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A high-throughput platform for real-time analysis of membrane fission reactions reveals dynamin function
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A high-throughput platform for real-time analysis of membrane fission reactions reveals dynamin function
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Journal Article
A high-throughput platform for real-time analysis of membrane fission reactions reveals dynamin function
2015
Overview
Dynamin, the paradigmatic membrane fission catalyst, assembles as helical scaffolds that hydrolyse GTP to sever the tubular necks of clathrin-coated pits. Using a facile assay system of supported membrane tubes (SMrT) engineered to mimic the dimensions of necks of clathrin-coated pits, we monitor the dynamics of a dynamin-catalysed tube-severing reaction in real time using fluorescence microscopy. We find that GTP hydrolysis by an intact helical scaffold causes progressive constriction of the underlying membrane tube. On reaching a critical dimension of 7.3 nm in radius, the tube undergoes scission and concomitant splitting of the scaffold. In a constant GTP turnover scenario, scaffold assembly and GTP hydrolysis-induced tube constriction are kinetically inseparable events leading to tube-severing reactions occurring at timescales similar to the characteristic fission times seen
in vivo
. We anticipate SMrT templates to allow dynamic fluorescence-based detection of conformational changes occurring in self-assembling proteins that remodel membranes.
Pucadyil and colleagues develop an
in vitro
technique to analyse the conformational dynamics of dynamin during membrane fission events in a real-time, high-throughput manner, using fluorescence microscopy.
Publisher
Nature Publishing Group UK,Nature Publishing Group
Subject
/ Cell Membrane - ultrastructure
/ Clathrin
/ Cleavage
/ Coated Pits, Cell-Membrane - chemistry
/ Coated Pits, Cell-Membrane - metabolism
/ Dynamin
/ Fission
/ Fluorescence Recovery After Photobleaching
/ Guanosine Triphosphate - chemistry
/ Guanosine Triphosphate - metabolism
/ Humans
/ Membrane Lipids - metabolism
/ Methods
/ Microscopy, Electron, Scanning - methods
/ Mutation
/ Phosphatidylinositol 4,5-Diphosphate - chemistry
/ Phosphatidylinositol 4,5-Diphosphate - metabolism
/ Pits
/ Time-Lapse Imaging - methods
/ Tubes
