Nedd4-2-dependent regulation of astrocytic Kir4.1 and Connexin43 controls neuronal network activity.

Nedd4-2 is an E3 ubiquitin ligase in which missense mutation is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of γ-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of the Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy.

Drebrins and Connexins: A Biomedical Perspective.

In this chapter we summarize knowledge on the role of drebrin in cell-cell communications. Specifically, we follow drebrin-connexin-43 interactions and drebrin behavior at the cell-cell interface described earlier. Drebrin is a part of the actin cytoskeleton which is a target of numerous bacteria and viruses invading mammalian cells. Drebrin phosphorylation, self-inhibition and transition between filaments, particles, and podosomes underlie cellular mechanisms involved in diseases and cognitive disorders. Cytoskeletal rearrangements influence the state of gap junction contacts which regulate cell signaling and metabolic flow of information across cells in tissues. Taking into account that connexin-43 (Cx43) (together with Cx30) is heavily expressed in astrocytes and that drebrin supports cell-cell contacts, the understanding of details of how brain cells live and die reveals molecular pathology involved in neurodegeneration, Alzheimer's disease (AD), other cognitive disorders, and aging.Bidirectional connexin channels are permeable to Ca2+ ions, IP3, ATP, and cAMP. Connexin hemichannels are important for paracrine regulation and can release and exchange energy with other cells using ATP to transfer information and to support damaged cells. Connexin channels, hemichannels, and adhesion plaques are regulated by assembly and disassembly of the actin cytoskeleton. Drebrin degradation can alter gap junction communication, and drebrin level is decreased in the brain of AD patients. The diversity of drebrin functions in neurons, astrocytes, and non-neuronal cells still remains to be revealed. We believe that the knowledge on drebrin summarized here will contribute to key questions, "covering the gap" between cell-cell communications and the submembrane cytoskeleton.

The murine cytomegalovirus immunoevasin gp40 binds MHC class I molecules to retain them in the early secretory pathway.

In the presence of the murine cytomegalovirus (mCMV) gp40 (m152) protein, murine major histocompatibility complex (MHC) class I molecules do not reach the cell surface but are retained in an early compartment of the secretory pathway. We find that gp40 does not impair the folding or high-affinity peptide binding of the class I molecules but binds to them, leading to their retention in the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, most likely by retrieval from the cis-Golgi to the ER. We identify a sequence in gp40 that is required for both its own retention in the early secretory pathway and for that of class I molecules.

Structural basis for the binding of tryptophan-based motifs by δ-COP.

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding ßγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αß'ε-COP B-subcomplex. We present the structure of the C-terminal µ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP µ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1-6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing.

Drebrin-like protein DBN-1 is a sarcomere component that stabilizes actin filaments during muscle contraction.

Actin filament organization and stability in the sarcomeres of muscle cells are critical for force generation. Here we identify and functionally characterize a Caenorhabditis elegans drebrin-like protein DBN-1 as a novel constituent of the muscle contraction machinery. In vitro, DBN-1 exhibits actin filament binding and bundling activity. In vivo, DBN-1 is expressed in body wall muscles of C. elegans. During the muscle contraction cycle, DBN-1 alternates location between myosin- and actin-rich regions of the sarcomere. In contracted muscle, DBN-1 is accumulated at I-bands where it likely regulates proper spacing of α-actinin and tropomyosin and protects actin filaments from the interaction with ADF/cofilin. DBN-1 loss of function results in the partial depolymerization of F-actin during muscle contraction. Taken together, our data show that DBN-1 organizes the muscle contractile apparatus maintaining the spatial relationship between actin-binding proteins such as α-actinin, tropomyosin and ADF/cofilin and possibly strengthening actin filaments by bundling.

Fast structural responses of gap junction membrane domains to AB5 toxins.

Gap junctions (GJs) represent connexin-rich membrane domains that connect interiors of adjoining cells in mammalian tissues. How fast GJs can respond to bacterial pathogens has not been known previously. Using Bessel beam plane illumination and confocal spinning disk microscopy, we found fast (~500 ms) formation of connexin-depleted regions (CDRs) inside GJ plaques between cells exposed to AB5 toxins. CDR formation appears as a fast redistribution of connexin channels within GJ plaques with minor changes in outline or geometry. CDR formation does not depend on membrane trafficking or submembrane cytoskeleton and has no effect on GJ conductance. However, CDR responses depend on membrane lipids, can be modified by cholesterol-clustering agents and extracellular K(+) ion concentration, and influence cAMP signaling. The CDR response of GJ plaques to bacterial toxins is a phenomenon observed for all tested connexin isoforms. Through signaling, the CDR response may enable cells to sense exposure to AB5 toxins. CDR formation may reflect lipid-phase separation events in the biological membrane of the GJ plaque, leading to increased connexin packing and lipid reorganization. Our data demonstrate very fast dynamics (in the millisecond-to-second range) within GJ plaques, which previously were considered to be relatively stable, long-lived structures.

Anti-inflammatory activity of IgG1 mediated by Fc galactosylation and association of FcγRIIB and dectin-1.

Complement is an ancient danger-sensing system that contributes to host defense, immune surveillance and homeostasis. C5a and its G protein­coupled receptor mediate many of the proinflammatory properties of complement. Despite the key role of C5a in allergic asthma, autoimmune arthritis, sepsis and cancer, knowledge about its regulation is limited. Here we demonstrate that IgG1 immune complexes (ICs), the inhibitory IgG receptor FcγRIIB and the C-type lectin­like receptor dectin-1 suppress C5a receptor (C5aR) functions. IgG1 ICs promote the association of FcγRIIB with dectin-1, resulting in phosphorylation of Src homology 2 domain­containing inositol phosphatase (SHIP) downstream of FcγRIIB and spleen tyrosine kinase downstream of dectin-1. This pathway blocks C5aR-mediated ERK1/2 phosphorylation, C5a effector functions in vitro and C5a-dependent inflammatory responses in vivo, including peritonitis and skin blisters in experimental epidermolysis bullosa acquisita. Notably, high galactosylation of IgG N-glycans is crucial for this inhibitory property of IgG1 ICs, as it promotes the association between FcγRIIB and dectin-1. Thus, galactosylated IgG1 and FcγRIIB exert anti-inflammatory properties beyond their impact on activating FcγRs.

Limiting transport steps and novel interactions of Connexin-43 along the secretory pathway.

Connexins are four-transmembrane-domain proteins expressed in all vertebrates which form permeable gap junction channels that connect cells. Here, we analysed Connexin-43 (Cx43) transport to the plasma membrane and studied the effects of small GTPases acting along the secretory pathway. We show that both GTP- and GDP-restricted Sar1 prevents exit of Cx43 from the endoplasmic reticulum (ER), but only GTP-restricted Sar1 arrests Cx43 in COP II-coated ER exit sites and accumulates 14-3-3 proteins in the ER fraction. FRET-FLIM data confirm that already in ER exit sites Cx43 exists in oligomeric form, suggesting an in vivo role for 14-3-3 in Cx43 oligomerization. Exit of Cx43 from the ER can be blocked by other factors--such as expression of the beta subunit of the COP I coat or p50/dynamitin that acts on the microtubule-based dynein motor complex. GTP-restricted Arf1 blocks Cx43 in the Golgi. Lastly, we show that GTP-restricted Arf6 removes Cx43 gap junction plaques from the cell-cell interface and targets them to degradation. These data provide a molecular explanation of how small GTPases act to regulate Cx43 transport through the secretory pathway, facilitating or abolishing cell-cell communication through gap junctions.

Two human ARFGAPs associated with COP-I-coated vesicles.

ADP-ribosylation factors (ARFs) are critical regulators of vesicular trafficking pathways and act at multiple intracellular sites. ADP-ribosylation factor-GTPase-activating proteins (ARFGAPs) are proposed to contribute to site-specific regulation. In yeast, two distinct proteins, Glo3p and Gcs1p, together provide overlapping, essential ARFGAP function required for coat protein (COP)-I-dependent trafficking. In mammalian cells, only the Gcs1p orthologue, named ARFGAP1, has been characterized in detail. However, Glo3p is known to make the stronger contribution to COP I traffic in yeast. Here, based on a conserved signature motif close to the carboxy terminus, we identify ARFGAP2 and ARFGAP3 as the human orthologues of yeast Glo3p. By immunofluorescence (IF), ARFGAP2 and ARFGAP3 are closely colocalized with coatomer subunits in NRK cells in the Golgi complex and peripheral punctate structures. In contrast to ARFGAP1, both ARFGAP2 and ARFGAP3 are associated with COP-I-coated vesicles generated from Golgi membranes in the presence of GTP-gamma-S in vitro. ARFGAP2 lacking its zinc finger domain directly binds to coatomer. Expression of this truncated mutant (DeltaN-ARFGAP2) inhibits COP-I-dependent Golgi-to-endoplasmic reticulum transport of cholera toxin (CTX-K63) in vivo. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 in HeLa cells does not decrease cell viability. However, silencing all three ARFGAPs causes cell death. Our data provide strong evidence that ARFGAP2 and ARFGAP3 function in COP I traffic.

Peptide-receptive major histocompatibility complex class I molecules cycle between endoplasmic reticulum and cis-Golgi in wild-type lymphocytes.

Prior to binding to a high affinity peptide and transporting it to the cell surface, major histocompatibility complex class I molecules are retained inside the cell by retention in the endoplasmic reticulum (ER), recycling through the ER-Golgi intermediate compartment and possibly the cis-Golgi, or both. Using fluorescence microscopy and a novel in vitro COPII (ER-to-ER-Golgi intermediate compartment) vesicle formation assay, we find that in both lymphocytes and fibroblasts that lack the functional transporter associated with antigen presentation, class I molecules exit the ER and reach the cis-Golgi. Intriguingly, in wild-type T1 lymphoma cells, peptide-occupied and peptide-receptive class I molecules are simultaneously exported from ER membranes with similar efficiencies. Our results suggest that binding of high affinity peptide and exit from the ER are not coupled, that the major histocompatibility complex class I quality control compartment extends into the Golgi apparatus under standard conditions, and that peptide loading onto class I molecules may occur in post-ER compartments.

Many faces of drebrin: from building dendritic spines and stabilizing gap junctions to shaping neurite-like cell processes.

In this review we consider the multiple functions of developmentally regulated brain protein (drebrin), an actin-binding protein, in the formation of cellular polarity in different cell types. Drebrin has a well-established role in the morphogenesis, patterning and maintenance of dendritic spines in neurons. We have recently shown that drebrin also stabilizes Connexin-43 containing gap junctions at the plasma membrane. The latest literature and our own data suggest that drebrin may be broadly involved in shaping cell processes and in the formation of stabilized plasma membrane domains, an effect that is likely to be of crucial significance for formation of cell polarity in both neuronal and non-neuronal types.

Crn7 interacts with AP-1 and is required for the maintenance of Golgi morphology and protein export from the Golgi.

Crn7 is a novel cytosolic mammalian WD-repeat protein of unknown function that associates with Golgi membranes. Here, we demonstrate that Crn7 knockdown by small interfering RNA results in dramatic changes in the Golgi morphology and function. First, the Golgi ribbon is disorganized in Crn7 KD cells. Second, the Golgi export of several marker proteins including VSV envelope G glycoprotein is greatly reduced but not the retrograde protein import into the Golgi complex. We further establish that Crn7 co-precipitates with clathrin adaptor AP-1 but is not required for AP-1 targeting to Golgi membranes. We identify tyrosine 288-based motif as part of a canonical YXXPhi sorting signal and a major mu1-adaptin binding site in vitro. This study provides the first insight into the function of mammalian Crn7 protein in the Golgi complex.

Deleterious and protective properties of an aggregate-prone protein with a polyalanine expansion.

Many aggregate-prone proteins, including proteins with long polyglutamine or polyalanine tracts, cause human diseases. Polyalanine proteins may also be present in the tissue of polyglutamine diseases as a result of frameshifting of the primary polyglutamine-encoding (CAG)n repeat mutation. We have generated a Drosophila model expressing green fluorescent protein tagged to 37 alanines that manifests both toxicity and inclusion formation in various tissues. Surprisingly, we show that this aggregate-prone protein with a polyalanine expansion can also protect against polyglutamine toxicity, which can be explained by induction of heat-shock response. A heat-shock response was also seen in an oculopharyngeal muscular dystrophy mouse model expressing an authentic polyalanine-expanded protein. We also show that long polyalanines can protect against a pro-apoptotic stimulus or the toxicity caused by the long polyalanines themselves. Thus, overexpression of an aggregate-prone protein without any normal functions can result in both pathogenic and protective effects in cell culture and in vivo.

Visualization of SHP-1-target interaction.

Signaling of receptor tyrosine kinases (RTKs) is regulated by protein-tyrosine phosphatases (PTPs). We previously discovered the efficient downregulation of Ros RTK signaling by the SH2 domain PTP SHP-1, which involves a direct interaction of both molecules. Here, we studied the mechanism of this interaction in detail. Phosphopeptides representing the SHP-1 candidate binding sites in the Ros cytoplasmic domain, pY2267 and pY2327, display high affinity binding to the SHP-1 N-terminal SH2 domain (Kd=217 nM and 171 nM, respectively). Y2327 is, however, a poor substrate of Ros kinase and, therefore, contributes little to SHP-1 binding in vitro. To explore the mechanism of association in intact cells, functional fluorescent fusion proteins of Ros and SHP-1 were generated. Complexes of both molecules could be detected by Förster resonance energy transfer (FRET) in intact HEK293 and COS7 cells. As expected, the association required the functional SHP-1 N-terminal SH2 domain. Unexpectedly, pY2267 and pY2327 both contributed to the association. Mutation of Y2327 reduced constitutive association in COS7 cells. Ligand-dependent association was abrogated upon mutation of Y2267 but remained intact when Y2327 was mutated. A phosphopeptide representing the binding site pY2267 was a poor substrate for SHP-1, whereas Ros activation loop phosphotyrosines were effectively dephosphorylated. We propose a model for SHP-1-Ros interaction in which ligand-stimulated phosphorylation of Ros Y2267 by Ros, phosphorylation of Y2327 by a heterologous kinase, and inactivation of Ros by SHP-1-mediated dephosphorylation play a role in the regulation of complex stability.

Coronin 7, the mammalian POD-1 homologue, localizes to the Golgi apparatus.

Coronins constitute an evolutionary conserved family of WD-repeat actin-binding proteins. Their primary function is thought to be regulating the actin cytoskeleton. Apart from that, several coronins were indirectly shown to participate in vesicular transport, establishment of cell polarity and cytokinesis. Here, we report a novel mammalian protein, coronin 7 (crn7), which is significantly different from other mammalian coronins in its domain architecture. Crn7 possesses two stretches of WD repeats in contrast to the other coronins only having one. The protein is expressed throughout the mouse embryogenesis and is strongly upregulated in brain and developing structures of the immune system in the course of development. In adult animals, both crn7 mRNA and protein are abundantly present in most organs, with significantly higher amounts in brain, kidney, thymus and spleen and lower amounts in muscle. At the subcellular level, the bulk of the protein appears to be present in the cytosol and in large cytosolic complexes. However, a significant portion of the protein is detected on vesicle-like cytoplasmic structures as well as on the cis-Golgi. In the Golgi region, crn7 staining appears broader than that of the cis-Golgi markers Erd2p and beta-COP, still, the trans-Golgi network appears predominantly crn7-negative. Importantly, the membrane-associated form of crn7 protein is phosphorylated on tyrosine residues, whereas the cytosolic form is not. Crn7 is the first coronin protein proven to localize to the Golgi membrane. We conclude that it plays a role in the organization of intracellular membrane compartments and vesicular trafficking rather than in remodeling the cytoskeleton.

Analysing the action of bacterial toxins in living cells with fluorescence resonance energy transfer (FRET).

Bacterial toxins represent small molecules produced by microorganisms. Different toxins act on specific target molecules in mammalian cells. Once discovered, bacterial toxins have been providing tools to study cellular functions and often helped the dissection of complex cellular pathways, e.g. endocytic or secretory trafficking or signal transduction, by virtue of the fact that they either block or activate their specific cellular target molecules. Purified bacterial toxins have also allowed to address many basic biological questions and have provided tools for in vitro and in vivo experimental approaches in many fields of modern biology. The understanding of how bacterial toxins act in living cells often depends on our ability to visualize the trafficking and signaling pathways of these molecules. Fluorescence microscopy and other imaging tools are essential to provide insights into the functional changes induced by these pathogens at the level of individual host cells or single target proteins. Inside a single cell we can measure and quantify the effects of bacterial toxins on specific cellular proteins by microscopic and spectroscopic techniques. Fluorescence resonance energy transfer (FRET) is a high-resolution technique that allows to study protein-protein interactions. FRET can provide distance information in the range of 3- 7 nm between fluorescently labeled bacterial proteins in the live cell and cellular target proteins expressed as chimeras with green fluorescent protein (GFP), or spectrally shifted variants thereof. The purpose of this review is to introduce readers to the main experimental setups for analyses of protein-protein interactions using FRET as well as some applications.

Drebrin is a novel connexin-43 binding partner that links gap junctions to the submembrane cytoskeleton.

BACKGROUND: Connexins form gap junctions that mediate the transfer of ions, metabolites, and second messengers between contacting cells. Many aspects of connexin function, for example cellular transport, plaque assembly and stability, and channel conductivity, are finely tuned and likely involve proteins that bind to connexins' cytoplasmic domains. However, little is known about such regulatory proteins. To identify novel proteins that interact with the COOH-terminal domain of Connexin-43 (Cx43), the most widely expressed connexin family member, we applied a proteomics approach to screen fractions of mouse tissue homogenates for binding partners. RESULTS: Drebrin was recovered as a binding partner of the Cx43 COOH-terminal domain from mouse brain homogenate. Drebrin had previously been described as an actin binding protein that diminishes in brains during Alzheimer's disease. The novel Drebrin-Cx43 interaction identified by proteomics was confirmed by colocalization of endogenous proteins in astrocytes and Vero cells, coimmunoprecipitation, electron microscopy, electrophysiology, coexpression of both proteins with fluorescent tags, and live-cell FRET analysis. Depletion of Drebrin in cells with siRNA results in impaired cell-cell coupling, internalization of gap junctions, and targeting of Cx43 to a degradative pathway. CONCLUSIONS: We conclude that Drebrin is required for maintaining Cx43-containing gap junctions in their functional state at the plasma membrane. It is thus possible that Drebrin may interact with gap junctions in zones of cell-cell contacts in a regulated fashion in response to extracellular signals. The rearrangement or disruption of interactions between connexins and the Drebrin-containing submembrane cytoskeleton directs connexins to degradative cellular pathways.

Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis.

Fluorescence (auto)correlation spectroscopy (FCS) has developed into a widely used method for investigating molecular dynamics and mobility of molecules in vitro and in vivo. Dual-color cross-correlation, an extension of this technique, also assesses the concomitant movement of two spectrally distinguishable fluorescent molecules and has therefore proven superior to autocorrelation analysis to study interactions between different molecular species in solution. Here we explore the benefits of cross-correlation analysis when applied to live cells, by demonstrating its potential in analyzing endocytic processes. Bacterial cholera toxin (CTX) was labeled with Cy2 and Cy5 dyes on different subunits of the same holotoxin. Along the endocytic pathway, positive cross-correlation between the A and B subunits was first preserved, later followed by a loss in cross-correlation upon their separation in the Golgi. Furthermore, endocytosis of a mixture of only Cy2- and only Cy5-labeled holotoxins also gave rise to cross-correlation. Our results suggest that cross-correlation may be used to recognize whether different cargoes use the same endocytic pathway. Additionally, we show that cross-correlation is applicable to two-dimensional membrane diffusion. CTX bound to GM1-containing artificial giant unilamellar vesicles was diffusible, whereas CTX bound to the plasma membrane was immobile on the FCS time-scale, possibly because of raft-association of GM1.

Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy.

Fluorescence resonance energy transfer (FRET) resolved by multifocal multiphoton microscopy (MMM) was successfully used to measure transport phenomena in living cells. We expressed different pairs of CFP-/YFP-fusion proteins involved in retrograde Golgi-to-ER transport to analyze sorting of the occupied KDEL-receptor into retrograde transport vesicles triggered by application of the external cholera toxin mutant CTXK63. FRET observed as a sensitized emission of the acceptor was confirmed by acceptor photobleaching and the dequenching of the donor was measured. FRET-MMM data obtained from single cells were compared with bulk cell experiments employing spectrofluorimetry. The importance of controlling the degree of overexpression of CFP-/YFP-fusion proteins for FRET analysis is stressed in this article. Using MMM we showed for the first time that FRET can be measured across the Golgi membrane. Finally, FRET-MMM records performed continuously over 2 h allowed to analyze intracellular retrograde transport and sorting events and to discuss these mechanisms on a single cell level.

Differential expression of receptors for Shiga and Cholera toxin is regulated by the cell cycle.

Cholera and Shiga toxin bind to the cell surface via glycolipid receptors GM1 and Gb3, respectively. Surprisingly, the majority of Vero cells from a non-synchronized population bind either Cholera or Shiga toxin but not both toxins. The hypothesis that the differential expression of toxin receptors is regulated by the cell cycle was tested. We find that Cholera toxin binds preferentially in G0/G1, with little binding through S-phase to telophase, whereas Shiga toxin binds maximally through G2 to telophase but does not bind during G0/G1 and S-phase. The changes result from the corresponding changes in Gb3 and GM1 synthesis, not from variations of receptor transport to the cell surface. The changes do not reflect competition of Gb3 and GM1 synthesis for lactosylceramide. Cells as diverse as Vero cells, PC12 cells and astrocytes show the same cell-cycle-dependent regulation of glycosphingolipid receptors, suggesting that this novel phenomenon is based on a conserved regulatory mechanism.