Visualizing molecules of functional human profilin

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.

Purification of Human β- and γ-actin from Budding Yeast

“…Under these conditions, α-, β-, or γ-actin show similar means of nucleation ranging from 15 ± 5.2 to 60.7 ± 23.8 filaments per field of view (P ≥ 0.9939)(Figure 3B). Control α-actin filaments (RMA) elongated at a rate of 10.0 subunits s -1 μM -1 ± 0.3 (SE), consistent with other studies (Figure 3C)(Liu et al, 2022; Pimm et al, 2022). β-actin and γ-actin filaments polymerized at mean rates of 11.1 subunits s-1 μM-1 ± 0.3 (SE) and 12.7 subunits s -1 μM -1 ± 0.2, respectively (Figure 3C).…”

“…In contrast, mixing of actins prior to polymerization, followed by Rh-phalloidin stabilization, produced uniformly labeled actin filaments in both wavelengths, consistent with co-polymerization (Figure 2D-E 3B). Control α-actin filaments (RMA) elongated at a rate of 10.0 subunits s -1 µM -1 ± 0.3 (SE), consistent with other studies (Figure 3C) (Liu et al, 2022;Pimm et al, 2022). β-actin and γ-actin filaments polymerized at mean rates of 11.1 subunits s-1 µM-1 ± 0.3 (SE) and 12.7 subunits s -1 µM -1 ± 0.2, respectively (Figure 3C).…”

“…We assessed each human actin made in yeast for Tβ4 binding using fluorescence polarization (Figure 4A). GFP-Tβ4 bound β-actin (k D = 1.05 ± 0.30 nM (SE)) and γ-actin (k D = 0.94 ± 0.09 nM) with similar affinities, which were each significantly stronger than skeletal muscle actin (k D = 7.71 nM ± 0.40; P < 0.002) (Figure 4A)(Pimm et al, 2022). This reinforces the notion that actin-binding proteins may have different functions with specific isoforms of actin.…”

“…Profilin-1 was dialyzed into 2 mM Tris (pH 7.5), 0.1 mM CaCl 2 , and 0.2 mM DTT. Rabbit muscle actin ((RMA); α-actin), mDia1(FH1-C) (amino acids 571-1255), GFP-thymosin-β4, profilin-1, and fascin used in TIRF or anisotropy assays were purified as described (Jansen et al, 2011; Liu et al, 2022; Pimm et al, 2022). Actin isoforms were fluorescently labeled with pyrene-iodoacetamide or Oregon Green 488-iodoacetamide as described (Aggeli et al, 2014).…”

“…Rabbit muscle actin (RMA; α-actin), mDia1(FH1-C) (amino acids 571-1255), GFP-thymosin-β4, profilin-1, and fascin used in TIRF or anisotropy assays were purified as described (Jansen et al, 2011; Liu et al, 2022; Pimm et al, 2022). Actin was labeled with Oregon Green iodoacetamide or Alexa Fluor NHS-Ester (Aggeli et al, 2014; Hert-zog and Carlier, 2005; Kuhn and Pollard, 2005).…”