Retromer has a selective function in cargo sorting via endosome transport carriers.

Retromer is a peripheral membrane protein complex that coordinates multiple vesicular trafficking events within the endolysosomal system. Here, we demonstrate that retromer is required for the maintenance of normal lysosomal morphology and function. The knockout of retromer subunit Vps35 causes an ultrastructural alteration in lysosomal structure and aberrant lysosome function, leading to impaired autophagy. At the whole-cell level, knockout of retromer Vps35 subunit reduces lysosomal proteolytic capacity as a consequence of the improper processing of lysosomal hydrolases, which is dependent on the trafficking of the cation-independent mannose 6-phosphate receptor (CI-M6PR). Incorporation of CI-M6PR into endosome transport carriers via a retromer-dependent process is restricted to those tethered by GCC88 but not golgin-97 or golgin-245. Finally, we show that this retromer-dependent retrograde cargo trafficking pathway requires SNX3, but not other retromer-associated cargo binding proteins, such as SNX27 or SNX-BAR proteins. Therefore, retromer does contribute to the retrograde trafficking of CI-M6PR required for maturation of lysosomal hydrolases and lysosomal function.

Sorting nexin 27 (SNX27) regulates the trafficking and activity of the glutamine transporter ASCT2.

Alanine-, serine-, cysteine-preferring transporter 2 (ASCT2, SLC1A5) is responsible for the uptake of glutamine into cells, a major source of cellular energy and a key regulator of mammalian target of rapamycin (mTOR) activation. Furthermore, ASCT2 expression has been reported in several human cancers, making it a potential target for both diagnostic and therapeutic purposes. Here we identify ASCT2 as a membrane-trafficked cargo molecule, sorted through a direct interaction with the PDZ domain of sorting nexin 27 (SNX27). Using both membrane fractionation and subcellular localization approaches, we demonstrate that the majority of ASCT2 resides at the plasma membrane. This is significantly reduced within CrispR-mediated SNX27 knockout (KO) cell lines, as it is missorted into the lysosomal degradation pathway. The reduction of ASCT2 levels in SNX27 KO cells leads to decreased glutamine uptake, which, in turn, inhibits cellular proliferation. SNX27 KO cells also present impaired activation of the mTOR complex 1 (mTORC1) pathway and enhanced autophagy. Taken together, our data reveal a role for SNX27 in glutamine uptake and amino acid-stimulated mTORC1 activation via modulation of ASCT2 intracellular trafficking.

Laser-mediated rupture of chlamydial inclusions triggers pathogen egress and host cell necrosis.

Remarkably little is known about how intracellular pathogens exit the host cell in order to infect new hosts. Pathogenic chlamydiae egress by first rupturing their replicative niche (the inclusion) before rapidly lysing the host cell. Here we apply a laser ablation strategy to specifically disrupt the chlamydial inclusion, thereby uncoupling inclusion rupture from the subsequent cell lysis and allowing us to dissect the molecular events involved in each step. Pharmacological inhibition of host cell calpains inhibits inclusion rupture, but not subsequent cell lysis. Further, we demonstrate that inclusion rupture triggers a rapid necrotic cell death pathway independent of BAK, BAX, RIP1 and caspases. Both processes work sequentially to efficiently liberate the pathogen from the host cytoplasm, promoting secondary infection. These results reconcile the pathogen's known capacity to promote host cell survival and induce cell death.

Structural basis for the hijacking of endosomal sorting nexin proteins by Chlamydia trachomatis.

During infection chlamydial pathogens form an intracellular membrane-bound replicative niche termed the inclusion, which is enriched with bacterial transmembrane proteins called Incs. Incs bind and manipulate host cell proteins to promote inclusion expansion and provide camouflage against innate immune responses. Sorting nexin (SNX) proteins that normally function in endosomal membrane trafficking are a major class of inclusion-associated host proteins, and are recruited by IncE/CT116. Crystal structures of the SNX5 phox-homology (PX) domain in complex with IncE define the precise molecular basis for these interactions. The binding site is unique to SNX5 and related family members SNX6 and SNX32. Intriguingly the site is also conserved in SNX5 homologues throughout evolution, suggesting that IncE captures SNX5-related proteins by mimicking a native host protein interaction. These findings thus provide the first mechanistic insights both into how chlamydial Incs hijack host proteins, and how SNX5-related PX domains function as scaffolds in protein complex assembly.

A molecular code for endosomal recycling of phosphorylated cargos by the SNX27-retromer complex.

Recycling of internalized receptors from endosomal compartments is essential for the receptors' cell-surface homeostasis. Sorting nexin 27 (SNX27) cooperates with the retromer complex in the recycling of proteins containing type I PSD95-Dlg-ZO1 (PDZ)-binding motifs. Here we define specific acidic amino acid sequences upstream of the PDZ-binding motif required for high-affinity engagement of the human SNX27 PDZ domain. However, a subset of SNX27 ligands, such as the ß2 adrenergic receptor and N-methyl-D-aspartate (NMDA) receptor, lack these sequence determinants. Instead, we identified conserved sites of phosphorylation that substitute for acidic residues and dramatically enhance SNX27 interactions. This newly identified mechanism suggests a likely regulatory switch for PDZ interaction and protein transport by the SNX27-retromer complex. Defining this SNX27 binding code allowed us to classify more than 400 potential SNX27 ligands with broad functional implications in signal transduction, neuronal plasticity and metabolite transport.

MTMR4 Is Required for the Stability of the Salmonella-Containing Vacuole.

The intracellular pathogen Salmonella enterica servovar Typhimurium (S.typhimurium) modulates the host cell's phosphoinositide (PI) metabolism to establish its intracellular replicative niche, the Salmonella-containing vacuole (SCV). Upon invasion, phosphoinositide 3-phosphate (PI(3)P) and other early endosomal markers are rapidly recruited to and remain associated with the SCV throughout its early maturation. While the phosphoinositide 3-phosphatase myotubularin 4 (MTMR4) has an established role in regulating autophagy and cellular PI(3)P-content, two processes associated with the intracellular survival of S. typhimurium, a direct role for MTMR4 in Salmonella biology has not been examined. Here we demonstrate that GFP-tagged MTMR4 is recruited to the SCV and infection of cells depleted of endogenous MTMR4 results in a decrease in viable intracellular Salmonella. This reflects a significant increase in the proportion of SCVs with compromised integrity, which targets the compartment for autophagy and consequent bacterial cell death. These findings highlight the importance of PI(3)P regulation to the integrity of the SCV and reveal a novel role for the myotubularins in bacterial pathogenesis.

Chlamydia infection depends on a functional MDM2-p53 axis.

Chlamydia, a major human bacterial pathogen, assumes effective strategies to protect infected cells against death-inducing stimuli, thereby ensuring completion of its developmental cycle. Paired with its capacity to cause extensive host DNA damage, this poses a potential risk of malignant transformation, consistent with circumstantial epidemiological evidence. Here we reveal a dramatic depletion of p53, a tumor suppressor deregulated in many cancers, during Chlamydia infection. Using biochemical approaches and live imaging of individual cells, we demonstrate that p53 diminution requires phosphorylation of Murine Double Minute 2 (MDM2; a ubiquitin ligase) and subsequent interaction of phospho-MDM2 with p53 before induced proteasomal degradation. Strikingly, inhibition of the p53-MDM2 interaction is sufficient to disrupt intracellular development of Chlamydia and interferes with the pathogen's anti-apoptotic effect on host cells. This highlights the dependency of the pathogen on a functional MDM2-p53 axis and lends support to a potentially pro-carcinogenic effect of chlamydial infection.

Patched1 is required in neural crest cells for the prevention of orofacial clefts.

Defects such as cleft lip with or without cleft palate (CL/P) are among the most common craniofacial birth defects in humans. In many cases, the underlying molecular and cellular mechanisms that result in these debilitating anomalies remain largely unknown. Perturbed hedgehog (HH) signalling plays a major role in craniofacial development, and mutations in a number of pathway constituents underlie craniofacial disease. In particular, mutations in the gene encoding the major HH receptor and negative regulator, patched1 (PTCH1), are associated with both sporadic and familial forms of clefting, yet relatively little is known about how PTCH1 functions during craniofacial morphogenesis. To address this, we analysed the consequences of conditional loss of Ptch1 in mouse neural crest cell-derived facial mesenchyme. Using scanning electron microscopy (SEM) and live imaging of explanted facial primordia, we captured defective nasal pit invagination and CL in mouse embryos conditionally lacking Ptch1. Our analysis demonstrates interactions between HH and FGF signalling in the development of the upper lip, and reveals cell-autonomous and non-autonomous roles mediated by Ptch1. In particular, we show that deletion of Ptch1 in the facial mesenchyme alters cell morphology, specifically in the invaginating nasal pit epithelium. These findings highlight a critical link between the neural crest cells and olfactory epithelium in directing the morphogenesis of the mammalian lip and nose primordia. Importantly, these interactions are critically dependent on Ptch1 function for the prevention of orofacial clefts.

A Novel Type III Endosome Transmembrane Protein, TEMP.

As part of a high-throughput subcellular localisation project, the protein encoded by the RIKEN mouse cDNA 2610528J11 was expressed and identified to be associated with both endosomes and the plasma membrane. Based on this, we have assigned the name TEMP for Type III Endosome Membrane Protein. TEMP encodes a short protein of 111 amino acids with a single, alpha-helical transmembrane domain. Experimental analysis of its membrane topology demonstrated it is a Type III membrane protein with the amino-terminus in the lumenal, or extracellular region, and the carboxy-terminus in the cytoplasm. In addition to the plasma membrane TEMP was localized to Rab5 positive early endosomes, Rab5/Rab11 positive recycling endosomes but not Rab7 positive late endosomes. Video microscopy in living cells confirmed TEMP's plasma membrane localization and identified the intracellular endosome compartments to be tubulovesicular. Overexpression of TEMP resulted in the early/recycling endosomes clustering at the cell periphery that was dependent on the presence of intact microtubules. The cellular function of TEMP cannot be inferred based on bioinformatics comparison, but its cellular distribution between early/recycling endosomes and the plasma membrane suggests a role in membrane transport.

Vps26A and Vps26B subunits define distinct retromer complexes.

The trimeric Vps29-Vps35-Vps26 sub-complex of retromer mediates retrograde transport of transmembrane proteins from endosomes to the trans-Golgi network. Our group has recently identified a Vps26 paralogue, Vps26B, which is able to suppress the expression of Vps26A when exogenously expressed in mammalian cells and defines a distinct retromer complex (Vps26B-retromer) in vivo and in vitro. In this study, we use HEK293 cells stably expressing either Vps26A-myc or Vps26B-myc to address the role of retromer cargo transport and subcellular localization of the two core retromer complexes as defined by the two mammalian-specific Vps26 paralogues. Vps26B-retromer, like Vps26A-retromer, associates with TBC1D5 and GOLPH3. In contrast, no interaction between Vps26B-retromer and cation-independent mannose 6-phosphate receptor (CI-M6PR) was detected, leading to a degradation of this receptor and an increase in cathepsin D secretion. Colocalization of Vps26 paralogues with different endosomally located Rab proteins shows prolonged association of Vps26B-retromer with maturing endosomes relative to Vps26A-retromer. Interestingly, the cycling of CI-M6PR is restored upon deletion of the variable Vps26B C-terminal region indicating that this region is directly responsible for the differential function of the two paralogues. In summary, we show that the two distinct retromer complexes defined by different Vps26 paralogues are not functionally equivalent and that the Vps26B C-terminal region can control cargo selection of the Vps26B-retromer.

Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides.

Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.

The SNX-PX-BAR family in macropinocytosis: the regulation of macropinosome formation by SNX-PX-BAR proteins.

BACKGROUND: Macropinocytosis is an actin-driven endocytic process, whereby membrane ruffles fold back onto the plasma membrane to form large (>0.2 µm in diameter) endocytic organelles called macropinosomes. Relative to other endocytic pathways, little is known about the molecular mechanisms involved in macropinocytosis. Recently, members of the Sorting Nexin (SNX) family have been localized to the cell surface and early macropinosomes, and implicated in macropinosome formation. SNX-PX-BAR proteins form a subset of the SNX family and their lipid-binding (PX) and membrane-curvature sensing (BAR) domain architecture further implicates their functional involvement in macropinosome formation. METHODOLOGY/PRINCIPAL FINDINGS: We exploited the tractability of macropinosomes through image-based screening and systematic overexpression of SNX-PX-BAR proteins to quantitate their effect on macropinosome formation. SNX1 (40.9+/-3.19 macropinosomes), SNX5 (36.99+/-4.48 macropinosomes), SNX9 (37.55+/-2.4 macropinosomes), SNX18 (88.2+/-8 macropinosomes), SNX33 (65.25+/-6.95 macropinosomes) all exhibited statistically significant (p<0.05) increases in average macropinosome numbers per 100 transfected cells as compared to control cells (24.44+/-1.81 macropinosomes). SNX1, SNX5, SNX9, and SNX18 were also found to associate with early-stage macropinosomes within 5 minutes following organelle formation. The modulation of intracellular PI(3,4,5)P(3) levels through overexpression of PTEN or a lipid phosphatase-deficient mutant PTEN(G129E) was also observed to significantly reduce or elevate macropinosome formation respectively; coexpression of PTEN(G129E) with SNX9 or SNX18 synergistically elevated macropinosome formation to 119.4+/-7.13 and 91.4+/-6.37 macropinosomes respectively (p<0.05). CONCLUSIONS/SIGNIFICANCE: SNX1, SNX5, SNX9, SNX18, and SNX33 were all found to elevate macropinosome formation and (with the exception of SNX33) associate with early-stage macropinosomes. Moreover the effects of SNX9 and SNX18 overexpression in elevating macropinocytosis is likely to be synergistic with the increase in PI(3,4,5)P(3) levels, which is known to accumulate on the cell surface and early-stage macropinocytic cups. Together these findings represent the first systematic functional study into the impact of the SNX-PX-BAR family on macropinocytosis.

Inhibition of the PtdIns(5) kinase PIKfyve disrupts intracellular replication of Salmonella.

3-phosphorylated phosphoinositides (3-PtdIns) orchestrate endocytic trafficking pathways exploited by intracellular pathogens such as Salmonella to gain entry into the cell. To infect the host, Salmonellae subvert its normal macropinocytic activity, manipulating the process to generate an intracellular replicative niche. Disruption of the PtdIns(5) kinase, PIKfyve, be it by interfering mutant, siRNA-mediated knockdown or pharmacological means, inhibits the intracellular replication of Salmonella enterica serovar typhimurium in epithelial cells. Monitoring the dynamics of macropinocytosis by time-lapse 3D (4D) videomicroscopy revealed a new and essential role for PI(3,5)P(2) in macropinosome-late endosome/lysosome fusion, which is distinct from that of the small GTPase Rab7. This PI(3,5)P(2)-dependent step is required for the proper maturation of the Salmonella-containing vacuole (SCV) through the formation of Salmonella-induced filaments (SIFs) and for the engagement of the Salmonella pathogenicity island 2-encoded type 3 secretion system (SPI2-T3SS). Finally, although inhibition of PIKfyve in macrophages did inhibit Salmonella replication, it also appears to disrupt the macrophage's bactericidal response.

Trafficking, development and hedgehog.

Embryogenesis is mediated by a relatively small number of developmental signaling pathways, and the morphogens, receptors and transcription factors integral to these cascades are considered the master regulators of development. However, superimposed on this is an additional layer of control by complex intracellular trafficking networks. The importance of trafficking in controlling the processes of morphogenesis and development is highlighted by recent data regarding the transport and localisation of the morphogen sonic hedgehog (Shh) and the machinery that leads to its secretion, modification, cellular internalisation and signal transduction. Here we review the regulation of hedgehog signaling by intracellular trafficking, including the role of the primary cilium and lipids in mediating pathway activity.

Statistical and visual differentiation of subcellular imaging.

BACKGROUND: Automated microscopy technologies have led to a rapid growth in imaging data on a scale comparable to that of the genomic revolution. High throughput screens are now being performed to determine the localisation of all of proteins in a proteome. Closer to the bench, large image sets of proteins in treated and untreated cells are being captured on a daily basis to determine function and interactions. Hence there is a need for new methodologies and protocols to test for difference in subcellular imaging both to remove bias and enable throughput. Here we introduce a novel method of statistical testing, and supporting software, to give a rigorous test for difference in imaging. We also outline the key questions and steps in establishing an analysis pipeline. RESULTS: The methodology is tested on a high throughput set of images of 10 subcellular localisations, and it is shown that the localisations may be distinguished to a statistically significant degree with as few as 12 images of each. Further, subtle changes in a protein's distribution between nocodazole treated and control experiments are shown to be detectable. The effect of outlier images is also examined and it is shown that while the significance of the test may be reduced by outliers this may be compensated for by utilising more images. Finally, the test is compared to previous work and shown to be more sensitive in detecting difference. The methodology has been implemented within the iCluster system for visualising and clustering bio-image sets. CONCLUSION: The aim here is to establish a methodology and protocol for testing for difference in subcellular imaging, and to provide tools to do so. While iCluster is applicable to moderate (<1000) size image sets, the statistical test is simple to implement and will readily be adapted to high throughput pipelines to provide more sensitive discrimination of difference.

Defining macropinocytosis.

Macropinocytosis represents a distinct pathway of endocytosis in mammalian cells. This actin-driven endocytic process is not directly co-ordinated by the presence of cargo but can be induced upon activation of growth factor signalling pathways. The capacity to dissect the contribution of macropinocytosis to cellular processes has been hampered by a lack of unique molecular markers and defining features. While aspects of macropinosome formation and maturation are common to those shared by the other endocytic pathways, a number of key differences have recently begun to emerge and will be discussed in this study. It is now well established that macropinocytosis significantly contributes to antigen presentation by the immune system and is exploited by a range of pathogens for cellular invasion and avoidance of immune surveillance.

A role for SNX5 in the regulation of macropinocytosis.

BACKGROUND: The mechanisms and components that regulate macropinocytosis are poorly understood. Here we have investigated the role of sorting nexin 5 (SNX5) in the regulation of macropinocytic activity. RESULTS: SNX5 is abundantly expressed in macrophages, cells very active in macropinocytosis, and is recruited onto newly-formed macropinosomes. LPS treatment of bone marrow-derived macrophages resulted in a 2.5 fold decrease in macropinosome formation that correlates with a reduction in the levels of SNX5. To investigate the relationship between SNX5 levels and macropinocytic activity we examined the formation of macropinosomes in HEK-FlpIn cells stably expressing GFP-SNX5. Constitutive macropinocytosis was increased approximately 2 fold in HEK-GFP-SNX5 cells compared with parental HEK-FlpIn cells. Furthermore, EGF stimulation resulted in a significant increase in macropinocytosis and there was also a 2.0 fold increase in the generation of macropinosomes in HEK-GFP-SNX5 cells compared with parental HEK-FlpIn cells. SNX5, which interacts specifically with PtdIns(3)P and PtdIns(3,4)P2 through its PX domain, was recruited to regions on the plasma membrane containing EGF receptor or positive for PtdIns(3,4)P2 as detected with the PH domain of TAPP1. Treatment with AG1478, an EGF receptor specific tyrosine kinase inhibitor, prevented the recruitment of SNX5 to the cytosolic face of the plasma membrane and inhibited the formation of macropinosomes in response to EGF treatment. CONCLUSION: Based on these data, we propose that SNX5 requires the generation of phosphoinositides for recruitment to the plasma membrane and, moreover, influences the level of macropinocytic activity.

Structure of Vps26B and mapping of its interaction with the retromer protein complex.

Retromer is a heteromeric protein complex with important roles in endosomal membrane trafficking, most notably in the retrograde transport of lysosomal hydrolase receptors from endosomes to the Golgi. The core of retromer is composed of three subunits vacuolar protein sorting (Vps)35, Vps26 and Vps29, and in mammals, there are two paralogues of the medium subunit Vps26A and Vps26B. We find that both Vps26A and Vps26B bind to Vps35/Vps29 with nanomolar affinity and compete for a single-binding site to define distinct retromer complexes in vitro and in vivo. We have determined the crystal structure of mouse Vps26B and compare this structure with that of Vps26A. Vps26 proteins have a striking similarity to the arrestin family of proteins that regulate the signalling and endocytosis of G-protein-coupled receptors, although we observe that surface residues involved in arrestin function are not conserved in Vps26. Using structure-based mutagenesis, we show that both Vps26A and Vps26B are incorporated into retromer complexes through binding of Vps35 to a highly conserved surface patch within the C-terminal subdomain and that this interaction is required for endosomal recruitment of the proteins.

EGF induces macropinocytosis and SNX1-modulated recycling of E-cadherin.

In epithelia, junction proteins are endocytosed for modulation of cell-cell adhesion and cell polarity. In response to growth factors, the cell-cell adhesion protein E-cadherin is internalized from the cell surface with degradation or recycling as potential fates. However, the cellular machinery involved in cadherin internalization and recycling remains controversial. Here we investigated EGF-induced E-cadherin internalization. EGF stimulation of MCF-7 cells resulted in Rac1-modulated macropinocytosis of the E-cadherin-catenin complex into endosomal compartments that colocalized with EEA1 and the sorting nexin, SNX1. Depletion of cellular SNX1 levels by siRNA resulted in increased intracellular accumulation and turnover of E-cadherin internalized from the cell surface in response to EGF. Moreover, SNX1 was also required for efficient recycling of internalized E-cadherin and re-establishment of epithelial adhesion. Together, these findings demonstrate a role for SNX1 in retrieval of E-cadherin from a degradative endosomal pathway and in membrane trafficking pathways that regulate E-cadherin recycling.

Analyzing real-time video microscopy: the dynamics and geometry of vesicles and tubules in endocytosis.

With the advent of live cell imaging microscopy, new types of mathematical analyses and measurements are possible. Many of the real-time movies of cellular processes are visually very compelling, but elementary analysis of changes over time of quantities such as surface area and volume often show that there is more to the data than meets the eye. This unit outlines a geometric modeling methodology and applies it to tubulation of vesicles during endocytosis. Using these principles, it has been possible to build better qualitative and quantitative understandings of the systems observed, as well as to make predictions about quantities such as ligand or solute concentration, vesicle pH, and membrane trafficked. The purpose is to outline a methodology for analyzing real-time movies that has led to a greater appreciation of the changes that are occurring during the time frame of the real-time video microscopy and how additional quantitative measurements allow for further hypotheses to be generated and tested.