Axial protocadherin is a mediator of prenotochord cell sorting in Xenopus

Prenotochord cell sorting is regarded as one of the first cell sorting events in early chordate development. We recently demonstrated that this sorting event occurs in vitro, although the mediator of this activity remains unidentified. Herein, we report the isolation of a full-length cDNA clone of Axial protocadherin (AXPC), the homologue of human protocadherin-1 (PCD1). AXPC encodes a transmembrane protein (AXPC) that is expressed exclusively in the notochord at the neurula stage and in the pronephros, somites, heart, optic vesicle, otic vesicle, and distinct parts of the brain at the tailbud stage. Cell dissociation and reaggregation assays and in vivo microinjection experiments demonstrated that cells overexpressing a membrane-tethered form of AXPC (MT-AXPC) acquired the same adhesive properties as prenotochord cells. Moreover, microinjection of either mRNA encoding the dominant negative form of AXPC (DN-AXPC) or morpholino oligonucleotides interferes with the sorting activity of prenotochord cells and normal axis formation. This study suggests that AXPC is necessary and sufficient for prenotochord cell sorting in the gastrulating embryo, and may also mediate sorting events later in development.     

Paraxial protocadherin mediates cell sorting and tissue morphogenesis by regulating C-cadherin adhesion activity

Little is known about how protocadherins function in cell adhesion and tissue development. Paraxial protocadherin (PAPC) controls cell sorting and morphogenetic movements in the Xenopus laevis embryo. We find that PAPC mediates these functions by down-regulating the adhesion activity of C-cadherin. Expression of exogenous C-cadherin reverses PAPC-induced cell sorting and gastrulation defects. Moreover, loss of endogenous PAPC results in elevated C-cadherin adhesion activity in the dorsal mesoderm and interferes with the normal blastopore closure, a defect that can be rescued by a dominant-negative C-cadherin mutant. Importantly, activin induces PAPC expression, and PAPC is required for activin-induced regulation of C-cadherin adhesion activity and explant morphogenesis. Signaling through Frizzled-7 is not required for PAPC regulation of C-cadherin, suggesting that C-cadherin regulation and Frizzled-7 signaling are two distinct branches of the PAPC pathway that induce morphogenetic movements. Thus, spatial regulation of classical cadherin adhesive function by local expression of a protocadherin is a novel mechanism for controlling cell sorting and tissue morphogenesis.     

Mouse paraxial protocadherin is expressed in trunk mesoderm and is not essential for mouse development

Paraxial protocadherin (PAPC) is a cell adhesion molecule that marks cells undergoing convergence-extension cell movements in Xenopus and zebrafish gastrulating embryos. Here a mouse homologue (mpapc) was identified and characterized. During early- to mid-gastrulation, mpapc is expressed in the primitive streak as the trunk mesoderm undergoes morphogenetic cell movements. At head-fold stage mpapc expression becomes localized to paraxial regions in which somites are formed in the segmental plate. At later stages, mpapc displays a complex expression pattern in cerebral cortex, olfactory bulb, inferior colliculus, and in longitudinal stripes in hindbrain. To analyze the effect of the loss of PAPC function during mouse development, a null allele of the mouse papc gene was generated. Homozygous animals show no defects in their skeleton and are viable and fertile.     

Sodium-dependent neurotransmitter transporters: oligomerization as a determinant of transporter function and trafficking

Constitutive oligomer formation appears to be the rule for the neurotransmitter:sodium symporter (NSS) family of proteins. The propensity to form oligomers is a prerequisite for NSS proteins to pass the rigid mechanisms of quality control in the endoplasmic reticulum. Moreover, recent findings suggest that correct trafficking to the plasma membrane appears to rely on the interaction of NSS homo-oligomers with components of the COPII-vesicle machinery. The transporters present at the plasma membrane are most likely organized in a tetrameric arrangement, as a dimer of dimers. In this review, we will address ongoing efforts to unravel the underlying mechanisms of oligomer formation at the molecular and cellular levels, and we will discuss oligomerization in terms of transporter function.     

The sorting and trafficking of lysosomal proteins

For a long time lysosomes were considered terminal organelles involved in the degradation of different substrates. However, this view is rapidly changing by evidence demonstrating that these organelles and their content display specialized functions in addition to the degradation of substances. Many lysosomal proteins have been implicated in specialized cellular functions and disorders such as antigen processing, targeting of surfactant proteins, and most lysosomal storage disorders. To date, about fifty lysosomal hydrolases have been identified, and the majority of them are targeted to the lysosomes via the mannose-6-phosphate receptor (M6P-Rc). However, recent studies on the intracellular trafficking of the non-enzymic lysosomal proteins prosaposin and GM2 activator (GM2AP) demonstrated that they use an alternative receptor termed "sortilin". Existing evidence suggests that some hydrolases traffic to the lysosomes in a mannose 6-phophate-indepentend manner. The possibility that sortilin is implicated in the targeting of some soluble hydrolases, as well as the consequences of this process, is addressed in the present review.     

The N-terminal and transmembrane domains of Commissureless are necessary for its function and trafficking within neurons

Commissureless (Comm) is a novel transmembrane molecule necessary both for commissural axons to cross the midline of the Drosophila central nervous system and normal synaptogenesis. Comm is able to reduce cell surface levels of Roundabout (Robo), a receptor for the midline repellent Slit, on commissural axons and unknown inhibitors of synaptogenesis expressed on muscle cells. Comm is expressed dynamically and is found at the cell surface and within intracellular vesicles. Comm can bind Robo and when the proteins are co-expressed Robo is found co-localised with Comm intracellularly. Here we show that the ability of Comm to localise intracellularly and hence regulate Robo surface levels requires sequences in both the N-terminal and transmembrane domains. We also show that Comm can dimerise via its N-terminal domain. Furthermore, absence of the Comm N-terminal and transmembrane regions results in the protein being restricted to the neuron soma.     

Functions of sorting nexin 17 domains and recognition motif for P-selectin trafficking

SNX17 is a member of the sorting nexin family (SNX), a group of hydrophilic proteins whose common characteristic property is a phox homology (PX) domain. The PX domain directs SNXs to phosphatidylinositides containing membranes of the endosomal compartment, where the SNXs are involved in the sorting of transmembrane proteins. SNX17 is known to interact with P-selectin and the LDL receptor family. Here, we report that the PX domain of SNX17 specifically binds to phosphatidylinositol 3-phosphate-containing membranes. The functional part of SNX17 that binds P-selectin or Patched (PTCH) consists of a truncated FERM domain and a unique C terminus together (FC-unit). In a yeast two-hybrid analysis a putative recognition motif for the FC-unit was revealed within P-selectin as FxNaa(F/Y). When HepG2 cells overexpress P-selectin together with SNX17, SNX17 changes its distribution from early endosomes to lysobisphosphatidic acid-containing late endosomes. Furthermore, overexpressed SNX17 restrains P-selectin in the outer membrane of the late endosomal compartment, thus preventing the normal lysosomal accumulation of P-selectin. These results suggest that the PX domain is necessary for the intracellular localisation, while the FC-unit is required for cargo recognition. We hypothesise that the expression level of SNX17 may regulate the lysosomal degradation, at least for P-selectin, by suppressing its entry into the inner vesicles of the multi-vesicular bodies (MVBs).     

Structural remodeling, trafficking and functions of glycosylphosphatidylinositol-anchored proteins

Glycosylphosphatidylinositol (GPI) is a glycolipid that is covalently attached to proteins as a post-translational modification. Such modification leads to the anchoring of the protein to the outer leaflet of the plasma membrane. Proteins that are decorated with GPIs have unique properties in terms of their physical nature. In particular, these proteins tend to accumulate in lipid rafts, which are critical for the functions and trafficking of GPI-anchored proteins (GPI-APs). Recent studies mainly using mutant cells revealed that various structural remodeling reactions occur to GPIs present in GPI-APs as they are transported from the endoplasmic reticulum to the cell surface. This review examines the recent progress describing the mechanisms of structural remodeling of mammalian GPI-anchors, such as inositol deacylation, glycan remodeling and fatty acid remodeling, with particular focus on their trafficking and functions, as well as the pathogenesis involving GPI-APs and their deficiency.     

Protein oligomerization modulates raft partitioning and apical sorting of GPI-anchored proteins

An essential but insufficient step for apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) in epithelial cells is their association with detergent-resistant microdomains (DRMs) or rafts. In this paper, we show that in MDCK cells both apical and basolateral GPI-APs associate with DRMs during their biosynthesis. However, only apical and not basolateral GPI-APs are able to oligomerize into high molecular weight complexes. Protein oligomerization begins in the medial Golgi, concomitantly with DRM association, and is dependent on protein-protein interactions. Impairment of oligomerization leads to protein missorting. We propose that oligomerization stabilizes GPI-APs into rafts and that this additional step is required for apical sorting of GPI-APs. Two alternative apical sorting models are presented.     

Regulation of opioid receptor trafficking and morphine tolerance by receptor oligomerization

The utility of morphine for the treatment of chronic pain is hindered by the development of tolerance to the analgesic effects of the drug. Morphine is unique among opiates in its ability to activate the mu opioid receptor (MOR) without promoting its desensitization and endocytosis. Here we demonstrate that [D-Ala(2)-MePhe(4)-Gly(5)-ol] enkephalin (DAMGO) can facilitate the ability of morphine to stimulate MOR endocytosis. As a consequence, rats treated chronically with both drugs show reduced analgesic tolerance compared to rats treated with morphine alone. These results demonstrate that endocytosis of the MOR can reduce the development of tolerance, and hence suggest an approach for the development of opiate analogs with enhanced efficacy for the treatment of chronic pain.     

Lipid raft association of carboxypeptidase E is necessary for its function as a regulated secretory pathway sorting receptor

Membrane carboxypeptidase E (CPE) is a sorting receptor for targeting prohormones, such as pro-opiomelanocortin, to the regulated secretory pathway in endocrine cells. Its membrane association is necessary for it to bind a prohormone sorting signal at the trans-Golgi network (TGN) to facilitate targeting. In this study, we examined the lipid interaction of CPE in bovine pituitary secretory granule membranes, which are derived from the TGN. We show that CPE is associated with detergent-resistant lipid domains, or rafts, within secretory granule membranes. Lipid analysis revealed that these rafts are enriched in glycosphingolipids and cholesterol. Pulse-chase and subcellular fractionation experiments in AtT-20 cells show that the association of CPE with membrane rafts occurred only after it reached the Golgi. Cholesterol depletion resulted in dissociation of CPE from secretory granule membranes and decreased the binding of prohormones to membranes. In vivo cholesterol depletion using lovastatin resulted in the lack of sorting of CPE and its cargo to the regulated secretory pathway. We propose that the sorting receptor function of CPE necessitates its interaction with glycosphingolipid-cholesterol rafts at the TGN, thereby anchoring it in position to bind to its prohormone cargo.     

Enumeration of the simian virus 40 early region elements necessary for human cell transformation

While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.     

Differential beta-arrestin trafficking and endosomal sorting of somatostatin receptor subtypes

The physiological responses of somatostatin are mediated by five different G protein-coupled receptors. Although agonist-induced endocytosis of the various somatostatin receptor subtypes (sst(1)-sst(5)) has been studied in detail, little is known about their postendocytic trafficking. Here we show that somatostatin receptors profoundly differ in patterns of beta-arrestin mobilization and endosomal sorting. The beta-arrestin-dependent trafficking of the sst(2A) somatostatin receptor resembled that of a class B receptor in that upon receptor activation, beta-arrestin and the receptor formed stable complexes and internalized together into the same endocytic vesicles. This pattern was dependent on GRK2 (G protein-coupled receptor kinase 2)-mediated phosphorylation of a cluster of phosphate acceptor sites within the cytoplasmic tail of the sst(2A) receptor. Unlike other class B receptors, however, the sst(2A) receptor was rapidly resensitized and recycled to the plasma membrane. The beta-arrestin mobilization of the sst(3) and the sst(5) somatostatin receptors resembled that of a class A receptor in that upon receptor activation, beta-arrestin and the receptor formed relatively unstable complexes that dissociated at or near the plasma membrane. Consequently, beta-arrestin was excluded from sst(3)-containing vesicles. Unlike other class A receptors, a large proportion of sst(3) receptors was subject to ubiquitin-dependent lysosomal degradation and did not rapidly recycle to the plasma membrane. The sst(4) somatostatin receptor is unique in that it did not exhibit agonist-dependent receptor phosphorylation and beta-arrestin recruitment. Together, these findings may provide important clues about the regulation of receptor responsiveness during long-term administration of somatostatin analogs.     

Mechanisms of procollagen and HSP47 sorting during ER-to-Golgi trafficking

Efficient quality control and export of procollagen from the cell is crucial for extracellular matrix homeostasis, yet it is still incompletely understood. One of the debated questions is the role of a collagen-specific ER chaperone HSP47 in these processes. Most ER chaperones preferentially bind to unfolded polypeptide chains, enabling selective export of natively folded proteins from the ER after chaperone release. In contrast, HSP47 preferentially binds to the natively folded procollagen and is believed to be released only in the ER-Golgi intermediate compartment (ERGIC) or cis-Golgi. HSP47 colocalization with procollagen in punctate structures observed by immunofluorescence imaging of fixed cells has thus been interpreted as evidence for HSP47 export from the ER together with procollagen in transport vesicles destined for ERGIC or Golgi. To understand the mechanism of this co-trafficking and its physiological significance, we imaged the dynamics of fluorescently tagged type I procollagen and HSP47 punctate structures in live MC3T3 murine osteoblasts with up to 120 nm spatial and 500 ms time resolution. Contrary to the prevailing model, we discovered that most bona fide carriers delivering procollagen from ER exit sites (ERESs) to Golgi contained no HSP47, unless the RDEL signal for ER retention in HSP47 was deleted or mutated. These transport intermediates exhibited characteristic rapid, directional motion along microtubules, while puncta with colocalized HSP47 and procollagen similar to the ones described before had only limited, stochastic motion. Live cell imaging and fluorescence recovery after photobleaching revealed that the latter puncta (including the ones induced by ARF1 inhibition) were dilated regions of ER lumen, ERESs, or autophagic structures surrounded by lysosomal membranes. Procollagen was colocalized with HSP47 and ERGIC53 at ERESs. It was colocalized with ERGIC53 but not HSP47 in Golgi-bound transport intermediates. Our results suggest that procollagen and HSP47 sorting occurs at ERES before procollagen is exported from the ER in Golgi-bound transport intermediates, providing new insights into mechanisms of procollagen trafficking.     

Multicolour imaging of post-Golgi sorting and trafficking in live cells

The biogenesis and maintenance of asymmetry is crucial to many cellular functions including absorption and secretion, signalling, development and morphogenesis. Here we have directly visualized the segregation and trafficking of apical (glycosyl phosphatidyl inositol-anchored) and basolateral (vesicular stomatitis virus glycoprotein) cargo in living cells using multicolour imaging of green fluorescent protein variants. Apical and basolateral cargo segregate progressively into large domains in Golgi/trans-Golgi network structures, exclude resident proteins, and exit in separate transport containers. These remain distinct and do not merge with endocytic structures suggesting that lateral segregation in the trans-Golgi network is the primary sorting event. Fusion with the plasma membrane was detected by total internal reflection microscopy and reveals differences between apical and basolateral carriers as well as new 'hot spots' for exocytosis.     

Structural basis of Vta1 function in the multivesicular body sorting pathway

The MVB pathway plays essential roles in several eukaryotic cellular processes. Proper function of the MVB pathway requires reversible membrane association of the ESCRTs, a process catalyzed by Vps4 ATPase. Vta1 regulates the Vps4 activity, but its mechanism of action was poorly understood. We report the high-resolution crystal structures of the Did2- and Vps60-binding N-terminal domain and the Vps4-binding C-terminal domain of S. cerevisiae Vta1. The C-terminal domain also mediates Vta1 dimerization and both subunits are required for its function as a Vps4 regulator. Emerging from our analysis is a mechanism of regulation by Vta1 in which the C-terminal domain stabilizes the ATP-dependent double ring assembly of Vps4. In addition, the MIT motif-containing N-terminal domain, projected by a long disordered linker, allows contact between the Vps4 disassembly machinery and the accessory ESCRT-III proteins. This provides an additional level of regulation and coordination for ESCRT-III assembly and disassembly.     

Overexpression of a novel sorting nexin, SNX15, affects endosome morphology and protein trafficking

Sorting nexin (SNX) 15 is a novel member of the SNX family of proteins. Although the functions of most SNXs have not yet been determined, several family members (e.g., SNX1, SNX2, SNX3, and SNX8) are orthologs of yeast proteins involved in protein trafficking. Overexpression of myc-tagged SNX15 in COS-7 cells altered the morphology of several endosomal compartments. In transient transfection experiments, myc-SNX15 was first seen in small punctate spots and small ring structures. Later, myc-SNX15 was found in larger rings. Finally, myc-SNX15 was observed in large, amorphous membrane-limited structures. These structures contained proteins from lysosomes, late endosomes, early endosomes, and the trans-Golgi network. However, the morphology of the endoplasmic reticulum and Golgi was not affected by overexpression of myc-SNX15. In myc-SNX15-overexpressing cells, the endocytosis of transferrin was severely inhibited and endocytosis of tac-trans-Golgi network (TGN) 38 and tac-furin was slowed. In addition, the recycling of internalized tac-TGN38 and tac-furin was also inhibited. Both the morphological and biochemical data indicate that SNX15 plays a crucial role in trafficking through the endocytic pathway. This is the first demonstration that a mammalian SNX protein is involved in protein trafficking.     

Amyloid precursor protein trafficking, processing, and function

Intracellular trafficking and proteolytic processing of amyloid precursor protein (APP) have been the focus of numerous investigations over the past two decades. APP is the precursor to the amyloid beta-protein (Abeta), the 38-43-amino acid residue peptide that is at the heart of the amyloid cascade hypothesis of Alzheimer disease (AD). Tremendous progress has been made since the initial identification of Abeta as the principal component of brain senile plaques of individuals with AD. Specifically, molecular characterization of the secretases involved in Abeta production has facilitated cell biological investigations on APP processing and advanced efforts to model AD pathogenesis in animal models. This minireview summarizes salient features of APP trafficking and amyloidogenic processing and discusses the putative biological functions of APP.