Visualizing molecules of functional human profilin
Year: 2022
Type: article
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 (k D = 1.89 μM) and the sides of microtubules in vitro. Titrations of self-labeling Haloligands 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.
Source: eLife
DOI: 10.7554/elife.76485
Institution: SUNY Upstate Medical University
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
Funders: ALS Association, State University of New York, University of Washington, University of Otago, European Molecular Biology Laboratory, National Institute for Health and Care Research, Universiteit van Amsterdam, Simon Fraser University, Université de Genève, National Institutes of Health, University of California, San Francisco, National Institute of General Medical Sciences
OpenAlex ID: W4281653741