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Genetic analysis of stoned B/stonin 2 function in vivo / Genetische Analyse der stoned B/stonin Funktion in vivoDiril, Muhammed Kasim 04 July 2004 (has links)
No description available.
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Recognition of basic sorting motifs within synaptic membrane cargo proteins by the clathrin-adaptor complex AP-2 / Die Erkennung basischer Sortierungsmotive in synaptischen Membranproteinen durch den Clathrin-Adaptor-Komplex AP-2Kastning, Kathrin 29 June 2005 (has links)
No description available.
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Endozytose der inneren Haarzelle / Endocytosis of inner hair cellsLenz, Christine 13 December 2011 (has links)
No description available.
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Proteomic analysis of the sorting machineries involved in vesicular traffic between the biosynthetic and endosomal compartmentsBaust, Thorsten Gerhard 05 September 2006 (has links)
Vesicular traffic along the biosynthetic and endocytic pathways is essential for homeostasis of eukaryotic cells. However, it raised the question of how the proteins characteristic for each compartment are transported to their destination (Bonifacino and Glick, 2004). This study is especially focusing on the connection between the Golgi apparatus and the endosomal compartment, mediated by two parallel trafficking pathways regulated by the clathrin adaptors AP-1A and AP-3 (Owen et al., 2004). Typical cargo molecules sorted along the AP-1A regulated pathway are mannose 6-phosphate receptors (MPRs) (Ghosh et al., 2003) or the gpI envelop glycoprotein of the Vesicular Zoster virus (Alconada et al., 1996), while sorting of lysosomal membrane proteins like Lamp-1 and LimpII is AP-3 regulated (Eskelinen et al., 2003). To study how AP-1A and AP-3 coats are stabilized on membranes and to identify the protein networks involved, a liposome based in vitro assay that recapitulates the fidelity of protein sorting in vivo was developed and combined with proteomic screens. Therefore, liposomes carrying cytoplasmic domains of gpI or Lamp-1/LimpII were used as affinity matrix to recruit selectively AP-1A or AP-3 and associated protein machineries. The coated liposomes were then analyzed by mass spectrometry. Using the in vitro recruitment assay, it was possible to demonstrate that efficient and selective recruitment of AP-1A and AP-3 coats depends on the presence of several low affinity binding sites on membranes. Thus, AP-1A and AP-3 recognize their target membranes by activated Arf1 GTPases, organelle specific phosphoinositides, PI-4P and PI-3P respectively, and distinct cargo molecules carrying intact signals in their cytoplasmic domains. The implication of PI-3P in AP-3 recruitment was further supported by in vivo experiments. During the biochemical characterization of the assay, several lines of evidence indicated that cargo tails containing intact sorting signals stabilize not only AP-1A and AP-3 coats on membranes but also influence the membrane recruitment of Arf1. It is possible that cargo molecules indirectly drive an Arf1 amplification loop, thereby ensuring efficient AP coat assembly. The proteomic screens identified protein networks of ≈40 proteins selectively recruited on AP-1A coated structures. The most appealing result of the analysis was the presence of two additional protein machineries, one involved in actin nucleation the other involved membrane fusion. More precisely, the AP-1A analysis identified the selective recruitment of the AP-1A subunits and interacting molecules (clathrin, g-synergin), Arf1 and Arf1 effectors (Big2, Git1), Rac1 including Rac1 effectors (b-PIX, RhoGEF7) and a Rac1 dependent actin nucleation machinery (Wave/Scar complex, Arp2/3 complex, associated effectors) as well as members of a Rab machinery (Rab11, Rab14). This finding was further supported by in vivo colocalization studies of the AP-1A cargo CI-MPR with CYFIP2, a protein of the Wave/Scar complex, and the localization of Big2 and Git1 on Rab11 positive membranes (Matafora et al., 2001; Shin et al., 2004). The biochemical characterization revealed that the stabilization of AP-1A coats, most probably driven by cargo molecules that stabilize AP-1A and Arf1 on membranes, leads as well to the stabilization of the two other machineries. Thus, the results support the notion that cargo sorting, vesicular movement and membrane fusion are coordinated during early steps of vesicular traffic. In analogy, the proteomic screens on AP-3 coated structures identified as well ≈40 selectively recruited proteins, which constituted a similar supramolecular network of protein machineries involved in coat formation, action nucleation and membrane fusion via Rab proteins. Thus, beside the AP-3 coat including the AP-3 subunits, Arf1 and Arf effectors (Big1, ARAP1, AGAP1), members of the septin family involved in actin rearrangements and most of the already described effectors of Rab5 microdomains (EEA1, Rabaptin-5, Rabex-5, Vps45) involved in early endosomal dynamics were selectively recruited together with Rab5 and Rab7. Thus, the proteomic analysis of AP-1A and AP-3 coated structures suggest that both AP coats use similar principles - coats, actin nucleation devices and Rab fusion machineries - to assemble supramolecular structures needed for membrane traffic. Although we do not have the ultimate proves yet, it seems as AP-1A and AP-3 use different members of subcomplexes, hence different GTPase effectors, different actin nucleation machineries and different Rab GTPases, to regulate their specific transport pathways and to link the different protein machineries. The proteomic analysis revealed for example that they probably use different Arf and Rho GTPase effectors to link the coat with actin nucleation. However, this has to be proven experimentally. In order to understand the networks of protein interactions, bioinformatic tools were used as a first approach. Even though some clues about the overall organization of the supramolecular protein complexes were provided, the direct links to the Rab machinery are still elusive. Maybe the proteins with thus far unknown functions could be involved. The biochemical analysis, especially the role of PIPs, and the Rab GTPases identified in the context of AP-1A and AP-3, provide indications about AP-1A and AP-3 function in vivo. The results could be interpreted in a way that AP-1A functions either in traffic from PI-4P positive membranes towards Rab11/Rab14 positive membranes or AP-1A coats assemble on PI-4P and Rab11 or Rab14 positive membranes, hence, TGN to endosomes traffic. The same holds true for AP-3, the results either suggest AP-3 mediates traffic from PI-3P positive towards Rab5/Rab7 positive membranes or they could be interpreted in a way that AP-3 assembles on PI-3P and Rab5 positive membranes for subsequent transport to Rab7 positive membranes, thus traffic from early to late endosomes. Overall, the results of this thesis research provided important insight into the formation of AP-1A and AP-3 coated structures and the potential interconnection between AP coats, actin nucleation and membrane fusion machineries. Alconada, A., U. Bauer, and B. Hoflack. 1996. A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network. Embo J. 15:6096-110. Bonifacino, J.S., and B.S. Glick. 2004. The mechanisms of vesicle budding and fusion. Cell. 116:153-66. Eskelinen, E.L., Y. Tanaka, and P. Saftig. 2003. At the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol. 13:137-45. Ghosh, P., N.M. Dahms, and S. Kornfeld. 2003. Mannose 6-phosphate receptors: new twists in the tale. Nat Rev Mol Cell Biol. 4:202-12. Matafora, V., S. Paris, S. Dariozzi, and I. de Curtis. 2001. Molecular mechanisms regulating the subcellular localization of p95-APP1 between the endosomal recycling compartment and sites of actin organization at the cell surface. J Cell Sci. 114:4509-20. Owen, D.J., B.M. Collins, and P.R. Evans. 2004. Adaptors for clathrin coats: structure and function. Annu Rev Cell Dev Biol. 20:153-91. Shin, H.W., N. Morinaga, M. Noda, and K. Nakayama. 2004. BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity. Mol Biol Cell. 15:5283-94.
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Engagement of T cells with Antigen Presenting Cells is Dependent on Clathrin-Independent Endocytic Trafficking: The Role of Arf6 and Rab22Johnson, Debra L. January 2016 (has links)
The clathrin-independent endosomal system is required for cellular homeostasis and specialized modifications of the plasma membrane such as cell spreading and polarization. Clathrin-independent endocytosis (CIE) has been demonstrated in adherent cells including fibroblasts and epithelial cells. However, non-adherent cells also have highly dynamic clathrin-independent pathways, which have not been well described. Here, I have characterized Arf6-associated clathrin-independent endocytosis (CIE) in the human T cell line Jurkat and identified it's importance in immunological synapse formation. Our findings indicate that the CIE pathway is similar in Jurkat cells as compared to other cell types including rates of endocytosis and sorting after internalization. Two GTPases, Arf6 and Rab22, have been shown to regulate the clathrin-independent endosomal system and play a role in cell spreading. We found that wild type and constitutively active Arf6 co-localized with CIE cargo in resting T cells. Arf6 constitutively active mutant inhibited CIE cargo internalization but not internalization of CME cargo. Rab22 co-localized with CIE cargo at the endocytic-recycling compartment. Expression of the dominant negative Rab22 mutant also inhibited internalization of MHCI indicating it plays a direct role in CIE cargo internalization. T cells must modify their membranes to specifically interact with antigen presenting cells. To establish the role of CIE in this process, we then examined the role of Arf6 and Rab22 in T cell/antigen presenting cell conjugate formation. Both expression of dominant negative or constitutively active mutants of Arf6 reduced T cell conjugate formation while expression of only the Rab22 dominant negative mutant inhibited T cell/APC conjugate formation. Furthermore, T cells expressing the dominant negative mutant of Rab22 were not able to spread on antibody-coated coverslips that normally cause T cell activation. These results indicate that the clathrin independent endosomal system is required for membrane remodeling events necessary for T cell conjugate formation and T cell spreading during activation. I also conducted a proteomics screen to identify binding partners of CIE cargo proteins. I identified multiple proteins that could possibly play a role in CIE internalization and discovered a subset of proteins that specifically interact with A cargo proteins and not B cargo proteins. It is possible they could play a role in cargo retention at the plasma membrane or sorting after internalization. Three proteins of interest that interact with A cargo include NHERF-1 and ezrin, which participate in actin arrangements, and Dlg-1, a known scaffolding protein for synaptic vesicles. Ezrin and Dlg-1 co-localize with the CIE cargo protein CD98 in HeLa cells indicating that they could be interacting in cells.
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Characterizing putative cellular mediators of West Nile virus infections in bird and mosquito tissuesPartridge, Alison 14 September 2015 (has links)
West Nile virus (WNV) is a mosquito-borne virus that infects many bird species. Examination of American crows and house sparrows from the Winnipeg region confirmed that WNV levels were at least 1000 times higher in crows than sparrows. No species differences were observed in the level of transcripts encoding a putative WNV receptor, β3 integrin. Differences in mosquito vector competence can be due to differences in the ability of WNV to enter mosquito cells. Using RNAi techniques, the role of two clathrin coat adaptor proteins in facilitating WNV infections in mosquito cells was examined, and the findings suggest that these proteins may act as resistance factors in Aedes aegypti, and as susceptibility factors in Culex quinquefasciatus. These findings will contribute to our understanding of the molecular basis of vector competence in different mosquitoes, and may help us determine whether other species could serve as potential vectors of this health-threatening virus. / October 2015
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Rôle de la clathrine dans le processus infectieux du champignon phytopathogène Botrytis cinerea / Role of clathrin in infection process of fungal plant pathogen Botrytis cinereaSouibgui, Eytham 04 May 2017 (has links)
Les champignons sont les principaux agents pathogènes des plantes. Leur étude est donc essentielle pour contrôler les maladies et maintenir un bon rendement de production agricole. La nutrition de ces pathogènes est basée sur l'absorption de nutriments, préalablement dégradés par un arsenal d'enzymes lytiques secrétées. La sécrétion des protéines est assurée par le trafic intracellulaire mettant en jeu de nombreuses vésicules. Chez les champignons filamenteux, ces vésicules ont été visualisées en microscopie électronique mais le processus mis en jeu pour leur biogénèse n'est toujours pas élucidé. L'identification de ce mécanisme est un donc un prérequis pour comprendre la sécrétion de facteurs de virulence. Dans ce but, un mutant non pathogène altéré au niveau de l'expression du gène codant la chaine lourde de la clathrine a été sélectionné parmi une banque de mutants générés chez le champignon nécrotrophe Botrytis cinerea. Le gène codant pour la chaine lourde de la clathrine est essentiel chez de nombreux organismes, ainsi un mutant dominant négatif de la chaine lourde de la clathrine a été généré et confirme la perte de pathogénicité. La caractérisation du mutant par une approche de protéomique a mis en évidence un défaut de sécrétion de 82 protéines incluant des facteurs de virulence connus. Un défaut de production de vésicules intracellulaires a également été constaté. Par ailleurs, le marquage de la clathrine à la GFP a permis de préciser sa localisation dans les cellules fongiques. Enfin, de façon surprenante, aucun défaut d'endocytose n'a été constaté au sein des mutants déficients en clathrine. Cette étude met en évidence pour la première fois le rôle essentiel de la clathrine dans le processus infectieux d'un champignon pathogène ainsi que son rôle dans a sécrétion de facteurs de virulence / Fungi are the most important plant pathogens on agricultural and horticultural crops. Study of fungal pathogens remains essential to understand pathogenic process and control plant diseases. These organisms secrete high amount of degrading enzymes involved in plant decomposition and they feed by absorption of degraded nutriments. Secretory proteins were described to be transported form Endoplasmic Reticulum and Golgi apparatus to extracellular space through intracellular vesicles. In filamentous fungi, intracellular vesicles were observed using electron microscopy but their biogenesis process is still unknown. Therefore, elucidation of the process and the identification of proteins involved in secretory vesicles biogenesis remains a challenge to understand virulence factors delivery. A nonpathogenic mutant altered in the expression of the gene coding for clathrin heavy chain was selected in a random mutant library generated in the necrotrophic pathogen Botrytis cinerea,. This gene is essential in many organisms, thus a clathrin dominant negative mutant was generated and confirming the nonpathogenic phenotype observed on several host plant. In eukaryotic cells, clathrin heavy chain is mainly described to be involved in endocytosis, but it is also essential for high density secretory vesicles formation in yeast. Characterization of the mutants using a proteomic approach revealed a secretion defect of 82 proteins including known virulence factors, as Plant Cell Wall Degrading Enzymes and elicitors. Furthermore, the clathrin mutant revealed a strong reduction of intracellular vesicles production. Clathrin was also localized in living cells using fluorescent GFP-tag protein. Endocytosis was also studied and surprisingly, any observable defect was observed for clathrin mutants. This study demonstrated for the first time the essential role of clathrin in the infectious process of a fungal pathogen and its role in virulence factors secretion
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Synaptic vesicle recycling in preclinical models of intellectual disability, autism spectrum disorder and epilepsyBonnycastle, Katherine January 2018 (has links)
The development of the central nervous system is dysregulated in neurodevelopmental disorders such as intellectual disability, autism spectrum disorder, and epilepsy. These three disorders have different clinical features, yet there is high comorbidity between them. They can be difficult to study due to their highly complex aetiologies, however there are various monogenic diseases that can cause all of them, including SYNGAP1 haploinsufficiency where the synaptic guanosine triphosphatase (GTPase)-activating protein (SYNGAP) protein levels are highly reduced; Fragile X syndrome where the fragile X mental retardation protein (FMRP) is no longer translated; and DNM1 epileptic encephalopathy where mutations in the Dynamin1 gene alter the protein function. These monogenic conditions are synaptopathies as the proteins affected play important roles in synapse stability and neurotransmission. Because of the high comorbidity between these disorders, it is hypothesised that there may be a common mechanism underlying them. We hypothesise that a deficit in presynaptic vesicle recycling may be part of a common mechanism underlying intellectual disability, autism spectrum disorder, and epilepsy especially in SYNGAP1 haploinsufficiency, Fragile X syndrome, and DNM1 epileptic encephalopathy. Using various fluorescent presynaptic activity reporters including synaptic pHluorins, tetramethylrhodamine dextran and calcium dyes to compare presynaptic activity in in vitro models of these monogenic conditions, we found differences in synaptic vesicle (SV) endocytosis in the genetically altered conditions compared to wildtype controls. We observed various SV endocytosis defects in clathrin-mediated endocytosis (CME) or activity-dependent bulk endocytosis (ADBE) in our models. We observed enhanced CME in SynGAP1 KO mouse hippocampal neurons. This enhanced SV endocytosis was accompanied by decreased SV cargo on the plasma membrane. Rat SynGAP1 KO hippocampal neurons did not display enhanced SV endocytosis, nor did neurons with the GTPase-activating (GAP) domain of SynGAP deleted. This was perhaps due to the altered time course of development between these rodent species. In mouse and rat models of Fragile X syndrome, CME was not altered compared to wildtype controls. However, in a rat model, we observed fewer nerve terminals undergoing ADBE which is the dominant SV endocytosis mode during elevated neuronal activity. De novo epileptic encephalopathy-associated mutations in DNM1 had differential effects on SV recycling through both CME and ADBE. Mouse hippocampal neurons overexpressing Dyn1R237W, Dyn1I289F and Dyn1H396D all showed less CME compared to overexpression of Dyn1WT. Moreover, fewer nerve terminals overexpressing Dyn1H396D were found to undergo ADBE. We also found that a large-conductance potassium (BK) channel opener can accelerate clathrin-mediated endocytosis and thus may be able to rescue the impaired SV endocytosis caused by these mutants. Although there is not yet a common underlying pathway at the presynaptic level between these conditions, SV recycling dysfunction is present across all of these models. Furthermore, we propose an axis of pathophysiology model where optimal SV endocytosis is required for optimised neural performance. We propose that either decreased or increased SV endocytosis can lead to the synaptic dysfunction observed in these models.
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Viral subversion of host cell membrane traffickingMuenzner, Julia January 2017 (has links)
Enveloped viruses acquire their membrane coat from the plasma membrane or intracellular organelles and rely on cellular machinery to facilitate envelopment and egress of virus progeny. This thesis examines egress-related interactions between host cell factors and proteins of two different enveloped viruses: hepatitis D virus (HDV) and herpes simplex virus 1 (HSV-1). HDV is a small RNA virus causing fulminant hepatitis or severely aggravating cirrhosis and hepatocellular carcinoma. HSV-1 is a large DNA virus infecting epithelial and neuronal cells. Infection with HSV-1 not only triggers the development of recurring sores on oral or genital mucosa, but can also cause severe disease in neonates and immunocompromised patients. The interaction between the large antigen of HDV (HDAg-L) and the N-terminal domain (NTD) of clathrin, a protein crucial for endocytosis and intracellular vesicular trafficking, was examined by structural, biochemical and biophysical techniques. Co-crystal structures of NTD bound to HDAg-L peptides derived from different HDV genotypes revealed that HDV interacts with multiple binding sites on NTD promiscuously, prompting re-evaluation of the binding between cellular peptides and NTD. Surprisingly, co-crystal structures and pull-down capture assays showed that cellular peptides containing clathrin-binding motifs can also bind multiple sites on the surface of NTD simultaneously. In addition, the structures of viral and cellular peptides bound to NTD enabled the molecular characterization of the fourth peptide binding site on NTD, the “Royle box”, and led to the identification of a novel binding mode at the “arrestin box” peptide binding site on NTD. The work in this thesis therefore not only identifies the molecular basis of HDV:clathrin interactions, but also furthers our understanding of basic clathrin biology. Even though many HSV-1 proteins have been implicated in the envelopment and egress of viral particles, only few interactions between HSV-1 and cellular proteins promoting these processes have been described. Therefore, the HSV-1 proteins gE, UL21 and UL56 were selected and characterized bioinformatically and/or biochemically. Cellular proteins interacting with UL56 were identified by yeast two-hybrid screening and quantitative mass spectrometry. Co-immunoprecipitation and pull-down experiments confirmed the Golgi-trafficking protein GOPC, components of the mammalian trafficking protein particle complex, and the ubiquitin ligase NEDD4 as novel binding partners of UL56, thereby suggesting exciting new avenues for the investigation of cellular mechanisms contributing to HSV-1 envelopment and egress.
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Clathrin Independent Carriers: Molecular characterisation of a novel clathrin-independent endocytic pathwayMark Howes Unknown Date (has links)
Endocytosis effectuates a critical interface between the eukaryotic cell and its apposing environment. It is, subsequently, paramount for many physiologically important processes and encompasses a diverse array of mechanisms and pathways. The classical endocytic routes mediated by clathrin and caveolin are the best understood and the molecular roles of their major regulators, such as dynamin, adaptor proteins and various lipid species, are the most comprehensively described. Recent identification of an assortment of constitutive, noncaveolar, clathrin-independent endocytic (CIE) pathways has expanded the endocytic system. Unlike the classical endocytic pathways, little is known about the guiding parameters of CIE routes. Consequently, it is not possible to understand the important cellular roles these pathways may be fulfilling. This study has begun to characterise the very basic parameters governing the morphologically striking Clathrin-Independent Carrier (CLIC) pathway. Development of a diverse molecular toolkit has now allowed the quantitation of endocytic capacity provided by CLICs, the visualisation of subtle sorting components of the CLIC pathway, the isolation of novel CLIC cargo and regulators, and has linked this mechanism to the critical cellular processes of cellular migration and membrane repair. Calculation of the individual capacity of endocytic routes provides important information about the contribution of each pathway to total plasma membrane (PM) uptake and turnover. Quantitation of the volume, surface area and number of structures forming per minute in this study shows that CLICs provide the vast majority of constitutive endocytosis, up to four times the capacity of the clathrin mediated endocytic (CME) pathway. As the equivalent of the entire PM area could pass through the CLIC pathway within 12 minutes it is evident that CLICs are fundamental housekeepers of bulk membrane internalisation. Thus, they are likely to be central regulators of PM homeostasis and turnover. High-resolution tomography, in conjunction with analysis of CLIC cargo trafficking, identifies these carriers as complex, pleiomorphic structures that sort the bulk of membrane to early endosomes and recycle cargo back to the cell surface. Such vast internalisation combined with an ability to rapidly recycle components quickly attributes the CLIC pathway as a complex sorting station. Isolation of novel cargo and regulators has identified a striking array of proteins now associated with the CLIC pathway for the first time. A significant proportion of identified targets localise to lipid-rafts and recycle from the PM, facets consistent with association to the CLIC pathway. Numerous targets have also been directly implicated in clathrin-independent endocytosis by independent groups. Verification of selected cargo, such as CD44, Thy-1 and myoferlin, showing specific internalisation through the CLIC pathway, has provided insight into the sorting ability of the CLIC pathway and links to adhesion turnover and membrane recycling. Consistent with a role in cellular adhesion turnover, it was found that CLICs become polarised within migrating cells. This has shown the first instance of spatial separation between three major endocytic routes, CLICs, caveolae and CME and highlights the important and coordinated roles of multiple endocytic pathways during physiologically significant processes. The specific internalisation of paxillin, Thy-1 and CD44 through CLICs at the leading edge of migrating cells suggests that CLICs rapidly turnover adhesion components for dynamic extracellular sensation during directional cell migration. Indeed, specific ablation of the CLIC pathway significantly impedes cellular migration, implying coordination with CME at the leading edge. This study has defined numerous parameters of the CLIC pathway, developing the current understanding of this poorly defined route and places the CLIC pathway as a unique player during critical cellular processes.
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