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Visualizing the GTPase activity of dynamin with nanobodies

Defense Thèse de doctorat : Univ. Genève, 2016 - Sc. 4905 - 2016/02/18
Abstract Dynamin is a large GTPase that mediates membrane fission at the last step of clathrin mediated endocytosis through helical polymerization and membrane constriction of the neck of the clathrin coated vesicle triggered by GTP hydrolysis. One current model states that membrane fission occurs with a concerted constriction of the dynamin polymer along the neck of the membrane which reduces its inner diameter by sliding of adjacent turns. In vitro studies have shown that fission events are stochastic and that dynamin has no cooperativity towards GTP hydrolysis, thus it is still unclear how dynamin can coordinate such constriction events in vivo and the dynamics of dynamin constriction events in cells have never been revealed. Here, we select a conformational-specific nanobody (dynact), able to bind preferentially to the GTP hydrolyzing/constricted state of dynamin 1 and we describe the occurrence of GTPase activity of dynamin 1 in mice fibroblasts. By analyzing the colocalization over time of dynamin 1 and dynact, we show that GTP hydrolysis/constriction events in cells are unpredictable and can occur at different times during dynamin permanence at the membrane and are not correlated with the size of the dynamin polymer, confirming the stochasticity of dynamin activity during the fission event.
Keywords DynaminEndocytosisNanobodiesPNACaprin-1Dynact
URN: urn:nbn:ch:unige-822956
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Research group Groupe Roux
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GALLI, Valentina. Visualizing the GTPase activity of dynamin with nanobodies. Université de Genève. Thèse, 2016. https://archive-ouverte.unige.ch/unige:82295

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Deposited on : 2016-04-04

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