Visualizing molecules of functional human profilin

  1. Morgan L Pimm
  2. Xinbei Liu
  3. Farzana Tuli
  4. Jennifer Heritz
  5. Ashley Lojko
  6. Jessica L Henty-Ridilla  Is a corresponding author
  1. SUNY Upstate Medical University, United States

Abstract

Profilin-1 (PFN1) is a cytoskeletal protein that regulates the dynamics of actin and microtubule assembly. Thus, PFN1 is essential for the normal division, motility, and morphology of cells. Unfortunately, conventional fusion and direct labeling strategies compromise different facets of PFN1 function. As a consequence, the only methods used to determine known PFN1 functions have been indirect and often deduced in cell-free biochemical assays. We engineered and characterized two genetically encoded versions of tagged PFN1 that behave identical to each other and the tag-free protein. In biochemical assays purified proteins bind to PIP lipids, catalyze nucleotide exchange on actin monomers, stimulate formin-mediated actin filament assembly, and impact microtubule dynamics. Halo-PFN1 or mApple-PFN1 restored morphological and cytoskeletal functions in PFN1-deficient mammalian cells. In biochemical assays, mAp-PFN1 bound tubulin dimers (kD = 1.89 µM) and the sides of microtubules in vitro. Titrations of self-labeling Halo-ligands were used to visualize molecules of PFN1. This approach combined with specific function-disrupting point-mutants (Y6D and R88E) revealed PFN1 bound to microtubules in live cells. Cells expressing the ALS-associated G118V disease variant did not associate with actin filaments or microtubules. Thus, these tagged PFN1s are reliable tools for studying the dynamic interactions of PFN1 with actin or microtubules in vitro as well as in important cell processes or disease-states.

Data availability

Datasets for each figure have been uploaded.Original image files (very large size) have been deposited in the Zenodo Henty-Ridilla laboratory community, available here: http://doi.org/10.5281/zenodo.53295855 and http://doi.org/10.5281/zenodo.53295854. Access will be granted once users confirm they will comply with CC-BY licensing.

Article and author information

Author details

  1. Morgan L Pimm

    Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Xinbei Liu

    Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Farzana Tuli

    Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Jennifer Heritz

    Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Ashley Lojko

    Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Jessica L Henty-Ridilla

    Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, United States
    For correspondence
    ridillaj@upstate.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7203-8791

Funding

Sinsheimer Foundation (Scholar Award)

  • Jessica L Henty-Ridilla

ALS Association (20-IIP-506)

  • Jessica L Henty-Ridilla

National Institutes of Health (GM133485)

  • Jessica L Henty-Ridilla

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

Reviewing Editor

  1. Alphee Michelot, Institut de Biologie du Développement, France

Publication history

  1. Preprint posted: September 2, 2021 (view preprint)
  2. Received: December 17, 2021
  3. Accepted: June 6, 2022
  4. Accepted Manuscript published: June 6, 2022 (version 1)
  5. Version of Record published: July 1, 2022 (version 2)

Copyright

© 2022, Pimm 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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  1. Morgan L Pimm
  2. Xinbei Liu
  3. Farzana Tuli
  4. Jennifer Heritz
  5. Ashley Lojko
  6. Jessica L Henty-Ridilla
(2022)
Visualizing molecules of functional human profilin
eLife 11:e76485.
https://doi.org/10.7554/eLife.76485

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