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Single-molecule in vitro reconstitution assay for kinesin-1-driven membrane dynamics

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Abstract

Intracellular membrane dynamics, especially the nano-tube formation, plays important roles in vesicle transportation and organelle biogenesis. Regarding the regulation mechanisms, it is well known that during the nano-tube formation, motor proteins act as the driven force moving along the cytoskeleton, lipid composition and its associated proteins serve as the linkers and key mediators, and the vesicle sizes play as one of the important regulators. In this review, we summarized the in vitro reconstitution assay method, which has been applied to reconstitute the nano-tube dynamics during autophagic lysosomal regeneration (ALR) and the morphology dynamics during mitochondria network formation (MNF) in a mimic and pure in vitro system. Combined with the single-molecule microscopy, the advantage of the in vitro reconstitution system is to study the key questions at a single-molecule or single-vesicle level with precisely tuned parameters and conditions, such as the motor mutation, ion concentration, lipid component, ATP/GTP concentration, and even in vitro protein knockout, which cannot easily be achieved by in vivo or intracellular studies.

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Acknowledgments

This work was supported by the University of Technology Sydney’s Grant for IBMD (Q.P.S.).

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Author notes

  1. Wanqing Du and Qian Peter Su contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Membrane Biology, Tsinghua University-Peking University Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China

    Wanqing Du

  2. Institute for Biomedical Materials & Devices (IBMD), Faculty of Science, University of Technology Sydney, Ultimo, NSW, 2007, Australia

    Qian Peter Su

Authors
  1. Wanqing Du
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  2. Qian Peter Su
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Contributions

W.D. and Q.P.S. contributed equally, prepared figures, and wrote and edited the manuscript together.

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Correspondence to Qian Peter Su.

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Conflict of interest

Wanqing Du declares that he has no conflict of interest. Qian Peter Su declares that he has no conflict of interest.

Additional information

This article is part of a Special Issue dedicated to the “2018 Joint Conference of the Asian Biophysics Association and Australian Society for Biophysics” edited by Kuniaki Nagayama, Raymond Norton, Kyeong Kyu Kim, Hiroyuki Noji, Till Böcking, and Andrew Battle.

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Du, W., Su, Q.P. Single-molecule in vitro reconstitution assay for kinesin-1-driven membrane dynamics. Biophys Rev 11, 319–325 (2019). https://doi.org/10.1007/s12551-019-00531-4

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