A functional family of fluorescent nucleotide analogues to investigate actin dynamics and energetics

Abstract

Actin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of high-quality probes. Most current probes affect dynamics of actin or its interactions with actin-binding proteins (ABPs), and cannot track the bound nucleotide. Here, we identify a family of highly sensitive fluorescent nucleotide analogues structurally compatible with actin. We demonstrate that these fluorescent nucleotides bind to actin, maintain functional interactions with a number of essential ABPs, are hydrolyzed within actin filaments, and provide energy to power actin-based processes. These probes also enable monitoring actin assembly and nucleotide exchange with single-molecule microscopy and fluorescence anisotropy kinetics, therefore providing robust and highly versatile tools to study actin dynamics and functions of ABPs.

Visualizing Molecules of FunctionalHumanProfilin

“…PFN1 is a well-characterized, high affinity (k D = 0.1 μM) actin monomer binding protein that catalyzes nucleotide exchange and suppresses actin filament nucleation (Mockrin and Korn, 1980; Goldschmidt-Clermont et al, 1992; Eads et al, 1998; Vinson et al, 1998; Wolven et al, 2000; Wen et al, 2008; Blanchoin et al, 2014; Colombo et al, 2021). To assess whether mAp-PFN1 binds actin monomers with similar affinity as untagged PFN1, we performed fluorescence polarization assays (Figure 3 and Figure S3).…”

“…These values are similar to those measured from direct binding assays (k D = 105.2 ± 26.9 nM) (Figure S3B) (Wen et al 2008). To assess whether mAp-PFN1 is capable of catalyzing nucleotide exchange with similar efficiency as untagged PFN1, we performed time resolved fluorescence polarization assays containing ATP-ATTO-488, unlabeled actin monomers, and either PFN1 (Colombo et al, 2021). Each PFN1 catalyzed and accelerated the nucleotide exchange on actin monomers at similar rates ( p = 0.1; ANOVA) (PFN1: k = 18.7 ± 7.5 nM or mAp-PFN1: k = 13.2 ± 9.7 nM), but were accelerated compared to control reactions lacking either PFN1 (actin alone: k = 514.3 ± 61.6 nM) (Figure 3C).…”

“…No microtubules were found when the contents of the highest tubulin-containing reaction were spotted onto coverslips and screened for microtubules by epifluorescence microscopy (561 filter) at the end of the experiment. Time-lapse polarization was performed to determine the rate of nucleotide exchange on actin using 2 µM actin (unlabeled), 500 nM ATP-ATTO-488, and 1 µM PFN1 or mAp-PFN1 in NFG (5 mM Tris (pH 8), 0.2 mM CaCl 2 , 0.5 mM DTT) supplemented with MEI (1 mM MgCl 2 , 1 mM EGTA, and 10 mM imidazole-HCl (pH 7.0)), similar to (Colombo et al, 2021). Polarization was recorded every 7.5-8 s for 30 min.…”

“…Profilin (PFN1) is a small (∼15 kDa) cytosolic protein that interacts with actin monomers, poly- L -proline (PLP) containing ligands, phosphoinositide (PIP) lipids, and microtubules to regulate many essential cell behaviors (Davey and Moens, 2020; Pimm et al, 2020; Karlsson and Dráber, 2021). It maintains cellular reserves of unassembled actin monomers, catalyzes nucleotide exchange, and sterically inhibits new filament formation (Goldschmidt-Clermont et al, 1992; Kaiser et al, 1999; Wolven et al, 2000; Suarez et al, 2015; Pimm et al, 2020; Colombo et al, 2021). In contrast, PFN1 can drastically stimulate actin assembly when paired with certain PLP-containing ligands (e.g., formins or Ena/VASP) (Manchester et al, 1998; Romero et al, 2004; Kovar et al, 2006; Breitsprecher and Goode, 2013; Henty-Ridilla and Goode, 2015; Funk et al, 2019; Zweifel and Courtemanche, 2020).…”

Celebrating 20 years of live single-actin-filament studies with five golden rules

“…New ways to fluorescently label actin filaments have been proposed, offering interesting possibilities; the fluorescently labeled Calponin Homology domains of utrophin, which efficiently decorate actin filaments, have been used to successfully monitor the rapid elongation of filaments exposed to over 100 µM unlabeled monomeric actin (i.e., concentrations where the background signal from labeled monomers would preclude imaging filaments, even in TIRF) ( 82 ). Another study has shown that the fluorescent nucleotide analog ATP-ATTO-488 could be used as an indirect yet reliable way to label actin monomers, with little impact on their kinetic rates and interaction with ABPs ( 83 ). Additional developments are likely to emerge in the future, such as new fluorescent probes or fully automated image analysis, and will certainly continue to expand our ability to monitor individual reactions on dynamic actin filaments.…”