Visualizing Molecules of FunctionalHumanProfilin

Abstract

Profilin is an essential regulator of actin and microtubule dynamics and therefore a critical control point for the normal division, motility, and morphology of cells. Most studies of profilin have focused on biochemical investigations using purified protein because high cellular concentrations (121 uM) present challenges for conventional imaging modalities. In addition, past studies that employed direct labeling or conventional fusion protein strategies compromised different facets of profilin function. We engineered a fluorescently-labeled profilin that retains native activities with respect to phosphoinositide lipids, actin monomers, formin-mediated actin assembly, and microtubule polymerization. This fluorescent profilin directly binds to dimers of tubulin (kD = 1.7 uM) and the microtubule lattice (kD = 10 uM) to stimulate microtubule assembly. In cells, our tagged profilin fully rescues profilin-1(-/-) cells from knockout-induced perturbations to cell shape, actin filament architecture, and microtubule arrays. Thus, this labeled profilin-1 is a reliable tool to investigate the dynamic interactions of profilin with actin or microtubules in live cell and in vitro applications.

Biochemical characterization of actin assembly mechanisms with ALS-associated profilin variants

“…What does the observation that certain profilin variants prefer specific formins mean for profilin in neurons or neurodegenerative disease states? In addition to its role with the actin cytoskeleton or with various cellular lipids, profilin also directly binds and regulates the dynamics of tubulin and microtubules (Henty-Ridilla et al, 2017; Pimm et al, 2021). Although C71G has not been tested, both M114T and G118V lack for microtubule-based activities (Henty-Ridilla et al, 2017).…”

“…A study linking the M114T and G118V variants to formins did not detect tubulin or microtubules from immunoprecipitation experiments with wild-type profilin or the ALS-associated variants (Schmidt et al, 2021). This is surprising because genetic and biochemical evidence strongly suggest profilin-microtubule interactions, but might be explained from temperature-dependent effects, lysis conditions or sensitivity of profilin to N-terminal tags which can influence binding interactions (Figley et al, 2014; Nejedla et al, 2017, 2016; Nejedlá et al, 2021; Pimm et al, 2021; Schmidt et al, 2021; Witke et al, 1998; Wittenmayer et al, 2004). A newly developed tool permits the visualization of a subset of cellular profilin molecules (Pimm et al, 2021).…”

“…Rabbit skeletal muscle actin (RMA), Oregon-Green (OG) labeled-actin, N-(1-pyrenyl)iodoacetamide (pyrene) actin, and Alexa-488 actin were purified from acetone powder and labeled as described in detail (Cooper et al, 1984; Henty-Ridilla et al, 2017; Pimm et al, 2021; Spudich and Watt, 1971). Biotin-labeled actin was purchased from Cytoskeleton, Inc (Denver, CO).…”

“…A modified pET23b vector containing the DNA sequence of either a constitutively active fragment of the formin mDia1 (mDia1 FH1-C) (amino acids 571-1255) or GFP-Tβ4 (Pimm et al, 2021) was transformed, induced, and collected as described for profilin (above). Cell pellets were resuspended in lysis buffer (2× PBS (pH 8.0) (2.8 M NaCl, 50 mM KCl, 200 mM sodium dibasic, 35 mM potassium monobasic), 20 mM imidazole (pH 7.4), 500 mM NaCl, 0.1% Triton-X 100, 14 mM BME) and lysed.…”

“…Given the increasing evidence of an active role for profilin in the regulation of cytoskeletal proteins beyond actin assembly (i.e., tubulin dimers and microtubules), we sought to more completely characterize the ALS profilin variants with respect to their actin-based interactions (Pimm et al, 2021; Pimm and Henty-Ridilla, 2021). This would allow us to determine which profilin variants are (and which are not) significantly altered for activities with different cytoskeletal systems.…”