Letter | Published:

Hemi-fused structure mediates and controls fusion and fission in live cells

Nature volume 534, pages 548552 (23 June 2016) | Download Citation

Abstract

Membrane fusion and fission are vital for eukaryotic life1,2,3,4,5. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission1,4,6,7,8,9,10. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed2,11,12,13,14,15. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic β-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.

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Acknowledgements

We thank T. Balla, G. Miesenböck, and D.-S. Wang for providing the construct containing the GFP-fused PH domain of PLCδ1, the VAMP2–pHluorin construct, and dynamin siRNA, respectively; C. Smith for technical support with STED microscopy; and S. Cheng and V. Crocker for their electron microscopy technical support. This work was supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program (ZIA NS003009-13 and ZIA NS003105-08) and National Institute on Deafness and Other Communication Disorders (NIDCD) Intramural Research Program (Z01-DC000002 and NIDCD Advanced Imaging Core ZIC DC000081).

Author information

Author notes

    • Hsueh-Cheng Chiang

    Present address: Department of Pharmacology, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan City, Taiwan 701.

    • Wei-Dong Zhao
    •  & Edaeni Hamid

    These authors contributed equally to this work.

Affiliations

  1. National Institute of Neurological Disorders and Stroke, 35 Convent Drive, Room 2B-1012, Bethesda, Maryland 20892, USA

    • Wei-Dong Zhao
    • , Edaeni Hamid
    • , Wonchul Shin
    • , Peter J. Wen
    • , Seth A. Villarreal
    • , Hsueh-Cheng Chiang
    •  & Ling-Gang Wu
  2. National Institute on Deafness and other Communication Disorders, 35A Convent Drive, Room 3D-824, Bethesda, Maryland 20892, USA

    • Evan S. Krystofiak
    •  & Bechara Kachar

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Contributions

W.-D.Z, E.H. and W.S. performed and analysed most experiments. P.J.W. and H.-C.C. initiated STED imaging of PH–eGFP. E.S.K., S.A.V. and B.K. performed electron microscopy. L.-G.W. designed experiments and wrote the manuscript with help from all authors. W.-D.Z. and E.H. participated in designing experiments and writing the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Ling-Gang Wu.

Reviewer Information Nature thanks A. T. Brunger, V. Haucke, M. M. Kozlov and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains the uncropped scans from Extended Data Figure 9a with size marker indications.

Videos

  1. 1.

    A same-onset spot

    A real-time video of a same-onset spot (same as in Fig. 1d) induced by depol1s at the cell bottom: PH-EGFP (green, left) and A655 (red, right) fluorescence increased simultaneously. Each frame is an average of 15 single images collected every 33 ms. Thus, the interval between frames is 495 ms. The whole video takes 20 s. The horizontal length of each image is 1.15 μ.

  2. 2.

    A PH-earlier spot

    A real-time video of a PH-earlier spot induced by depol1s at the cell bottom: PH-EGFP fluorescence increase (green, left) preceded A655 fluorescence (red, right) increase. Each frame is an average of 15 single images collected every 33 ms. Thus, the interval between frames is 495 ms. The whole video takes 12.5 s. The vertical length of each image is 1.05 μ.

  3. 3.

    A PH-only spot

    A real-time video of a PH-only spot induced by depol1s at the cell bottom: PH-EGFP fluorescence increase (green, left) was not accompanied by A655 fluorescence (red, right) increase. Each frame is an average of 15 single images collected every 33 ms. Thus, the interval between frames is 495 ms. The whole video takes 24.5 s. The vertical length of each image is 1.1 μ.

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DOI

https://doi.org/10.1038/nature18598

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