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Flippase-mediated phospholipid asymmetry promotes fast Cdc42 recycling in dynamic maintenance of cell polarity

Nature Cell Biology volume 14, pages 304–310 (2012)Cite this article

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Abstract

Lipid asymmetry at the plasma membrane is essential for such processes as cell polarity, cytokinesis and phagocytosis1,2,3. Here we find that a lipid flippase complex, composed of Lem3, Dnf1 or Dnf2 (ref. 4), has a role in the dynamic recycling of the Cdc42 GTPase, a key regulator of cell polarity5, in yeast. By using quantitative microscopy methods, we show that the flippase complex is required for fast dissociation of Cdc42 from the polar cortex by the guanine nucleotide dissociation inhibitor. A loss of flippase activity, or pharmacological blockage of the inward flipping of phosphatidylethanolamine, a phospholipid with a neutral head group, disrupts Cdc42 polarity maintained by guanine nucleotide dissociation inhibitor-mediated recycling. Phosphatidylethanolamine flipping may reduce the charge interaction between a Cdc42 carboxy-terminal cationic region with the plasma membrane inner leaflet, enriched for the negatively charged lipid phosphatidylserine. Using a reconstituted system with supported lipid bilayers, we show that the relative composition of phosphatidylethanolamine versus phosphatidylserine directly modulates Cdc42 extraction from the membrane by guanine nucleotide dissociation inhibitor.

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Figure 1: Δlem3 suppresses the growth and Cdc42 localization defects due to RDI1 overexpression.
Figure 2: Lem3 regulates Rdi1-mediated Cdc42 internalization and polar-cap morphology.
Figure 3: The Lem3–Dnf1/Dnf2 complex regulates the polar Cdc42 dynamics and phosphatidylserine distribution.
Figure 4: The influence of the charge property of a Cdc42 carboxy-terminal region on Cdc42 dynamics.
Figure 5: Lipid composition directly regulates Rdi1-mediated Cdc42 extraction.

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Acknowledgements

The authors thank W. Wiegraebe (Stowers Institute for Medical Research) for advice on imaging, K. Lee (Harvard Medical School) for advice on supported lipid bilayer preparation and N. Pavelka for assistance in genome screening data analysis. This study was done to fulfil, in part, requirements for A.D.’s PhD thesis as a student registered with the Open University. This work was supported by NIH grant RO1-GM057063 to R.L.

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  1. Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110, USA

    Arupratan Das, Brian D. Slaughter, Jay R. Unruh, William D. Bradford, Richard Alexander, Boris Rubinstein & Rong Li

  2. Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, USA

    Rong Li

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Contributions

A.D. and R.L. designed the experiments; A.D. carried out all experiments and prepared the manuscript, with help from B.D.S. and J.R.U.; R.A. and W.D.B. assisted in the whole genome screening; B.R. assisted in data analysis; R.L. conceived and supervised the project and revised the manuscript.

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Correspondence to Rong Li.

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Das, A., Slaughter, B., Unruh, J. et al. Flippase-mediated phospholipid asymmetry promotes fast Cdc42 recycling in dynamic maintenance of cell polarity. Nat Cell Biol 14, 304–310 (2012). https://doi.org/10.1038/ncb2444

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