Irina Majoul

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‐γ‐S in vitro. ARFGAP2 lacking its zinc finger domain directly binds to coatomer. Expression of this truncated mutant (ΔN‐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.

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.

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.

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 β‐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.

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 YXXΦ sorting signal and a major μ1-adaptin binding site in vitro. This study provides the first insight into the function of mammalian Crn7 protein in the Golgi complex.

Practical Fluorescence Resonance Energy Transfer or Molecular Nanobioscopy of Living Cells

After formulating this philosophic question in a poetic form, Leonardo the Scientist, provides us with a real experimental (optical) setup. “As I propose to treat the nature of the moon, it is necessary that I first describe the perspective of mirrors, whether plane, concave, or convex,” (B.M.94r – Arundel MS in British Museum). Next, in the pages of Codex Atlanticus (C.A.190r), Leonardo invites us to “Construct the glasses to see the moon magnified” and half a millennium later we are still following him for, as Bulgakov famously said, “Manuscripts do not burn!”

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.