A cytosolic disulfide bridge‐supported dimerization is crucial for stability and cellular distribution of Coxsackievirus B3 protein 3A

RNA viruses in the Picornaviridae family express a large 250 kDa viral polyprotein that is processed by virus-encoded proteinases into mature functional proteins with specific functions for virus replication. One of these proteins is the highly conserved enteroviral transmembrane protein 3A that assists in reorganizing cellular membranes associated with the Golgi apparatus. Here, we studied the molecular properties of the Coxsackievirus B3 (CVB3) protein 3A with regard to its dimerization and its functional stability. By applying mutational analysis and biochemical characterization, we demonstrate that protein 3A forms DTT-sensitive disulfide-linked dimers via a conserved cytosolic cysteine residue at position 38 (Cys38). Homodimerization of CVB3 protein 3A via Cys38 leads to profound stabilization of the protein, whereas a C38A mutation promotes a rapid proteasome-dependent elimination of its monomeric form. The lysosomotropic agent chloroquine (CQ) exerted only minor stabilizing effects on the 3A monomer but resulted in enrichment of the homodimer. Our experimental data demonstrate that disulfide linkages via a highly conserved Cys-residue in enteroviral protein 3A have an important role in the dimerization of this viral protein, thereby preserving its stability and functional integrity

A spontaneous missense mutation in the chromodomain helicase DNA‐binding protein 8 (CHD8) gene: a novel association with congenital myasthenic syndrome

Aims: Congenital myasthenic syndromes (CMS) are characterized by muscle weakness, ptosis and episodic apnoea. Mutations affect integral protein components of the neuromuscular junction (NMJ). Here we searched for the genetic basis of CMS in female monozygotic twins. Methods: We employed whole-exome sequencing for mutation detection and Sanger sequencing for segregation analysis. Immunohistology was done with antibodies against CHD8, rapsyn, β-catenin (βCAT) and golgin on fi-bro-blasts, human and mouse muscle. We recorded superresolution images of the NMJ using 3D-structured illumination microscopy. Results: We discovered a spontaneous missense mutation in CHD8 [chr14:g.21,884,051G>A, GRCh37.p11 | c.1732C>T, NM_00117062 | p.(R578C)], the gene encoding chromodomain helicase DNA-binding protein 8. This is the first missense mutation affecting Duplin, the short 110 kDa isoform of CHD8. It is known that CHD8/Duplin negatively regulates βCAT signalling in the WNT pathway and plays a role in chromatin remodelling. Inactivating CHD8 mutations are associated with autism spectrum disorder and intellectual disability in combination with facial dysmorphism, overgrowth and macrocephalus. No muscle-specific phenotype has been reported to date. Co-immunostaining with rapsyn on human and mouse muscle revealed a strong presence of CHD8 at the NMJ being located towards the sarcoplasmic side of the rapsyn cluster, where it co-localizes with βCAT. Conclusion: We hypothesize CHD8 to have a role in the maintenance of the structural integrity and function of the NMJ. Both patients benefited from treatment with 3,4-diaminopyridine, a reversible blocker of voltage-gated potassium channels at the nerve terminal that prolongs the action potential and increases acetylcholine release

A Presynaptic Role for the Cytomatrix Protein GIT in Synaptic Vesicle Recycling

Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. Here, we identify the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ- associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D. melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery

Mikroskopie von einzelnen Fusionen an der Plasmamembrane mittels Totalreflexions-Fluoreszenzmikroskopie - Anwendungen im Bereich Vesikeltransport

Dissertation Titelblatt und Inhalt ReadMeVideo Videos3 Video3.3 Video3.3b Video3.4a Videos4 Video4.3a Video4.3d Video4.5b Video4.5c Video4.6a Video4.6b Video4.7a Video4.7c, slow Video4.7c Video4.7c, end, slow Video4.7c, end Video4.9b Videos5 Video5.2a, nonpol Video5.2a, pol Video5.2b, nonpol Video5.2b, pol Video5.2c Video5.4a Video5.4b Video5.5 Video5.6b Video5.6c Videos7 Video7.3a, slow Video7.3a Video7.3c, slow Video7.3cThe last stage of the constitutive secretory pathway is the delivery and fusion of post-Golgi transport vesicles (carriers) to the plasma membrane. However, it is still unclear where at the plasma membrane these carriers fuse, in a living cell. In this work we used total internal reflection fluorescence microscopy (TIR-FM) to study the delivery and fusion of post-Golgi carriers, as well as of recycling vesicles from the endosomal compartment, to the cell surface in live cells. TIR-FM is suited to image fluorescent molecules near the cell-substrate interphase, since it selectively illuminates the contact surface of cells. One aim of this work was to establish a quantitative method for the microscopic detection of single exocytic fusion events. Following the localization of fusion we answered basic, previously unresolved, questions in the field of membrane traffic. 1\. Imaging fusion of single post-Golgi carriers. A TIR-FM system was optimized to image exocytosis of single post- Golgi carriers. By labeling the carriers with a GFP-tagged membrane protein (vesicular stomatitis virus glycoprotein, VSVG), quantitative criteria for the microscopic detection of single carrier fusion events were established for the first time. Quantitative analysis of time-lapse images could clearly distinguish fusion of the carriers from both movement of carriers relative to the plasma membrane as well as lysis of carriers. The flattening of the carriers into the plasma membrane as well as the subsequent diffusion of the membrane cargo into the plasma membrane was resolved. The duration of the flattening process was found to depend on the size of the carrier, distinguishing small spherical from large tubular carriers. 2\. Role of microtubules in post-Golgi traffic of fibroblasts. The simul-taneous imaging of post-Golgi carriers and microtubules using a novel dual-color TIR-FM system showed that post-Golgi carriers are transported along microtubules to the fusion sites at the plasma membrane. The data strongly suggested that the carriers are capable of undergoing fusion while still attached to the microtubules and that the carriers do not have to reach the end of the microtubules in order to fuse. In contrast to stationary fibroblasts, migrating fibroblasts were shown to have a microtubule-mediated mechanism for polarized insertion of post-Golgi carriers (here LDL-receptor-GFP) close to the leading edge. Disrupting the microtubules restricted this directed delivery of the carriers to regions of the plasma membrane close to the Golgi complex, making the distribution of fusion sites in stationary and migrating cells indistinguishable. Disrupting the microtubules also decreased the overall fusion frequency, increased the frequency of "partial" fusions, and increased the amount of cargo delivered per fusion. We conclude that the microtubule cytoskeleton is necessary for the domain-specific delivery of post-Golgi membrane cargo in fibroblasts. 3\. Role of microtubules in post- Golgi traffic of polarized epithelial cells. Time-lapse fluorescence microscopy was used to analyze the delivery of apical and basolateral membrane proteins to the cell surface in both non-polarized and polarized epithelial cells. We demonstrated that post-Golgi carriers containing either apical or basolateral membrane proteins fuse to the basal membrane in non-polarized cells. Upon polarization, exocytosis of all carriers to the basal membrane was abrogated. Basolateral carriers were seen to fuse to sites at the lateral membrane, while apical carriers presumably fused to the apical membrane. This selective targeting is concomitant with redistribution of the t-SNAREs, syntaxin 3 and 4, upon polarization. Furthermore, we showed that both the targeted exocytosis of apical proteins and the exclusive localization of syntaxin 3 at the apical plasma membrane are dependent on intact microtubules in polarized epithelial cells. In contrast, targeted exocytosis of basolateral proteins and the basolateral distribution of syntaxin 4 and sec6 are maintained independently of microtubules in polarized cells. 4\. Insulin- regulated recycling of glucose transporter. We studied the insulin-regulated release of the glucose transporter (GLUT4) from the endosomal recycling compartment (ERC) in live cells. We show that GLUT4 is retained within the transferrin receptor-containing general ERC in fibroblasts. Using dual-color TIR-FM, we demonstrate that the transferrin receptor and GLUT4 are transported from the ERC in separate vesicles. This provides the first functional evidence for the formation of distinct classes of vesicles from the ERC. We propose that GLUT4 is dynamically retained within the ERC in fibroblasts because it is concentrated in vesicles that form more slowly than those that transport transferrin receptor.Der Transport von Post-Golgi-Vesikeln und deren Fusion mit der Plasmamembran sind die finalen Prozesse bei der konstitutiven Sekretion in lebenden Zellen. Der Transportweg zum Ort der Fusion dieser Vesikel mit der Plasmamembran wurde bisher nicht ausreichend untersucht. In der vorliegenden Arbeit wurde Totalreflexions-Fluoreszenzmikroskopie (TIR-FM) genutzt, um Transport und Fusion von Post-Golgi-Vesikeln, wie auch von Vesikeln des endosomalen "Recycling" Kompartiments, zu untersuchen. TIR-FM is speziell dafür geeignet, fluoreszente Moleküle am Zell-Substrat-Übergang bildhaft darzustellen, weil die Kontaktfläche der Zelle selektiv angeregt wird. Ein Ziel dieser Arbeit war es, eine quantitative Methode zur mikroskopischen Detektion einzelner Ereignisse bei der Exozytose zu etablieren. Aufbauend auf der Lokalisierung der Fusionen von Post-Golgi-Vesikeln wurden grundlegende offene Fragen auf dem Gebiet des Vesikeltransports beantwortet. 1\. Mikroskopische Darstellung von Fusionsereignissen einzelner Post-Golgi-Transportvesikel. Ein TIR-FM-Systems wurde optimiert, um die Exozytose einzelner Post-Golgi-Vesikel zu detektieren. Indem die Vesikel mit einem Membranprotein (vesikuläre stomatische virale Glykoprotein, VSVG), gekoppelt an GFP, markiert wurden, konnten erstmalig quantitative Kriterien für die mikroskopische Detektion von einzelnen Fusionen etabliert werden. Durch quantitative Bildanalyse des zeitlichen Ablaufes konnten die Fusionen sowohl von der Bewegung relativ zur Plasmamembran als auch von der lichtinduzierten Lyse unterschieden werden. Der Einbau der Vesikelmembran in die Plasmamembran und die darauffolgende Diffusion der Membranproteine konnten zeitlich aufgelöst werden. Kleinere, sphärische und größere, tubuläre Vesikel wurden anhand der Dauer des vesikulären Einbauprozesses in die Plasmamembran unterschieden. 2\. Funktion von Mikrotubuli im Post-Golgi-Vesikeltransport von Fibroblasten. Die gleichzeitige Beobachtung von Post-Golgi-Vesikeln und Mikrotubuli durch die Verwendung eines neuartigen Zweifarben-TIR-FM-Systems zeigte, daß die Vesikel an Mikrotubuli zum Ort ihrer Fusion transportiert werden. Dabei können die Vesikel noch zur Zeit der Fusion an die Mikrotubuli gebunden sein und müssen für die Fusion nicht notwendigerweise bis an das Ende der Mikrotubuli transportiert werden. Weiterhin zeigten unsere Ergebnisse, daß Exozytose von Vesikeln in wandernden Fibroblasten, im Gegensatz zu stationären Fibroblasten, bevorzugt in der Nähe des führenden Lamellipodiums ("leading edge") stattfindet. Nach Depolymerisierung der Mikrotubuli wurde dieser gerichtete Transport des Membranproteins (hier LDL-Rezeptor-GFP) unterbrochen und wandernde Fibroblasten konnten anhand der örtlichen Verteilung der Fusionen nicht mehr von stationären unterschieden werden. Die Depolymerisierung der Mikrotubuli führte außerdem zur Senkung der Fusionsfrequenz, zum Anstieg "unvollständiger" Fusionen und zum Anstieg der Menge an Membranproteinen, die während einzelner Fusionen an die Plasmamembran abgegeben wurden. Wir schließen daraus, daß Microtubuli für den gerichteten Transport von Membranproteinen in Post-Golgi- Vesikeln in Fibroblasten notwendig sind. 3\. Funktion von Mikrotubuli im Post- Golgi-Vesikeltransport von polari-sierten Epithelialzellen. Mittels zeitaufgelöster Fluoreszenzmikroskopie habe wir den Transport von apikalen und basolateralen Membranproteinen zur Plasmamembran sowohl in unpolarisierten als auch in polarisierten Epithelialzellen untersucht. Unsere Resultate zeigten, daß in unpolarisierten Zellen sowohl apikale als auch basolaterale Post-Golgi- Vesikel an der basalen Zellmembran fusionieren. Nach Polarisation der Zellen fand die basale Exozytose beider Vesikel nicht mehr statt. Stattdessen fusionierten basolaterale Vesikel direkt zu Regionen an der lateralen Membran, während apikale Vesikel zur apikalen Plasmamembran transportiert wurden. Dieser selektive Transport fand nach der Polarisation der Zellen statt, parallel zur Umverteilung der t-SNAREs (Syntaxin 3 und 4). Weitere Ergebnisse zeigten, daß die exklusive Lokalisierung von Syntaxin 3 und apikalen Membranproteinen intakte Mikrotubuli benötigt. Im Gegensatz dazu, ist die gerichtete Exozytose von basolateralen Membranproteinen und die Verteilung von Syntaxin 4 und sec6 in polarisierten Zellen unabhängig von dem Zustand der Mikrotubuli. 4\. Insulinabhängiges ?Recycling? des Glukosetransporters. Die insulin-abhängige Freigabe des Glukosetransporters GLUT4 vom endosomalen ?Recycling? Kompartiment (ERC für ?endosomal recycling compartment?) wurde untersucht. Die Ergebnisse zeigten, daß GLUT4 in Fibroblasten dynamisch in dem generellen ERC zurückgehalten wird, das auch den Transferrin-Rezeptor enthält. Mittels Zweifarben-TIR-FM demonstrieren wir, daß der Transferrin-Rezeptor und GLUT4 in separaten Vesikeln vom ERC zur Plasmamembran transportiert werden. Damit wurde der erste funktionellen Beweis für die Formation separater Vesikel vom ERC geliefert. Der langsame, insulinabhängige Transport der GLUT4-Vesikel kann dadurch erklärt werden, dass GLUT4 in Vesikel konzentriert wird, die sich langsamer bilden als Vesikel, die den Transferrin-Rezeptor beinhalten