1. Biochemistry and Chemical Biology
  2. Computational and Systems Biology
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A robust and tunable mitotic oscillator in artificial cells

  1. Ye Guan
  2. Zhengda Li
  3. Shiyuan Wang
  4. Patrick M Barnes
  5. Xuwen Liu
  6. Haotian Xu
  7. Minjun Jin
  8. Allen P Liu
  9. Qiong Yang  Is a corresponding author
  1. University of Michigan, United States
  2. University of Science and Technology of China, China
  3. Wayne State University, United States
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Cite this article as: eLife 2018;7:e33549 doi: 10.7554/eLife.33549

Abstract

Single-cell analysis is pivotal to deciphering complex phenomena like heterogeneity, bistability, and asynchronous oscillations, where a population ensemble cannot represent individual behaviors. Bulk cell-free systems, despite having unique advantages of manipulation and characterization of biochemical networks, lack the essential single-cell information to understand a class of out-of-steady-state dynamics including cell cycles. Here, by encapsulating Xenopus egg extracts in water-in-oil microemulsions, we developed artificial cells that are adjustable in sizes and periods, sustain mitotic oscillations for over 30 cycles, and function in forms from the simplest cytoplasmic-only to the more complicated ones involving nuclear dynamics, mimicking real cells. Such innate flexibility and robustness make it key to studying clock properties like tunability and stochasticity. Our results also highlight energy as an important regulator of cell cycles. We demonstrate a simple, powerful, and likely generalizable strategy of integrating strengths of single-cell approaches into conventional in vitro systems to study complex clock functions.

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Article and author information

Author details

  1. Ye Guan

    Department of Biophysics, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Zhengda Li

    Department of Biophysics, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Shiyuan Wang

    Department of Biophysics, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Patrick M Barnes

    Department of Physics, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Xuwen Liu

    Department of Physics, University of Science and Technology of China, Hefei, China
    Competing interests
    The authors declare that no competing interests exist.
  6. Haotian Xu

    Department of Computer Science, Wayne State University, Detroit, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Minjun Jin

    Department of Biological Chemistry, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Allen P Liu

    Department of Biophysics, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0309-7018
  9. Qiong Yang

    Department of Biophysics, University of Michigan, Ann Arbor, United States
    For correspondence
    qiongy@umich.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2442-2094

Funding

National Institutes of Health (MIRA #GM119688)

  • Qiong Yang

National Science Foundation (CAREER Grant #1553031)

  • Qiong Yang

Alfred P. Sloan Foundation (Sloan Research Fellowship)

  • Qiong Yang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Naama Barkai, Weizmann Institute of Science, Israel

Publication history

  1. Received: November 15, 2017
  2. Accepted: April 4, 2018
  3. Accepted Manuscript published: April 5, 2018 (version 1)
  4. Version of Record published: April 27, 2018 (version 2)

Copyright

© 2018, Guan et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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Further reading

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    Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitosis and kinetochore function; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.

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    Cells increase microtubule dynamics to make large rearrangements to their microtubule cytoskeleton during cell division. Changes in microtubule dynamics are essential for the formation and function of the mitotic spindle, and misregulation can lead to aneuploidy and cancer. Using in vitro reconstitution assays we show that the mitotic spindle protein Cytoskeleton-Associated Protein 2 (CKAP2) has a strong effect on nucleation of microtubules by lowering the critical tubulin concentration 100-fold. CKAP2 increases the apparent rate constant ka of microtubule growth by 50-fold and increases microtubule growth rates. In addition, CKAP2 strongly suppresses catastrophes. Our results identify CKAP2 as the most potent microtubule growth factor to date. These finding help explain CKAP2's role as an important spindle protein, proliferation marker, and oncogene.