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.

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.

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.

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.

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.

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.

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.

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.