Helical structure of actin stress fibers and its possible contribution to inducing their direction-selective disassembly upon cell shortening

Okamoto, Tatsuki; Matsui, Tsubasa S.; Ohishi, Taiki; Deguchi, Shinji

doi:10.1007/s10237-019-01228-z

Cited by 18 publications

(21 citation statements)

References 35 publications

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“…5D). This tendency is consistent with the view that NMII works as a major cross-linker to bundle actin filaments into SFs, in which the loss of the actin-myosin interaction causes the unbundling of SFs into individual actin filaments (Matsui et al 2011;Okamoto et al 2020;Saito et al 2020;Huang et al 2021) (Fig. 6).…”

Section: Discussionsupporting

confidence: 90%

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

Saitô

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.

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Section: Discussionsupporting

confidence: 90%

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

Saitô

Matsunaga

et al. 2021

Preprint

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“…3A). Specifically, cells were treated with hypotonic shock to finally remove most of the cell bodies according to our previously developed techniques (Matsui et al ., 2011; Okamoto et al ., 2020), leaving ventral SFs physically attaching to the substrate. These SFs remained unchanged in appearance during the hypotonic shock treatment as observed with YFP-actin transiently expressed in the cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In the present study, we took a similar approach by isolating SFs with hypotonic shock and more gentle fluid shearing forces on the same fibroblast cell line. This isolation method using a cytoskeleton-stabilizing buffer has been validated in many aspects including the maintained helical microstructures as well as contractile function (Matsui et al ., 2011; Deguchi et al ., 2012; Okamoto et al ., 2020). We consequently succeeded in specifying at least 263 proteins as the components of SFs, and among them 101 and 7 are upregulated and downregulated upon RS, respectively.…”

Section: Discussionmentioning

confidence: 99%

Proteome of actin stress fibers

Kang

et al. 2021

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Stress fibers (SFs), which are actomyosin structures, reorganize in response to various cues to maintain cellular homeostasis. Currently, the protein components of SFs are only partially identified, limiting our understanding of their responses. Here we isolate SFs from human fibroblasts HFF-1 to determine with proteomic analysis the whole protein components and how they change with replicative senescence (RS), a state where cells decline in ability to replicate after repeated divisions. We found that at least 263 proteins are associated with SFs, and 101 of them are upregulated with RS, by which SFs become larger in size. Among them, we focused on eEF2 (eukaryotic translation elongation factor 2) as it exhibited upon RS the most significant increase in abundance. We show that eEF2 is critical to the reorganization and stabilization of SFs in senescent fibroblasts. Our findings provide a novel molecular basis for SFs to be reinforced to resist cellular senescence.

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“…Pure diffusion properties, which are independent of the association/dissociation to the surrounding molecules and are thus intrinsic to the molecules, are also obtained in our analysis from the diffusive behavior of the non-reactive mutant. The described framework is universally applicable, in addition to transgelin-2 demonstrated here, to any protein bound to virtually immobile scaffolds like ventral SFs (Saito et al 2020;Okamoto et al 2020), thus providing a versatile approach to identifying the role of protein domains expressed within living cells.…”

Section: Discussionmentioning

confidence: 99%

“…6B), which is considerably below the effective range of FCS. Therefore, we instead focused on the same non-reactive mutant in the cytoplasm with looser actin meshwork, in which the molecules are supposed to diffuse faster compared to the inside of SFs composed of tighter actin bundles (Okamoto et al 2020 (Fig. 6B), the equilibrium dissociation…”

Section: Distinction Between the Effective And Pure Diffusions Enablementioning

confidence: 99%

Determining the inherent reaction-diffusion properties of actin-binding proteins in cells by incorporating genetic engineering to FRAP-based framework

Saitô

Matsunaga

Matsui

et al. 2020

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Proteins in cells undergo repeated association to other molecules, thereby reducing the apparent extent of their intracellular diffusion. While much effort has been made to analytically decouple these combined effects of pure diffusion and chemical reaction, it is difficult to attribute the measured quantities to the nature of specific domains of the probed proteins particularly if, as is often the case, the protein has multiple domains to independently interact with the same types but different molecules. Motivated by the common goal in cell signaling research aimed at identifying the protein domains responsible for particular intermolecular interactions, here we describe a new approach to determining the domain-level reaction and pure diffusion properties. To validate this methodology, we apply it to transgelin-2, an actin-binding protein whose intracellular dynamics remains elusive. We develop a fluorescence recovery after photobleaching (FRAP)-based framework, in which comprehensive combinations of domain-deletion mutants are created with genetic engineering, and the difference among the mutants in FRAP response is analyzed. We demonstrate that transgelin-2 in cells interacts with F-actin via two separate domains, and the chemical equilibrium constant of the interaction is determined at the individual domain levels. Its pure diffusion properties independent of the association to F-actin is also obtained. This approach requires some effort to construct the mutants, but instead enables in situ domain-level determination of the physicochemical properties, which will be useful, as specifically shown here for transgelin-2, in addressing the signaling mechanism of cellular proteins.

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Helical structure of actin stress fibers and its possible contribution to inducing their direction-selective disassembly upon cell shortening

Cited by 18 publications

References 35 publications

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

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

Proteome of actin stress fibers

Determining the inherent reaction-diffusion properties of actin-binding proteins in cells by incorporating genetic engineering to FRAP-based framework

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