Markus C. Kerr

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.

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.

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.

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)P2 in macropinosome‐late endosome/lysosome fusion, which is distinct from that of the small GTPase Rab7. This PI(3,5)P2‐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 macrophages bactericidal response.

Sorting nexin 5 is localized to a subdomain of the early endosomes and is recruited to the plasma membrane following EGF stimulation

Sorting nexins are a large family of proteins that contain the phosphoinositide-binding Phox homology (PX) domain. A number of sorting nexins are known to bind to PtdIns(3)P, which mediates their localization to membranes of the endocytic pathway. We show here that sorting nexin 5 (SNX5) can be recruited to two distinct membrane compartments. In non-stimulated cells, the PX domain was independently targeted to endosomal structures and colocalized with full-length SNX5. The membrane binding of the PX domain was inhibited by the PI 3-kinase inhibitor, wortmannin. Although SNX5 colocalized with a fluid-phase marker and was found predominantly within a PtdIns(3)P-rich endosomal domain, very little colocalization was observed between SNX5 and the PtdIns(3)P-binding protein, EEA1. Using liposome-based binding assays, we have shown that the PX domain of SNX5 interacts not only with PtdIns(3)P but also with PtdIns(3,4)P2. In response to EGF stimulation, either the SNX5-PX domain or full-length SNX5 was rapidly recruited to the plasma membrane. The localization of SNX1, which does not bind PtdIns(3,4)P2, was unaffected by EGF signalling. Therefore, SNX5 is localized to a subdomain of the early endosome distinct from EEA1 and, following EGF stimulation and elevation of PtdIns(3,4)P2, is also transiently recruited to the plasma membrane. These results indicate that SNX5 may have functions not only associated with endosomal sorting but also with the phosphoinositide-signalling pathway.

A Novel Hook-Related Protein Family and the Characterization of Hook-Related Protein 1

The spatial organization of organelles within a cell is dependent on microtubules. Recently, members of the Hook family of proteins have been proposed to function in linking organelles to microtubules. We report the identification of a completely novel protein family, the Hook‐related protein (HkRP) family, from which the Hook proteins have diverged. Bioinformatic analysis of the HkRP family revealed several conserved domains, including a unique C‐terminal HkRP domain. The central region of each protein is comprised of an extensive coiled‐coil domain, and the N‐terminus contains a putative microtubule‐binding domain. This domain has been shown to bind microtubules in the Hook protein and show that the HkRP1 protein is microtubule‐associated. While endogenous HkRP1 has no distinct organelle association, expression of the C‐terminal membrane‐binding domain suggests a function of the HkRP1 in early endosome. Ultrastructural studies reveal that expression of the C‐terminal HkRP1 domain causes an accumulation of internal membranes with an electron‐dense coat. Co‐localization studies show a concomitant redistribution of the early endosome marker sorting‐nexin 1 but not the early endosome antigen‐1 (EEA1). The steady‐state distribution of the epidermal growth factor receptor is also specifically disrupted by expression of the C‐terminal domain. We propose that HkRP1 is involved in the process of tubulation of sorting nexin‐1 positive membranes from early endosome subdomains.

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.

A novel mammalian retromer component, Vps26B.

The mammalian retromer protein complex, which consists of three proteins – Vps26, Vps29, and Vps35 – in association with members of the sorting nexin family of proteins, has been implicated in the trafficking of receptors and their ligands within the endosomal/lysosomal system of mammalian cells. A bioinformatic analysis of the mouse genome identified an additional transcribed paralog of the Vps26 retromer protein, which we termed Vps26B. No paralogs were identified for Vps29 and Vps35. Phylogenetic studies indicate that the two paralogs of Vps26 become evident after the evolution of the chordates. We propose that the chordate Vps26‐like gene published previously be renamed Vps26A to differentiate it from Vps26B. As for Vps26A, biochemical characterization of Vps26B established that this novel 336 amino acid residue protein is a peripheral membrane protein. Vps26B co‐precipitated with Vps35 from transfected cells and the direct interaction between these two proteins was confirmed by yeast 2‐hybrid analysis, thereby establishing Vps26B as a subunit of the retromer complex. Within HeLa cells, Vps26B was found in the cytoplasm with low levels at the plasma membrane, while Vps26A was predominantly associated with endosomal membranes. Within A549 cells, both Vps26A and Vps26B co‐localized with actin‐rich lamellipodia at the cell surface. These structures also co‐localized with Vps35. Total internal reflection fluorescence microscopy confirmed the association of Vps26B with the plasma membrane in a stable HEK293 cell line expressing cyan fluorescent protein (CFP)‐Vps26B. Based on these observations, we propose that the mammalian retromer complex is located at both endosomes and the plasma membrane in some cell types.

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.

A role for SNX5 in the regulation of macropinocytosis

BackgroundThe 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.ResultsSNX5 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 ~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.ConclusionBased 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.