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Nat Cell Biol. 2015 Dec;17(12):1588-96. doi: 10.1038/ncb3254. Epub 2015 Oct 19.

A high-throughput platform for real-time analysis of membrane fission reactions reveals dynamin function.

Author information

1
Indian Institute of Science Education and Research, Dr. Homi Bhabha Road Pashan, Pune 411008, Maharashtra, India.

Abstract

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.

PMID:
26479317
DOI:
10.1038/ncb3254
[Indexed for MEDLINE]

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