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

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.

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Figure 1: Supported membrane tubes.
Figure 2: Dynamin scaffold assembly.
Figure 3: GTP hydrolysis-induced tube constriction precedes tube scission.
Figure 4: Coordination between scaffold assembly and tube scission in constant presence of GTP.
Figure 5: Role of dynamin PHD in tube scission.
Figure 6: Proposed mechanism of dynamin-catalysed tube scission.

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Acknowledgements

We thank S. Schmid, R. Mallik and the Pucadyil laboratory members for discussions and critical comments on the manuscript, S. Holkar for the scanning electron microscopy, and V. Vitthal for confocal microscopy. S.D. and S.C.K. acknowledge the Council for Scientific and Industrial Research (CSIR) for fellowships. T.J.P. is an Intermediate Fellow of the Wellcome Trust-DBT India Alliance and thanks the Alliance and IISER Pune for funds.

Author information

S.D. and T.J.P. designed experiments. S.D. performed all experiments. S.D. and S.C.K. designed and standardized preparation of SMrT templates. S.D. and T.J.P analysed data and wrote the manuscript.

Correspondence to Thomas J. Pucadyil.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Calculation of calibration constant to equate tube fluorescence to physical dimensions.

Fluorescence micrographs of SMrT templates with preassembled scaffolds are acquired in the tube fluorescence channel (Step 1). The bimodal frequency distribution of pixel intensities along the tube length is fitted to a sum of 2 gaussian function (Step 2). The lower mean represents the tube fluorescence under the scaffold, which is then equated to the tube radius by I = KR2 where, I = tube fluorescence under scaffold; R = tube radius, 11.2 nm9 to calculate the calibration constant K.

Supplementary Figure 2 GTP hydrolysis-induced tube scission leads to splitting of scaffolds.

A panel of kymographs from time-lapse movies monitoring GTP addition to Alexa488-labeled dynamin scaffolds preassembled on SMrT templates.

Supplementary information

Supplementary Information

Supplementary Information (PDF 799 kb)

Dynamics of dynamin scaffold assembly on SMrT templates.

The movie shows tube fluorescence changes on scaffold assembly on SMrT templates. Scale bar = 5 μm. (MOV 4242 kb)

Dynamics of dynamin scaffold assembly on a freestanding membrane tether.

The movie shows tube fluorescence changes on scaffold assembly on a freestanding tether. Scale bar = 5 μm. (MOV 1716 kb)

Tube scission events with preassembed scaffolds on SMrT templates.

The movie shows tube fluorescence changes in response to GTP addition to preassembled scaffolds on SMrT templates. Scale bar = 5 μm. (MOV 501 kb)

Tube scission events with dynamin in the constant presence of GTP on SMrT templates.

The movie shows tube fluorescence changes in response to dynamin addition to SMrT templates bathed in excess (1 mM) GTP. Scale bar = 5 μm. (MOV 1237 kb)

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Dar, S., Kamerkar, S. & Pucadyil, T. A high-throughput platform for real-time analysis of membrane fission reactions reveals dynamin function. Nat Cell Biol 17, 1588–1596 (2015). https://doi.org/10.1038/ncb3254

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