi-related trafficking events impaired by pathogenic LRRK2.

GGA3 interacts with L-type prostaglandin D synthase and regulates the recycling and signaling of the DP1 receptor for prostaglandin D2 in a Rab4-dependent mechanism.

Mechanisms controlling the recycling of G protein-coupled receptors (GPCRs) remain largely unclear. We report that GGA3 (Golgi-associated, γ adaptin ear containing, ADP-ribosylation factor-binding protein 3) regulates the recycling and signaling of the PGD2 receptor DP1 through a new mechanism. An endogenous interaction between DP1 and GGA3 was detected by co-immunoprecipitation in HeLa cells. The interaction was promoted by DP1 agonist stimulation, which was supported by increased DP1-GGA3 colocalization in confocal microscopy. Pulldown assays showed that GGA3 interacts with the intracellular loop 2 and C-terminus of DP1, whereas the receptor interacts with the VHS domain of GGA3. The Arf-binding deficient GGA3 N194A mutant had the same effect as wild-type GGA3 on DP1 trafficking, suggesting a new mechanism for GGA3 in recycling. Depletion of Rab4 inhibited the GGA3 effect on DP1 recycling, revealing a Rab4-dependent mechanism. Interestingly, depletion of L-PGDS (L-type prostaglandin synthase, the enzyme that produces the agonist for DP1) impaired the ability of GGA3 to mediate DP1 recycling, while GGA3 knockdown prevented L-PGDS from promoting DP1 recycling, indicating that both proteins function interdependently. A novel interaction was observed between co-immunoprecipitated endogenous L-PGDS and GGA3 proteins in HeLa cells, and in vitro using purified recombinant proteins. Redistribution of L-PGDS towards GGA3- and Rab4-positive vesicles was induced by DP1 activation. Silencing of GGA3 inhibited ERK1/2 activation following DP1 stimulation. Altogether, our data reveal a novel function for GGA3, in a newly described association with L-PGDS, in the recycling and signaling of a GPCR, namely DP1.

Complex Rab4-Mediated Regulation of Endosomal Size and EGFR Activation.

Early sorting endosomes are responsible for the trafficking and function of transferrin receptor (TfR) and EGFR. These receptors play important roles in iron uptake and signaling and are critical for breast cancer development. However, the role of morphology, receptor composition, and signaling of early endosomes in breast cancer remains poorly understood. A novel population of enlarged early endosomes was identified in breast cancer cells and tumor xenografts but not in noncancerous MCF10A cells. Quantitative analysis of endosomal morphology, cargo sorting, EGFR activation, and Rab GTPase regulation was performed using super-resolution and confocal microscopy followed by 3D rendering. MDA-MB-231 breast cancer cells have fewer, but larger EEA1-positive early endosomes compared with MCF10A cells. Live-cell imaging indicated dysregulated cargo sorting, because EGF and Tf traffic together via enlarged endosomes in MDA-MB-231, but not in MCF10A. Large EEA1-positive MDA-MB-231 endosomes exhibited prolonged and increased EGF-induced activation of EGFR upon phosphorylation at tyrosine-1068 (EGFR-p1068). Rab4A overexpression in MCF10A cells produced EEA1-positive enlarged endosomes that displayed prolonged and amplified EGF-induced EGFR-p1068 activation. Knockdown of Rab4A lead to increased endosomal size in MCF10A, but not in MDA-MB-231 cells. Nevertheless, Rab4A knockdown resulted in enhanced EGF-induced activation of EGFR-p1068 in MDA-MB-231 as well as downstream signaling in MCF10A cells. Altogether, this extensive characterization of early endosomes in breast cancer cells has identified a Rab4-modulated enlarged early endosomal compartment as the site of prolonged and increased EGFR activation. IMPLICATIONS: Enlarged early endosomes play a Rab4-modulated role in regulation of EGFR activation in breast cancer cells.

Membrane trafficking of large conductance Ca2+- and voltage-activated K+ (BK) channels is regulated by Rab4 GTPase.

The large conductance voltage- and Ca2+-activated K+ (BK) channel is a major ionic determinant of vascular tone, vasodilation, and blood pressure. The activity of BK channels is regulated in part by membrane presentation. Rab GTPase (Rab) regulates important cellular processes, including ion channel membrane trafficking. We hypothesize that Rab4a participates in the regulation of BK channel α-subunit (BK-α) membrane trafficking. We found that vascular BK-α interacts physically with Rab4a. Co-expression of dominant-negative Rab4a reduced BK-α surface expression, whereas that of constitutively-active Rab4a augmented BK-α surface presentation. These novel findings suggest that vascular BK channel membrane expression is regulated by Rab4a through channel membrane trafficking.

Cytoskeleton Protein Filamin A Is Required for Efficient Somatostatin Receptor Type 2 Internalization and Recycling through Rab5 and Rab4 Sorting Endosomes in Tumor Somatotroph Cells.

The high expression of somatostatin receptor 2 (SST2) in growth hormone (GH)-secreting tumors represents the rationale for the clinical use of somatostatin analogs (SSAs) in acromegaly. Recently, the cytoskeletal protein Filamin A (FLNA) has emerged as key modulator of the responsiveness of GH-secreting pituitary tumors to SSAs by regulating SST2 signaling and expression. The aim of this study was to explore FLNA involvement in SST2 intracellular trafficking in tumor somatotroph cells. By biotinylation assay, we found that FLNA silencing abolished octreotide-mediated SST2 internalization in rat GH3 cell line (28.0 ± 2.7 vs. 4 ± 4.3% SST2 internalization, control versus FLNA small interfering RNAs (siRNA) cells, respectively, p < 0.001) and human GH-secreting primary cultured cells (70.3 ± 21.1 vs. 24 ± 19.2% SST2 internalization, control versus FLNA siRNA cells, respectively, p < 0.05). In addition, confocal imaging revealed impaired SST2 recycling to the plasma membrane in FLNA silenced GH3 cells. Coimmunoprecipitation and immunofluorescence experiments showed that FLNA, as well as ß-arrestin2, is timely dependent recruited to octreotide-stimulated SST2 receptors both in rat and human tumor somatotroph cells. Although FLNA expression knock down did not prevent the formation of ß-arrestin2-SST2 complex in GH3 cells, it significantly impaired efficient SST2 loading into cytosolic vesicles positive for the early endocytic and recycling markers Rab5 and 4, respectively (33.7 ± 8.9% down to 25.9 ± 6.9%, p < 0.05, and 28.4 ± 7.4% down to 17.6 ± 5.7%, p < 0.01, for SST2-Rab5 and SST2-Rab4 colocalization, respectively, in control versus FLNA siRNA cells). Altogether these data support an important role for FLNA in the mediation of octreotide-induced SST2 trafficking in GH-secreting pituitary tumor cells through Rab5 and 4 sorting endosomes.

L-type prostaglandin D synthase regulates the trafficking of the PGD2 DP1 receptor by interacting with the GTPase Rab4.

Accumulating evidence indicates that G protein-coupled receptors (GPCRs) interact with Rab GTPases during their intracellular trafficking. How GPCRs recruit and activate the Rabs is unclear. Here, we report that depletion of endogenous L-type prostaglandin D synthase (L-PGDS) in HeLa cells inhibited recycling of the prostaglandin D2 (PGD2) DP1 receptor (DP1) to the cell surface after agonist-induced internalization and that L-PGDS overexpression had the opposite effect. Depletion of endogenous Rab4 prevented l-PGDS-mediated recycling of DP1, and l-PGDS depletion inhibited Rab4-dependent recycling of DP1, indicating that both proteins are mutually involved in this pathway. DP1 stimulation promoted its interaction through its intracellular C terminus with Rab4, which was increased by l-PGDS. Confocal microscopy revealed that DP1 activation induces l-PGDS/Rab4 co-localization. l-PGDS/Rab4 and DP1/Rab4 co-immunoprecipitation levels were increased by DP1 agonist treatment. Pulldown assays with purified GST-l-PGDS and His6-Rab4 indicated that both proteins interact directly. l-PGDS interacted preferentially with the inactive, GDP-locked Rab4S22N variant rather than with WT Rab4 or with constitutively active Rab4Q67L proteins. Overexpression and depletion experiments disclosed that l-PGDS partakes in Rab4 activation following DP1 stimulation. Experiments with deletion mutants and synthetic peptides revealed that amino acids 85-92 in l-PGDS are involved in its interaction with Rab4 and in its effect on DP1 recycling. Of note, GTPγS loading and time-resolved FRET assays with purified proteins suggested that l-PGDS enhances GDP-GTP exchange on Rab4. Our results reveal how l-PGDS, which produces the agonist for DP1, regulates DP1 recycling by participating in Rab4 recruitment and activation.

WDFY2 restrains matrix metalloproteinase secretion and cell invasion by controlling VAMP3-dependent recycling.

Cancer cells secrete matrix metalloproteinases to remodel the extracellular matrix, which enables them to overcome tissue barriers and form metastases. The membrane-bound matrix metalloproteinase MT1-MMP (MMP14) is internalized by endocytosis and recycled in endosomal compartments. It is largely unknown how endosomal sorting and recycling of MT1-MMP are controlled. Here, we show that the endosomal protein WDFY2 controls the recycling of MT1-MMP. WDFY2 localizes to endosomal tubules by binding to membranes enriched in phosphatidylinositol 3-phosphate (PtdIns3P). We identify the v-SNARE VAMP3 as an interaction partner of WDFY2. WDFY2 knockout causes a strong redistribution of VAMP3 into small vesicles near the plasma membrane. This is accompanied by increased, VAMP3-dependent secretion of MT1-MMP, enhanced degradation of extracellular matrix, and increased cell invasion. WDFY2 is frequently lost in metastatic cancers, most predominantly in ovarian and prostate cancer. We propose that WDFY2 acts as a tumor suppressor by serving as a gatekeeper for VAMP3 recycling.

DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes.

Cancer cells degrade the extracellular matrix through actin-rich protrusions termed invadopodia. The formation of functional invadopodia requires polarized membrane trafficking driven by Rho GTPase-mediated cytoskeletal remodeling. We identify the Rho GTPase-activating protein deleted in liver cancer 3 (DLC3; also known as STARD8) as an integral component of the endosomal transport and sorting machinery. We provide evidence for the direct regulation of RhoB by DLC3 at endosomal membranes to which DLC3 is recruited by interacting with the sorting nexin SNX27. In TGF-ß-treated MCF10A breast epithelial cells, DLC3 knockdown enhanced metalloproteinase-dependent matrix degradation, which was partially rescued by RhoB co-depletion. This was recapitulated in MDA-MB-231 breast cancer cells in which early endosomes demonstrated aberrantly enriched F-actin and accumulated the metalloproteinase MT1-MMP (also known as MMP14) upon DLC3 knockdown. Remarkably, Rab4 (herein referring to Rab4A) downregulation fully rescued the enhanced matrix degradation of TGF-ß-treated MCF10A and MDA-MB-231 cells. In summary, our findings establish a novel role for DLC3 in the suppression of MT1-MMP-dependent matrix degradation by inactivating RhoB signaling at endosomal membranes. We propose that DLC3 function is required to limit endosomal actin polymerization, Rab4-dependent recycling of MT1-MMP and, consequently, matrix degradation mediated by invadopodial activity.

Protein interacting with Amyloid Precursor Protein tail-1 (PAT1) is involved in early endocytosis.

Protein interacting with Amyloid Precursor Protein (APP) tail 1 (PAT1) also called APPBP2 or Ara 67 has different targets such as APP or androgen receptor and is expressed in several tissues. PAT1 is known to be involved in the subcellular trafficking of its targets. We previously observed in primary neurons that PAT1 is poorly associated with APP at the cell surface. Here we show that PAT1 colocalizes with vesicles close to the cell surface labeled with Rab5, Rab4, EEA1 and Rabaptin-5 but not with Rab11 and Rab7. Moreover, PAT1 expression regulates the number of EEA1 and Rab5 vesicles, and endocytosis/recycling of the transferrin receptor. In addition, low levels of PAT1 decrease the size of transferrin-colocalized EEA1 vesicles with time following transferrin uptake. Finally, overexpression of the APP binding domain to PAT1 is sufficient to compromise endocytosis. Altogether, these data suggest that PAT1 is a new actor in transferrin early endocytosis. Whether this new function of PAT1 may have consequences in pathology remains to be determined.