Long-term molecular turnover of actin stress fibers revealed by advection-reaction analysis in fluorescence recovery after photobleaching

Abstract

Fluorescence recovery after photobleaching (FRAP) is a versatile technique to evaluate the intracellular molecular exchange called turnover. Physicochemical models of FRAP typically consider the molecular diffusion and chemical reaction that simultaneously occur on a time scale of seconds to minutes. Particularly for long-term measurements, however, an advection effect can no longer be ignored, which transports the proteins in specific directions within the cells and accordingly shifts the spatial distribution of the local chemical equilibrium. Nevertheless, existing FRAP models have not considered the spatial shift, and as such, the turnover rate is often analyzed without considering the spatiotemporally updated chemical equilibrium. Here we develop a new FRAP model aimed at long-term measurements to quantitatively determine the two distinct effects of the advection and chemical reaction, i.e., the different major sources of the change in fluorescence intensity. To validate this approach, we carried out FRAP experiments on actin in stress fibers over a time period of more than 900 s, and the advection rate was shown to be comparable in magnitude to the chemical dissociation rate. We further found that the actin-myosin interaction and actin polymerization differently affect the advection and chemical dissociation. Our results thus suggest that the distinction between the two effects is indispensable to extract the intrinsic chemical properties of the actin cytoskeleton from the observations of complicated turnover in cells.

Fluorescence Recovery after Photobleaching in Colloidal Science: Introduction and Application

“…127 The guar oligomer is retained by the hydrogel until it reaches the colonic environment when it is degraded by bacteria-secreted enzymes. The diffusion of guar oligomer inside the hydrogel was quantified by FRAP, which revealed that diffusion was greatly reduced compared with noninteracting 99 x Sustr et al 86,100 x Pinte et al 101 x Bruggen et al 102,103 x Nauman et al 104 x Prakash et al 105 x Balijepalli et al 106 x Jin-lei et al 107 x Grosskopf et al 16 x Govindaraj and Pos 4 x Hager et al 108 x Saito et al 109,110 x Sancataldo and Vetri 111 x Kvist et al 112 x Kindt et al 97 x Bui et al 113 x Kingsley al. 114 x Dey et al 115 x Devkota et al 116 x Waharte et al 117 x Sato et al 118 x Hashimoto et al 119 x probes and stayed constant over a couple of hours, resulting in a slow release.…”

Analysis of chemical and mechanical behaviors in living cells by continuum mechanics-based FRAP

“…Regarding the involvement of directed movements in FRAP response, advection-reaction-diffusion models were developed to analyze one-dimensional movements of motor proteins in relatively short-term recovery curves of < 3 min (22, 23). In addition, advection-reaction models were developed to analyze two-dimensional directed movements of actin molecules observed in long-term FRAP experiments of > 10 min where fast diffusion process is ignorable (24). However, these models assumed constant velocities of the movement, and therefore more general nonlinearly and temporarily changing movements were beyond the scope of the analysis.…”

“…Here we develop a continuum mechanics-based approach to simultaneously determine the molecular turnover and active movements that inevitably appear in long-term FRAP responses. Analyses for such long-term behaviors are indispensable particularly for analyzing the dynamics of actin molecules associated with stress fibers, a contractile apparatus consisting of actin filaments, non-muscle myosin II, and other cross-linking and actin-binding proteins, given its long lifetime that exceeds 10 min (24)(25)(26)(27). Because of the absence of appropriate FRAP analysis models for long-term behaviors, actin dynamics has not accurately been decomposed to evaluate the individual contribution of the two factors.…”

Nanocellulose Product Design Aided by Confocal Laser Scanning Microscopy

“…The FRAP behavior of CNC gel 27 is shown in Figure 6a. This report exemplified the capacity of FRAP to detect gel diffusion parameters when the microstructure of the gel altered owing 92 2D/circular Gaussian analytic Deschout et al 108 2D/linear rectangle analytic Roding et al 153 2D/none arbitrary analytic/ numeric Hinow et al 154 1D/linear rectangle analytic Dey et al 23 2D/linear rectangle analytic Saito et al 155 2D/linear rectangle analytic Dushek et al 156 2D/linear rectangle numeric Braga et al 157 2D/radial Gaussian numeric Kang et al 99 2D/radial Gaussian analytic a Dimension here refers to diffusion being considered in x-, y-, and zdirections. to gravity or more complex gelation.…”