Myosin-independent cytokinesis in Giardia utilizes flagella to coordinate force generation and direct membrane trafficking

Edited by Edward D. Korn, National Heart, Lung, and Blood Institute, Bethesda, MD, and approved June 13, 2017 (received for review March 28, 2017)
July 5, 2017
114 (29) E5854-E5863

Significance

Many protists, including Giardia, lack myosin II and thus are unlikely to use the canonical contractile mechanism of cell division. Giardia depends solely on its flagella for motility; here, we show that flagella function is also required to drive daughter cells in opposite directions for cytokinesis. Additionally, just before cytokinesis, Rab11 accumulated in the forming furrow and the nascent intracytoplasmic axonemes were oriented to deliver Rab11. This mechanism constitutes a means to mark the center of the cell and guide trafficking to the furrow. These results support an emerging view that flagella play a central role in cell division among protists that lack myosin II.

Abstract

Devoid of all known canonical actin-binding proteins, the prevalent parasite Giardia lamblia uses an alternative mechanism for cytokinesis. Unique aspects of this mechanism can potentially be leveraged for therapeutic development. Here, live-cell imaging methods were developed for Giardia to establish division kinetics and the core division machinery. Surprisingly, Giardia cytokinesis occurred with a median time that is ∼60 times faster than mammalian cells. In contrast to cells that use a contractile ring, actin was not concentrated in the furrow and was not directly required for furrow progression. Live-cell imaging and morpholino depletion of axonemal Paralyzed Flagella 16 indicated that flagella-based forces initiated daughter cell separation and provided a source for membrane tension. Inhibition of membrane partitioning blocked furrow progression, indicating a requirement for membrane trafficking to support furrow advancement. Rab11 was found to load onto the intracytoplasmic axonemes late in mitosis and to accumulate near the ends of nascent axonemes. These developing axonemes were positioned to coordinate trafficking into the furrow and mark the center of the cell in lieu of a midbody/phragmoplast. We show that flagella motility, Rab11, and actin coordination are necessary for proper abscission. Organisms representing three of the five eukaryotic supergroups lack myosin II of the actomyosin contractile ring. These results support an emerging view that flagella play a central role in cell division among protists that lack myosin II and additionally implicate the broad use of membrane tension as a mechanism to drive abscission.

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Acknowledgments

The authors thank B. Wakimoto, S. Parkhurst, C. Asbury, L. Wordeman, and J. Vicente for critical reading of the manuscript and E. Thomas, M. Steele-Ogus, and K. Hennessey for help with editing. This work was supported by National Institutes of Health Grant 5R01AI110708 (to A.R.P.) and National Science Foundation Grant GRFP DGE-1256082 (to W.R.H.).

Supporting Information

Supporting Information (PDF)
Dataset_S01 (XLSX)
Dataset_S02 (XLSX)
Movie S1.
Selected focal planes from 4D movie of mNG-Tub corresponding to the cell in Fig. 1. Movie is 15 min 56 s in real time.
Movie S2.
Time-lapse movie of mNG–β-tubulin showing flagella flexion and positioning during cytokinesis. Note that the caudal flagella flex to push daughter cells apart at the initiation of cytokinesis, and, when the flagella are no longer juxtaposed, the cells swim apart to complete cytokinesis. Movie is 87 s in real time.
Movie S3.
DIC time-lapse movie of a Giardia trophozoite progressing from interphase to cytokinesis. Movie is 9 min 40 s in real time.
Movie S4.
PF16-HA depletion results in failed cytokinesis. Movie is 23 min 47 s in real time.
Movie S5.
Actin-KD cell fails to divide as a result of physical blockage of furrow progression. Movie is 20 min 30 s in real time.
Movie S6.
Actin-depleted cell stalls at the tail-to-tail phase and completes cytokinesis after 60 min. Movie is 16 min 10 s in real time.
Movie S7.
Representative BFA-treated cell that arrested in cytokinesis. Movie is 17 min 58 s in real time.
Movie S8.
A 4D movie of mNG-Rab11. Movie is 56 s in real time.
Movie S9.
A 3D stack of fixed cells at different stages of cytokinesis: HA-Rab11 (red), actin (green), tubulin (grayscale), and DNA (blue). Note that actin and Rab11 colocalize at the ends of the forming posterolateral and ventral flagella.

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Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 114 | No. 29
July 18, 2017
PubMed: 28679631

Classifications

Submission history

Published online: July 5, 2017
Published in issue: July 18, 2017

Keywords

  1. actin
  2. Rab11
  3. PF16
  4. tubulin
  5. mitosis

Acknowledgments

The authors thank B. Wakimoto, S. Parkhurst, C. Asbury, L. Wordeman, and J. Vicente for critical reading of the manuscript and E. Thomas, M. Steele-Ogus, and K. Hennessey for help with editing. This work was supported by National Institutes of Health Grant 5R01AI110708 (to A.R.P.) and National Science Foundation Grant GRFP DGE-1256082 (to W.R.H.).

Notes

This article is a PNAS Direct Submission.

Authors

Affiliations

William R. Hardin
Department of Biology, University of Washington, Seattle, WA 98195;
Renyu Li
Department of Biology, University of Washington, Seattle, WA 98195;
Jason Xu
Department of Statistics, University of Washington, Seattle, WA 98195
Andrew M. Shelton
Department of Biology, University of Washington, Seattle, WA 98195;
Germain C. M. Alas
Department of Biology, University of Washington, Seattle, WA 98195;
Department of Biology, University of Washington, Seattle, WA 98195;
Department of Statistics, University of Washington, Seattle, WA 98195
Department of Biology, University of Washington, Seattle, WA 98195;

Notes

1
To whom correspondence should be addressed. Email: [email protected].
Author contributions: W.R.H. and A.R.P. designed research; W.R.H., R.L., A.M.S., and G.C.M.A. performed research; W.R.H. contributed new reagents/analytic tools; W.R.H., R.L., J.X., A.M.S., V.N.M., and A.R.P. analyzed data; and W.R.H. and A.R.P. wrote the paper.

Competing Interests

The authors declare no conflict of interest.

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    Myosin-independent cytokinesis in Giardia utilizes flagella to coordinate force generation and direct membrane trafficking
    Proceedings of the National Academy of Sciences
    • Vol. 114
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    • pp. 7469-E6026

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