Determining the domain-level reaction-diffusion properties of an actin-binding protein transgelin-2 within cells

Matsunaga

Deguchi

2022

Preprint

Self Cite

Fluorescence recovery after photobleaching (FRAP) is a common technique to analyze the turnover of molecules in living cells. Numerous physicochemical models have been developed to quantitatively evaluate the rate of turnover driven by chemical reaction and diffusion that occurs in a few seconds to minutes. On the other hand, they have limitations in interpreting long-term FRAP responses where intracellular active movement inevitably provides target molecular architectures with additional effects other than chemical reaction and diffusion, namely directed transport and structural deformation. To overcome the limitations, we develop a continuum mechanics-based model that allows for decoupling FRAP response into the intrinsic turnover rate and subcellular mechanical characteristics such as displacement vector and strain tensor. Our approach was validated using fluorescently-labeled beta-actin in an actomyosin-mediated contractile apparatus called stress fibers, revealing spatially distinct patterns of the multi-physicochemical events, in which the turnover rate of beta-actin was significantly higher at the center of the cell. We also found that the turnover rate is negatively correlated with the strain rate along stress fibers but, interestingly, not with the absolute strain magnitude. Moreover, stress fibers are subjected to centripetal flow as well as both contractile and tensile strains along them. Taken together, this novel framework for long-term FRAP analysis allows for unveiling the contribution of overlooked microscopic mechanics to molecular turnover in living cells.

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Matsunaga

Deguchi

2022

Preprint

Self Cite

“…To determine the specific form of ( , ), first, spatiotemporal fluorescence recovery in rectangular photobleached region is described by ), respectively (Deschout et al 2010); and, eff represents the effective diffusion coefficient defined as eff = pure /(1 + −1 ) where pure and represent the purediffusion coefficient of free actin molecules at unbound state and equilibrium dissociation constant, respectively (Crank 1975;Sprague et al 2004;Ait-Haddou et al 2010;Saito et al 2021). As we previously demonstrated (Saito et al 2021), actin in SFs is subjected to slow turnover and is predominantly at bound state, resulting in that ≪ 1 , and consequently eff ≈ 0. This drop of eff is reasonable because actin intensity in bleached region indeed recovers spatially uniformly in FRAP experiments (Supplementary Fig.…”

Section: Model Descriptionmentioning

confidence: 99%

“…The copyright holder for this preprint this version posted June 20, 2021. ; https://doi.org/10.1101/2021.06.19.449123 doi: bioRxiv preprint more complex cases where the diffusion and chemical reaction potentially contribute nearly equally to the turnover, elaborate physicochemical models have been used to separately characterize the two distinct factors (Sprague et al 2006;Mueller et al 2008). We also previously developed a platform based on FRAP analysis that allows, for the first time to our knowledge, to determine the protein domain-level chemical properties and pure diffusion coefficient of actin binding proteins (Saito et al 2021).…”

Section: Introductionmentioning

confidence: 99%

“…According to previous studies with single/double exponential fitting approaches (Campbell and Knight 2007), the turnover of actin in SFs takes a time constant of ~5 min, and the bleached region recovers spatially uniformly. The actin turnover is thus suggested to be dominated by chemical replacement rather than diffusion (Saito et al 2021). The relatively long time constant of minutes also suggests that proteins in (which was not certified by peer review) is the author/funder.…”

Section: Introductionmentioning

confidence: 99%

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

Matsunaga

et al. 2021

Preprint

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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.

CM‐FRAP–Continuum Mechanics‐Based Fluorescence Recovery After Photobleaching

Deguchi

2023

Current Protocols

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Fluorescence recovery after photobleaching (FRAP) is widely used to evaluate intracellular molecular turnover or repeated translocation of molecules using confocal laser scanning microscopy. While numerous models have been developed for the analysis of FRAP responses, in which chemical interactions and/or fast diffusion processes are involved, it is inherently difficult to evaluate the long-term behavior of molecular turnover because of the presence of intracellular flow and microscopic deformation of bleached regions. To overcome these difficulties, we have developed a novel continuum mechanics-based FRAP (CM-FRAP) approach that enables simultaneous evaluation of longterm molecular turnover and intracellular flow/deformation. Here we demonstrate the utility of CM-FRAP by using actin molecules associated with stress fibers in rat aortic smooth muscle cells with clarification of the experimental setup and data analysis.