Visualizing molecules of functional human profilin
- 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
Jessica L Henty-Ridilla
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.
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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Visualizing molecules of functional human profilin
eLife 11:e76485.
https://doi.org/10.7554/eLife.76485
