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Dynamin A as a one-component division machinery for synthetic cells

Nature Nanotechnology volume 19, pages 70–76 (2024)Cite this article

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Abstract

Membrane abscission, the final cut of the last connection between emerging daughter cells, is an indispensable event in the last stage of cell division and in other cellular processes such as endocytosis, virus release or bacterial sporulation. However, its mechanism remains poorly understood, impeding its application as a cell-division machinery for synthetic cells. Here we use fluorescence microscopy and fluorescence recovery after photobleaching measurements to study the in vitro reconstitution of the bacterial protein dynamin A inside liposomes. Upon external reshaping of the liposomes into dumbbells, dynamin A self-assembles at the membrane neck, resulting in membrane hemi-scission and even full scission. Dynamin A proteins constitute a simple one-component division machinery capable of splitting dumbbell-shaped liposomes, marking an important step towards building a synthetic cell.

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Fig. 1: DynA localizes at the necks of dumbbells and affects membrane connectivity.
Fig. 2: Quantification of membrane-remodelling events at necks in the presence or absence of DynA.
Fig. 3: Characterization of DynA-induced membrane hemi-scission events.
Fig. 4: Example of a full scission event that was captured live.
Fig. 5: DynA in membrane scission and fusion.

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Data availability

The data that support the findings of this study are available within the paper and its Supplementary Information. Other relevant data are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

The image analysis source code is available via Zenodo at https://zenodo.org/record/8056488.

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Acknowledgements

We thank S. J. Marrink and W. Pezeshkian for useful discussions, and M. Bramkamp for kindly providing the plasmid for B. subtilis DynA. We acknowledge funding support from the BaSyC program of NWO-OCW, from ERC Advanced Grant 883684 and from Regenerative Mechanisms for Health—ReMedy project MAB/2017/2.

Author information

Authors and Affiliations

  1. Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, The Netherlands

    Nicola De Franceschi, Roman Barth, Sabrina Meindlhumer, Alessio Fragasso & Cees Dekker

  2. IMol Polish Academy of Sciences, Warsaw, Poland

    Nicola De Franceschi

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  1. Nicola De Franceschi
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Contributions

C.D., N.D.F. and S.M. designed the study. N.D.F. and C.D. designed the experiments. N.D.F. and S.M. carried out the experiments. N.D.F., R.B. and A.F. analysed the data. C.D. supervised the study. C.D. and N.D.F. wrote the manuscript with input from all other authors. All the authors provided critical feedback on the research and the manuscript.

Corresponding author

Correspondence to Cees Dekker.

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The authors declare no competing interests.

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Nature Nanotechnology thanks Gerhard Gompper, Oskar Staufer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Membrane topologies.

Relative positions of membrane and protein machinery for the forward and reverse topology. The membrane is in magenta, proteins are in green.

Extended Data Fig. 2 Schematic depicting the expected values of NI.

Schematic depicting the expected values of NI for different amounts of membrane reservoir in control and bleached lobes. (a) equal amount; (b) larger reservoir in control lobe; (c) larger reservoir in the bleached lobe.

Extended Data Fig. 3 Recovery times in FRAP experiments and example of open neck in the absence of DynA.

(a) Median of the duration of recovery times in FRAP experiments. Acquisition was stopped when no further lipid recovery was apparent. This quantitation is obtained from datasets such as shown in Fig. 2a,b and Supplementary Fig. 5b. n = 190 necks from 24 independent experiments. Data are presented as median values +/− SEM. (b) example of FRAP experiment showing full lipid recovery (open neck) on a chain of dumbbells without reconstitution of DynA. The orange asterisk indicates the control lobe, the blue asterisk indicates the bleached lobe. Scale bar: 5µm.

Source data

Extended Data Fig. 4 Lobe detachment upon scission.

(a) Confocal images of a dumbbell with a DynA cluster (in green) localized at neck. The dumbbell was imaged just as scission was occurring and the two lobes are drifting apart. (b) same dumbbell as in panel a. After lobes separation, FRAP analysis confirmed that the membrane connection between the two lobes had been lost and that no lipid recovery occurred. The bleached lobe is indicated by a blue asterisk, and its position is revealed by the low fluorescent signal of DynA binding to the membrane in the overexposed image.

Extended Data Fig. 5 Quantification of the NI in different experimental conditions.

(a) Quantification of the NI of a large dataset of necks from chains of dumbbells. This plot shows the data pooled from the three experimental conditions shown in Fig. 2a,b and Supplementary Fig. 6b (n = 190 chains of dumbbells), and are decomposed into three peaks using a Gaussian Mixture Model. The three peaks correspond to open necks, hemi-scission and full scission. For each data point, the fluorescent lipids of one lobe were bleached and their recovery was followed over time until reaching the plateau. The final intensity was used for calculating and plotting the NI. (b) Plot showing the NI of dumbbells having only bare membrane (left), and pie-chart (right) indicating the fraction of open necks, hemi-scission and full scission events (BN; 63 necks from 5 independent preparations).

Source data

Extended Data Fig. 6 Membrane remodelling by DynA in different experimental conditions.

(a) pie-charts indicating the fraction of open necks, hemi-scission and full scission events in the presence or absence of GTP. Both samples include cholesterol-oligo, CN and DynA. With GTP, n = 59; without GTP n = 10. (b): lack of binding of DynA-D2 domain to membrane containing 7.5% DOPG in the absence of Mg++. Scale bar: 5µm. (c) DynA assembly at the neck results in neck constriction. The plot shows the neck diameter in the presence or absence of DynA, estimated based on dye recovery upon photobleaching. In open necks and in the absence of DynA, the average inner diameter of necks was 134 ± 108 nm (mean ± SD). In the presence of DynA, the width of open necks appeared more constricted, with an average diameter of 57 ± 33 nm (mean ± SD). n = 8 without DynA; n = 9 with DynA. The estimation has been performed according to the procedure detailed in Ref. 18.

Source data

Extended Data Fig. 7 Recovery in hemi-scission state is achieved via the outer leaflet.

(a) Full recovery of both fluorescent lipids (which are in both leaflets of the bilayer) and fluorescent nanostars (which attach to the outer leaflet only) upon photo-bleaching of a dumbbell liposome having an open neck (without DynA). Scale bar: 5µm. (b) Partial recovery of fluorescent lipids and full recovery of fluorescent nanostars upon photo-bleaching of a dumbbell liposome with a DynA-D2 cluster. The neck is in a hemi-scission state, as visualized by the partial lipid recovery. The green arrowhead indicates the position of the neck connecting the two lobes. Scale bar: 10µm.

Extended Data Fig. 8 Membrane remodelling by Dyna-D1 and DynA-D2 domains.

(a) DynA-D1 domain reconstituted at necks of dumbbell liposomes. (b) DynA-D2 domain reconstituted at necks of dumbbell liposomes. Arrowheads indicate protein clusters localized at necks. Scale bars: 10µm. (c) Pie-charts indicating the fraction of open necks, hemi-scission, and full scission events upon reconstitution of DynA D1 and D2 domains. n = 12 necks from 3 independent preparations (D1); n = 27 from 3 independent preparation (D2).

Source data

Extended Data Fig. 9 Lobe detachment upon scission and visualization of nanotube.

(a) confocal image of liposomes that underwent full scission (identical to Fig. 4e). (b) Same image as in (a) but with increased gain, causing overexposure of the liposome membrane. This condition confirms that the two lobes indeed underwent scission, since no nanotube is visibly connecting the two liposomes. Note that in panel d, a nanotube was visible upon overexposure. Scale bar: 10µm. (c) Confocal image of two liposomes under standard imaging conditions which allow to clearly see the liposome membrane without overexposure. Upon bleaching of one liposome (marked with a blue asterisk) full lipid recovery could be observed. (d) Same images as in panel c with increased gain, causing overexposure of the liposome membrane. Such conditions reveal that a membrane nanotube is connecting the two liposomes and allows full lipid recovery. Scale bar: 5µm.

Extended Data Table 1 Composition of inner and outer solutions
Full size table

Supplementary information

Supplementary Information

Supplementary Notes 1 and 2 and Figs. 1 and 2.

Supplementary Video 1

Liposome deformation into a chain of dumbbells. Video of a confocal plane across a liposome as it is being generated by the SMS approach. The video captures the progressive deformation of the initially oblate liposomes into a chain of dumbbells. Liposome drifting as it sinks is compensated by manually moving the stage. Lipid fluorescence is shown in magenta. Frame rate, one image per second.

Supplementary Video 2

Stable localization of DynA at neck of a dumbbell liposome. Video of a confocal plane across a liposome. The video shows a DynA cluster stably localized at the neck. Lipid fluorescence is shown in magenta and DynA in green. Frame rate, one image per second.

Supplementary Video 3

Full recovery of fluorescent lipids in dumbbell liposome. Video of a confocal plane across a liposome during the FRAP experiment. The video shows lipid bleaching and progressive full recovery of fluorescent lipids flowing from the adjacent lobes through the neck. Lipid fluorescence is shown in magenta and DynA in green. Frame rate, one image per second.

Supplementary Video 4

Partial recovery of fluorescent lipids in dumbbell liposome. Video of a confocal plane across a liposome during the FRAP experiment. The video shows lipid bleaching and progressive partial recovery of fluorescent lipids flowing from the adjacent lobes through the neck. Lipid fluorescence is shown in magenta and DynA in green. Frame rate, one image per second.

Supplementary Video 5

Lack of recovery of fluorescent lipids in dumbbell liposome. Video of a confocal plane across a liposome during the FRAP experiment. The video shows lipid bleaching and the lack of recovery of fluorescent lipids. Lipid fluorescence is shown in magenta and DynA in green. Frame rate, one image per second.

Supplementary Video 6

Scission event captured live. Video of a confocal plane across a dumbbell liposome during the FRAP experiment, following the steps denoted in Fig. 4. The video shows a partial lipid recovery after the photobleaching of one lobe and the subsequent detachment of another lobe that underwent scission. Frame rate, five images per second.

Source data

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Source Data Extended Data Fig. 3

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Source Data Extended Data Fig. 5

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Source Data Extended Data Fig. 6

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Source Data Extended Data Fig. 8

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De Franceschi, N., Barth, R., Meindlhumer, S. et al. Dynamin A as a one-component division machinery for synthetic cells. Nat. Nanotechnol. 19, 70–76 (2024). https://doi.org/10.1038/s41565-023-01510-3

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