A Cytohesin Homolog in Dictyostelium Amoebae

Background

Dictyostelium, an amoeboid motile cell, harbors several paralogous Sec7 genes that encode members of three distinct subfamilies of the Sec7 superfamily of Guanine nucleotide exchange factors. Among them are proteins of the GBF/BIG family present in all eukaryotes. The third subfamily represented with three members in D. discoideum is the cytohesin family that has been thought to be metazoan specific. Cytohesins are characterized by a Sec7 PH tandem domain and have roles in cell adhesion and migration.

Principal Findings

Dictyostelium SecG exhibits highest homologies to the cytohesins. It harbors at its amino terminus several ankyrin repeats that are followed by the Sec7 PH tandem domain. Mutants lacking SecG show reduced cell-substratum adhesion whereas cell-cell adhesion that is important for development is not affected. Accordingly, multicellular development proceeds normally in the mutant. During chemotaxis secG⁻ cells elongate and migrate in a directed fashion towards cAMP, however speed is moderately reduced.

Significance

The data indicate that SecG is a relevant factor for cell-substrate adhesion and reveal the basic function of a cytohesin in a lower eukaryote.     

Dictyostelium, a microbial model for brain disease

Background

Most neurodegenerative diseases are associated with mitochondrial dysfunction. In humans, mutations in mitochondrial genes result in a range of phenotypic outcomes which do not correlate well with the underlying genetic cause. Other neurodegenerative diseases are caused by mutations that affect the function and trafficking of lysosomes, endosomes and autophagosomes. Many of the complexities of these human diseases can be avoided by studying them in the simple eukaryotic model Dictyostelium discoideum.

Scope of review

This review describes research using Dictyostelium to study cytopathological pathways underlying a variety of neurodegenerative diseases including mitochondrial, lysosomal and vesicle trafficking disorders.

Major conclusions

Generalised mitochondrial respiratory deficiencies in Dictyostelium produce a consistent pattern of defective phenotypes that are caused by chronic activation of a cellular energy sensor AMPK (AMP-activated protein kinase) and not ATP deficiency per se. Surprisingly, when individual subunits of Complex I are knocked out, both AMPK-dependent and AMPK-independent, subunit-specific phenotypes are observed. Many nonmitochondrial proteins associated with neurological disorders have homologues in Dictyostelium and are associated with the function and trafficking of lysosomes and endosomes. Conversely, some genes associated with neurodegenerative disorders do not have homologues in Dictyostelium and this provides a unique avenue for studying these mutated proteins in the absence of endogeneous protein.

General significance

Using the Dictyostelium model we have gained insights into the sublethal cytopathological pathways whose dysregulation contributes to phenotypic outcomes in neurodegenerative disease. This work is beginning to distinguish correlation, cause and effect in the complex network of cross talk between the various organelles involved. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research     

Structure of an Arrestin2-Clathrin Complex Reveals a Novel Clathrin Binding Domain That Modulates Receptor Trafficking

Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin β-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formed by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I)₂GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.Many transmembrane signaling systems consist of specific G protein-coupled receptors (GPCRs)³ that transduce a diverse array of extracellular stimuli into intracellular signaling events (1). GPCRs modulate the activity of numerous effector molecules and regulate multiple biological functions including neurotransmission, sensory perception, cardiovascular function, development, and cell growth and differentiation (2). To ensure that extracellular stimuli are translated into intracellular signals of appropriate magnitude and duration, these signaling cascades are tightly regulated. GPCRs are subject to three principle modes of regulation; 1) desensitization, in which a receptor becomes refractory to continued stimuli; 2) endocytosis, where receptors are removed from the cell surface; 3) down-regulation, where total receptor levels are decreased (3, 4). Agonist-dependent regulation is primarily mediated by GPCR kinases that specifically phosphorylate activated GPCRs and initiate the recruitment of arrestins. Arrestins are divided into two major classes, visual and non-visual, based on their localization and function. The non-visual arrestins, arrestin2 and 3 (also termed β-arrestin1 and -2, respectively), are broadly distributed and function in multiple processes including GPCR desensitization, trafficking, and signaling (4–6).Initial structural insight on arrestins was provided by the x-ray crystal structure of bovine arrestin1 (7, 8), whereas the crystal structures of C-terminal-truncated (9) and wild type (10) bovine arrestin2 and salamander arrestin4 (11) have also been solved. In general, arrestins are composed of two major domains made up of β strands and connecting loops that are held together by a polar core region consisting of buried salt bridges. It has been proposed that arrestins adopt an active conformation upon binding to phosphorylated receptors, which disrupts the polar core resulting in the release of the C-terminal tail (12). Disruption of the polar core by point mutation of Arg-169 generates a constitutively active arrestin2, which mimics the active state. This mutated arrestin binds to the β₂-adrenergic receptor (β₂AR) in a phosphorylation-independent manner, induces internalization of a δ-opioid receptor lacking phosphorylation sites (13), and has increased binding to clathrin and AP-2 (14).A role for non-visual arrestins in GPCR endocytosis was first described for the β₂AR (15, 16), although it is now evident that arrestins regulate the trafficking of multiple GPCRs as well as additional classes of receptors (4). An early step in this process involves arrestin binding to an activated phosphorylated receptor that enhances arrestin interaction with the endocytic proteins, clathrin, and AP-2 (16, 17). An additional important step in this process involves arrestin interaction with phosphoinositides such as phosphatidylinositol diphosphate and trisphosphate (18). Although the dynamics of these interactions have not been studied, arrestin2 and -3 have been shown to interact specifically and stoichiometrically with clathrin (16). Furthermore, fluorescence microscopy reveals that activated β₂AR, arrestin2, clathrin, and AP-2 all colocalize upon receptor stimulation (16). The primary clathrin binding determinant in arrestin2, LIELD, spans residues 376–380 and is located in an extended disordered loop that immediately precedes the final C-terminal β-strand (10, 19). This region, the clathrin binding box, is consistent with a consensus motif, LϕXϕ(D/E) (where ϕ is a bulky hydrophobic residue, and X represents any polar amino acid), established in other clathrin-binding proteins including AP-2 (20), AP180 (21), amphiphysin (22), and epsin (23). Importantly, the mutation of this motif in arrestin3 and its deletion in arrestin2 significantly disrupts clathrin binding and receptor endocytosis (14, 19). A mutagenesis study of clathrin localized an arrestin binding site to the N-terminal domain of the clathrin heavy chain, specifically residues Glu-89, Lys-96, and Lys-98 (24). Moreover, a crystal structure of clathrin-(1–363) in complex with an arrestin3 peptide (residues 369–381) supports the mutagenesis data and the predicted location of the arrestin-clathrin interaction site (25).To further elucidate the mechanisms involved in mediating arrestin/clathrin interaction, we have determined the crystal structure of clathrin with the short (arrestin2S) and long (arrestin2L) isoforms of arrestin2, which differ by an 8-amino acid insert between β strands 18 and 19 (26). Our results identify an additional and unique interaction encoded in the arrestin2L isoform that is distinct from the previously well characterized interaction involving the LϕXϕ(D/E) motif. Specifically, we observe that the 8 amino acid splice loop in arrestin2L interacts with a pocket formed by blades 4 and 5 in clathrin. Biochemical and cell biological analysis confirm a role for both binding sites in arrestin2L/clathrin interaction and demonstrate an essential role of these interactions in arrestin-mediated GPCR endocytosis.     

Transcriptome-wide modulation of splicing by the exon junction complex

Background

The exon junction complex (EJC) is a dynamic multi-protein complex deposited onto nuclear spliced mRNAs upstream of exon-exon junctions. The four core proteins, eIF4A3, Magoh, Y14 and MLN51, are stably bound to mRNAs during their lifecycle, serving as a binding platform for other nuclear and cytoplasmic proteins. Recent evidence has shown that the EJC is involved in the splicing regulation of some specific events in both Drosophila and mammalian cells.

Results

Here, we show that knockdown of EJC core proteins causes widespread alternative splicing changes in mammalian cells. These splicing changes are specific to EJC core proteins, as knockdown of eIF4A3, Y14 and MLN51 shows similar splicing changes, and are different from knockdown of other splicing factors. The splicing changes can be rescued by a siRNA-resistant form of eIF4A3, indicating an involvement of EJC core proteins in regulating alternative splicing. Finally, we find that the splicing changes are linked with RNA polymerase II elongation rates.

Conclusion

Taken together, this study reveals that the coupling between EJC proteins and splicing is broader than previously suspected, and that a possible link exists between mRNP assembly and splice site recognition.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0551-7) contains supplementary material, which is available to authorized users.     

Identification and Evolution of FYVE Domain-Containing Proteins and Their Expression Patterns in Response to Abiotic Stresses in Rice

The FYVE domain is a typical zinc finger motif containing four conserved CxxC pairs and has been shown to specifically binds to PtdIns(3)P on the surface of cell membrane. FYVE domain-containing proteins are commonly distributed in eukaryotic cells and have been implicated in diverse functions like signal transduction, membrane trafficking, exocytosis and endocytosis, phosphoinositides (PIs) metabolism, and cytoskeletal regulation. Analysis of the rice genome using comprehensive online databases and research tools resulted in the identification of 19 putative rice FYVE (OsFVYE) proteins. Based on domain architectural and phylogenetic analyses, these OsFYVEs were further classified into six groups. Groups I–V were conserved in Arabidopsis (dicots) and rice (monocot) at both the genetic and protein levels, while group VI was widely present in plants but truncated in Arabidopsis and species of Brassicaceae. Comprehensive and comparative investigation of their expression profiles showed that FYVE genes in plant exhibited a variety of expression patterns during different developmental stages and in response to phytohormones and abiotic stresses. These findings indicated that both OsFYVE and AtFYVE genes may played potential roles in normal plant growth, hormone signal transduction, and abiotic stress tolerance. Results from our study shed light on the potential roles of FYVE proteins in plant growth, development, and stress responses.     

Ordered Patterns of Cell Shape and Orientational Correlation during Spontaneous Cell Migration

Background

In the absence of stimuli, most motile eukaryotic cells move by spontaneously coordinating cell deformation with cell movement in the absence of stimuli. Yet little is known about how cells change their own shape and how cells coordinate the deformation and movement. Here, we investigated the mechanism of spontaneous cell migration by using computational analyses.

Methodology

We observed spontaneously migrating Dictyostelium cells in both a vegetative state (round cell shape and slow motion) and starved one (elongated cell shape and fast motion). We then extracted regular patterns of morphological dynamics and the pattern-dependent systematic coordination with filamentous actin (F-actin) and cell movement by statistical dynamic analyses.

Conclusions/Significance

We found that Dictyostelium cells in both vegetative and starved states commonly organize their own shape into three ordered patterns, elongation, rotation, and oscillation, in the absence of external stimuli. Further, cells inactivated for PI3-kinase (PI3K) and/or PTEN did not show ordered patterns due to the lack of spatial control in pseudopodial formation in both the vegetative and starved states. We also found that spontaneous polarization was achieved in starved cells by asymmetric localization of PTEN and F-actin. This breaking of the symmetry of protein localization maintained the leading edge and considerably enhanced the persistence of directed migration, and overall random exploration was ensured by switching among the different ordered patterns. Our findings suggest that Dictyostelium cells spontaneously create the ordered patterns of cell shape mediated by PI3K/PTEN/F-actin and control the direction of cell movement by coordination with these patterns even in the absence of external stimuli.     

FYVE1 Is Essential for Vacuole Biogenesis and Intracellular Trafficking in Arabidopsis

The plant vacuole is a central organelle that is involved in various biological processes throughout the plant life cycle. Elucidating the mechanism of vacuole biogenesis and maintenance is thus the basis for our understanding of these processes. Proper formation of the vacuole has been shown to depend on the intracellular membrane trafficking pathway. Although several mutants with altered vacuole morphology have been characterized in the past, the molecular basis for plant vacuole biogenesis has yet to be fully elucidated. With the aim to identify key factors that are essential for vacuole biogenesis, we performed a forward genetics screen in Arabidopsis (Arabidopsis thaliana) and isolated mutants with altered vacuole morphology. The vacuolar fusion defective1 (vfd1) mutant shows seedling lethality and defects in central vacuole formation. VFD1 encodes a Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing protein, FYVE1, that has been implicated in intracellular trafficking. FYVE1 localizes on late endosomes and interacts with Src homology-3 domain-containing proteins. Mutants of FYVE1 are defective in ubiquitin-mediated protein degradation, vacuolar transport, and autophagy. Altogether, our results show that FYVE1 is essential for plant growth and development and place FYVE1 as a key regulator of intracellular trafficking and vacuole biogenesis.The plant vacuole is the largest organelle in a plant cell in which proteins, metabolites, and ions can be stored or sequestered. The vacuole is essential for plant development and growth and is directly or indirectly involved in various biotic and abiotic stress responses (Zhang et al., 2014). The vacuole is also the central organelle for degradation of endocytic and autophagic protein substrates through the activity of vacuolar proteases. In both degradation pathways, substrates are transported to the vacuole by intracellular membrane trafficking. In endocytic degradation, plasma membrane-localized proteins are targeted to the vacuole for degradation by endosomes (Reyes et al., 2011). This process is important, among others, to control the abundance of plasma membrane receptors and thus downstream signaling events. Autophagic degradation is mainly involved in nutrient recycling. During this process, cytosolic proteins and organelles are either selectively or nonselectively transported by double membrane autophagosomes to the vacuole to be degraded (Liu and Bassham, 2012). Vacuolar transport defines an intracellular transport pathway by which de novo synthesized proteins or metabolic compounds are carried to the vacuole by vesicle transport (Drakakaki and Dandekar, 2013).In yeast (Saccharomyces cerevisiae), forward genetic screens aimed at finding mutants with defective vacuolar transport or vacuolar morphology have identified more than 30 VACUOLAR PROTEIN SORTING (VPS) and VACUOLAR MORPHOLOGY (VAM) genes (Banta et al., 1988; Raymond et al., 1992; Wada and Anraku, 1992). Closer analyses have shown that many of these mutants have defects both in protein sorting and in vacuole biogenesis, suggesting a close link between these processes. vps and vam mutants were classified into six mutant classes according to their phenotypes. The strategic success of these screens has been confirmed when later studies revealed that many of the genes categorized in the same mutant class were coding for subunits of the same protein complexes. Among them were complexes important for membrane transport and fusion events, such as the endosomal sorting complex required for transport (ESCRT)-I to ESCRT-III (Henne et al., 2011) or the homotypic fusion and vacuole protein sorting (HOPS) complex (Balderhaar and Ungermann, 2013).Sequence homologs of most yeast VPS genes can be found in the Arabidopsis (Arabidopsis thaliana) genome (Sanderfoot and Raikhel, 2003; Bassham et al., 2008), and some of them were reported to be involved in intracellular trafficking as well as vacuole biogenesis. For example, the Arabidopsis vacuoleless (vcl)/vps16 mutant is embryo lethal and lacks lytic vacuoles (Rojo et al., 2001). VPS16 is a subunit of the HOPS complex, suggesting that membrane fusion events mediated by VCL/VPS16 are also important for plant vacuole biogenesis. Several other Arabidopsis vps mutants were also shown to have altered vacuole morphology at the mature embryo stage (Shimada et al., 2006; Sanmartín et al., 2007; Ebine et al., 2008, 2014; Yamazaki et al., 2008; Zouhar et al., 2009; Shahriari et al., 2010), showing that there is a conserved mechanism regulating vacuolar transport and vacuole biogenesis. However, in contrast to yeast, in which mutants without vacuole or severe biogenesis defects are viable, plant vacuoles seem to be essential for plant development.We have previously shown that defects in the deubiquitinating enzyme (DUB) ASSOCIATED MOLECULE WITH THE Src homology-3 DOMAIN OF STAM3 (AMSH3) also lead to a severe vacuole biogenesis defect (Isono et al., 2010). AMSH homologs do not exist in budding yeast but are conserved in animals and plants. Our previous studies have shown that AMSH3 can directly interact with ESCRT-III subunits (Katsiarimpa et al., 2013). ESCRT-III is a multiprotein complex that is essential for multivesicular body (MVB) sorting (Winter and Hauser, 2006) and hence for plant growth and development (Haas et al., 2007; Spitzer et al., 2009; Katsiarimpa et al., 2011; Cai et al., 2014). AMSH proteins regulate intracellular trafficking events, including endocytic degradation, vacuolar transport, and autophagic degradation through its interaction with ESCRT-III (Isono et al., 2010; Katsiarimpa et al., 2011, 2013, 2014). Prior to our characterization of the amsh3 mutant, AMSH proteins had not been implicated in vacuole biogenesis. Thus, we reasoned that there might be additional, yet unidentified, factors important for regulating vacuole biogenesis in plants. Further, we reasoned that other mutants with a defect in vacuole biogenesis, analogous to amsh3, might also exhibit seedling lethality.Thus, with the goal to identify and characterize these factors, we carried out a two-step mutant screen. We first selected seedling lethal mutants from an ethyl methansulfonate (EMS)-mutagenized population and then examined the vacuole morphology in these mutants. The isolated mutants were designated vacuolar fusion defective (vfd). vfd1 is affected in the expression of a functional Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing FYVE1 protein. FYVE1 was originally identified in silico as one of 16 FYVE domain-containing proteins in Arabidopsis with no apparent homologs in yeast and mammals (van Leeuwen et al., 2004). FYVE domains bind phosphatidylinositol 3-P, a phospholipid that is a major constituent of endosomal membranes. Hence, FYVE domain-containing proteins are implicated in intracellular trafficking (van Leeuwen et al., 2004; Wywial and Singh, 2010). In a previous work, we have shown that a null mutant of FYVE1, fyve1-1, is defective in IRON-REGULATED TRANSPORTER1 (IRT1) polarization and that FYVE1 is essential for plant growth and development (Barberon et al., 2014). A very recent publication describing the same mutant has shown that FYVE1/FYVE domain protein required for endosomal sorting1 (FREE1) is also important for the early and late endosomal trafficking events (Gao et al., 2014). In this study, we show that FYVE1 is also regulating ubiquitin-dependent membrane protein degradation, vacuolar transport, autophagy, and vacuole biogenesis. Altogether, our results point toward FYVE1 being a key component of the intracellular trafficking machinery in plants.     

Rab11 Helps Maintain Apical Crumbs and Adherens Junctions in the Drosophila Embryonic Ectoderm

Background

Tissue morphogenesis and organogenesis require that cells retain stable cell-cell adhesion while changing shape and moving. One mechanism to accommodate this plasticity in cell adhesion involves regulated trafficking of junctional proteins.

Methodology/Principal Findings

Here we explored trafficking of junctional proteins in two well-characterized model epithelia, the Drosophila embryonic ectoderm and amnioserosa. We find that DE-cadherin, the transmembrane protein of adherens junctions, is actively trafficked through putative vesicles, and appears to travel through both Rab5-positive and Rab11-positive structures. We manipulated the functions of Rab11 and Rab5 to examine the effects on junctional stability and morphogenesis. Reducing Rab11 function, either using a dominant negative construct or loss of function alleles, disrupts integrity of the ectoderm and leads to loss of adherens junctions. Strikingly, the apical junctional regulator Crumbs is lost before AJs are destabilized, while the basolateral protein Dlg remains cortical. Altering Rab5 function had less dramatic effects, not disrupting adherens junction integrity but affecting dorsal closure.

Conclusions/Significance

We contrast our results with what others saw when disrupting other trafficking regulators, and when disrupting Rab function in other tissues; together these data suggest distinct mechanisms regulate junctional stability and plasticity in different tissues.     

A RabGAP Regulates Life-Cycle Duration via Trimeric G-protein Cascades in Dictyostelium discoideum

Background

The life-cycle of cellular slime molds comprises chronobiologically regulated processes. During the growth phase, the amoeboid cells proliferate at a definite rate. Upon starvation, they synthesize cAMP as both first and second messengers in signalling pathways and form aggregates, migrating slugs, and fruiting bodies, consisting of spores and stalk cells, within 24 h. In Dictyostelium discoideum, because most growth-specific events cease during development, proliferative and heterochronic mutations are not considered to be interrelated and no genetic factor governing the entire life-cycle duration has ever been identified.

Methodology/Principal Findings

Using yeast 2-hybrid library screening, we isolated a Dictyostelium discoideum RabGAP, Dd Rbg-3, as a candidate molecule by which the Dictyostelium Gα2 subunit directs its effects. Rab GTPase-activating protein, RabGAP, acts as a negative regulator of Rab small GTPases, which orchestrate the intracellular membrane trafficking involved in cell proliferation. Deletion mutants of Dd rbg-3 exhibited an increased growth rate and a shortened developmental period, while an overexpression mutant demonstrated the opposite effects. We also show that Dd Rbg-3 interacts with 2 Gα subunits in an activity-dependent manner in vitro. Furthermore, both human and Caenorhabditis elegans rbg-3 homologs complemented the Dd rbg-3–deletion phenotype in D. discoideum, indicating that similar pathways may be generally conserved in multicellular organisms.

Conclusions/Significance

Our findings suggest that Dd Rbg-3 acts as a key element regulating the duration of D. discoideum life-span potentially via trimeric G-protein cascades.     

NtGNL1 plays an essential role in pollen tube tip growth and orientation likely via regulation of post-Golgi trafficking

Background

Tobacco GNOM LIKE 1 (NtGNL1), a new member of the Big/GBF family, is characterized by a sec 7 domain. Thus, we proposed that NtGNL1 may function in regulating pollen tube growth for vesicle trafficking.

Methodology/Principal Findings

To test this hypothesis, we used an RNAi technique to down-regulate NtGNL1 expression and found that pollen tube growth and orientation were clearly inhibited. Cytological observations revealed that both timing and behavior of endocytosis was disrupted, and endosome trafficking to prevacuolar compartments (PVC) or multivesicular bodies (MVB) was altered in pollen tube tips. Moreover, NtGNL1 seemed to partially overlap with Golgi bodies, but clearly colocalized with putative late endosome compartments. We also observed that in such pollen tubes, the Golgi apparatus disassembled and fused with the endoplasmic reticulum, indicating abnormal post-Golgi trafficking. During this process, actin organization was also remodeled.

Conclusions/Significance

Thus, we revealed that NtGNL1 is essential for pollen tube growth and orientation and it likely functions via stabilizing the structure of the Golgi apparatus and ensuring post-Golgi trafficking.     

The GIP Receptor Displays Higher Basal Activity than the GLP-1 Receptor but Does Not Recruit GRK2 or Arrestin3 Effectively

Background and Objectives

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of insulin secretion, and their functional loss is an early characteristic of type 2 diabetes mellitus (T2DM). Pharmacological levels of GLP-1, but not GIP, can overcome this loss. GLP-1 and GIP exert their insulinotropic effects through their respective receptors expressed on pancreatic β-cells. Both the GLP-1 receptor (GLP-1R) and the GIP receptor (GIPR) are members of the secretin family of G protein-coupled receptors (GPCRs) and couple positively to adenylate cyclase. We compared the signalling properties of these two receptors to gain further insight into why GLP-1, but not GIP, remains insulinotropic in T2DM patients.

Methods

GLP-1R and GIPR were transiently expressed in HEK-293 cells, and basal and ligand-induced cAMP production were investigated using a cAMP-responsive luciferase reporter gene assay. Arrestin3 (Arr3) recruitment to the two receptors was investigated using enzyme fragment complementation, confocal microscopy and fluorescence resonance energy transfer (FRET).

Results

GIPR displayed significantly higher (P<0.05) ligand-independent activity than GLP-1R. Arr3 displayed a robust translocation to agonist-stimulated GLP-1R but not to GIPR. These observations were confirmed in FRET experiments, in which GLP-1 stimulated the recruitment of both GPCR kinase 2 (GRK2) and Arr3 to GLP-1R. These interactions were not reversed upon agonist washout. In contrast, GIP did not stimulate recruitment of either GRK2 or Arr3 to its receptor. Interestingly, arrestin remained at the plasma membrane even after prolonged (30 min) stimulation with GLP-1. Although the GLP-1R/arrestin interaction could not be reversed by agonist washout, GLP-1R and arrestin did not co-internalise, suggesting that GLP-1R is a class A receptor with regard to arrestin binding.

Conclusions

GIPR displays higher basal activity than GLP-1R but does not effectively recruit GRK2 or Arr3.     

Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells

Background

ABCA3 transporter (ATP-binding cassette transporter of the A subfamily) is localized to the limiting membrane of lamellar bodies, organelles for assembly and storage of pulmonary surfactant in alveolar epithelial type II cells (AECII). It transports surfactant phospholipids into lamellar bodies and absence of ABCA3 function disrupts lamellar body biogenesis. Mutations of the ABCA3 gene lead to fatal neonatal surfactant deficiency and chronic interstitial lung disease (ILD) of children. ABCA3 mutations can result in either functional defects of the correctly localized ABCA3 or trafficking/folding defects where mutated ABCA3 remains in the endoplasmic reticulum (ER).

Methods

Human alveolar epithelial A549 cells were transfected with vectors expressing wild-type ABCA3 or one of the three ABCA3 mutant forms, R43L, R280C and L101P, C-terminally tagged with YFP or hemagglutinin-tag. Localization/trafficking properties were analyzed by immunofluorescence and ABCA3 deglycosylation. Uptake of fluorescent NBD-labeled lipids into lamellar bodies was used as a functional assay. ER stress and apoptotic signaling were examined through RT-PCR based analyses of XBP1 splicing, immunoblotting or FACS analyses of stress/apoptosis proteins, Annexin V surface staining and determination of the intracellular glutathion level.

Results

We demonstrate that two ABCA3 mutations, which affect ABCA3 protein trafficking/folding and lead to partial (R280C) or complete (L101P) retention of ABCA3 in the ER compartment, can elevate ER stress and susceptibility to it and induce apoptotic markers in the cultured lung epithelial A549 cells. R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling.

Conclusion

Our data suggest that expression of partially or completely ER localized ABCA3 mutant proteins can increase the apoptotic cell death of the affected cells, which are factors that might contribute to the pathogenesis of genetic ILD.     

Extracellular HCV-Core Protein Induces an Immature Regulatory Phenotype in NK Cells: Implications for Outcome of Acute Infection

Background

Hepatitis C viral (HCV) proteins, including core, demonstrate immuno-modulatory properties; however, the effect of extracellular core on natural killer (NK) cells has not previously been investigated.

Aims

To characterise NKs in acute HCV infection over time, and, to examine the effect of exogenous HCV-core protein on NK cell phenotype and function.

Methods

Acute HCV patients (n = 22), including 10 subjects who spontaneously recovered, were prospectively studied. Flow-cytometry was used to measure natural cytotoxicity and to phenotype NKs directly ex vivo and after culture with HCV-core protein. Microarray analysis was used to identify pathways involved in the NK cell response to exogenous HCV-core.

Results

Direct ex vivo analysis demonstrated an increased frequency of immature/regulatory CD56^bright NKs early in acute HCV infection per se which normalized with viral clearance. Natural cytotoxicity was reduced and did not recover after viral clearance. There was a statistically significant correlation between the frequency of CD56^bright NKs and circulating serum levels of HCV core protein. In vitro culture of purified CD56^bright NK cells with HCV-core protein in the presence of IL-15 maintained a significant proportion of NKs in the CD56^bright state. The in vitro effect of core closely correlates with NK characteristics measured directly ex vivo in acute HCV infection. Pathway analysis suggests that HCV-core protein attenuates NK interferon type I responses.

Conclusions

Our data suggest that HCV-core protein alters NK cell maturation and may influence the outcome of acute infection.     

Sortin2 enhances endocytic trafficking towards the vacuole in Saccharomyces cerevisiae

Background

A highly regulated trafficking of cargo vesicles in eukaryotes performs protein delivery to a variety of cellular compartments of endomembrane system. The two main routes, the secretory and the endocytic pathways have pivotal functions in uni- and multi-cellular organisms. Protein delivery and targeting includes cargo recognition, vesicle formation and fusion. Developing new tools to modulate protein trafficking allows better understanding the endomembrane system mechanisms and their regulation. The compound Sortin2 has been described as a protein trafficking modulator affecting targeting of the vacuolar protein carboxypeptidase Y (CPY), triggering its secretion in Saccharomyces cerevisiae.

Results

A reverse chemical-genetics approach was used to identify key proteins for Sortin2 bioactivity. A genome-wide Sortin2 resistance screen revealed six yeast deletion mutants that do not secrete CPY when grown at Sortin2 condition where the parental strain does: met18, sla1, clc1, dfg10, dpl1 and yjl175w. Integrating mutant phenotype and gene ontology annotation of the corresponding genes and their interactome pointed towards a high representation of genes involved in the endocytic process. In wild type yeast endocytosis towards the vacuole was faster in presence of Sortin2, which further validates the data of the genome-wide screen. This effect of Sortin2 depends on structural features of the molecule, suggesting compound specificity. Sortin2 did not affect endocytic trafficking in Sortin2-resistant mutants, strongly suggesting that the Sortin2 effects on the secretory and endocytic pathways are linked.

Conclusions

Overall, the results reveal that Sortin2 enhances the endocytic transport pathway in Saccharomyces cerevisiae. This cellular effect is most likely at the level where secretory and endocytic pathways are merged. Them Sortin2 specificity over the endomembrane system places it as a powerful biological modulator for cell biology.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0032-9) contains supplementary material, which is available to authorized users.     

A-RAF Kinase Functions in ARF6 Regulated Endocytic Membrane Traffic

Background

RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation.

Methodology/Principal Findings

Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149) that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment.

Conclusions/Significance

A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking.     

The Hybrid Type Polyketide Synthase SteelyA Is Required for cAMP Signalling in Early Dictyostelium Development

Background

In our previous study we found that the expression of stlA showed peaks both in the early and last stages of development and that a product of SteelyA, 4-methyl-5-pentylbenzene-1,3-diol (MPBD), controlled Dictyostelium spore maturation during the latter. In this study we focused on the role of SteelyA in early stage development.

Principal Findings

Our stlA null mutant showed aggregation delay and abnormally small aggregation territories. Chemotaxis analysis revealed defective cAMP chemotaxis in the stlA null mutant. cAMP chemotaxis was restored by MPBD addition during early stage development. Assay for cAMP relay response revealed that the stlA null mutant had lower cAMP accumulation during aggregation, suggesting lower ACA activity than the wild type strain. Exogenous cAMP pulses rescued the aggregation defect of the stlA null strain in the absence of MPBD. Expression analysis of cAMP signalling genes revealed lower expression levels in the stlA null mutant during aggregation.

Conclusion

Our data indicate a regulatory function by SteelyA on cAMP signalling during aggregation and show that SteelyA is indispensable for full activation of ACA.