Assembly Domain-Based Optogenetic System for the Efficient Control of Cellular Signaling

We previously developed the Magnet system, which consists of two distinct Vivid protein variants, one positively and one negatively charged, designated the positive Magnet (pMag) and negative Magnet (nMag), respectively. These two proteins bind to each other through electrostatic interactions, preventing unwanted homodimerization and providing selective light-induced heterodimerization. The Magnet system enables the manipulation of cellular functions such as protein–protein interactions and genome editing, although the system could be improved further. To enhance the ability of pMagFast2 (a pMag variant with fast kinetics) to bind nMag, we introduced several pMagFast2 modules in tandem into a single construct, pMagFast2(3×). However, the expression level of this construct decreased drastically with increasing number of pMagFast2 molecules integrated into a single construct. In the present study, we applied a new approach to improve the Magnet system based on an assembly domain (AD). Among several ADs, the Ca²⁺/calmodulin-dependent protein kinase IIα association domain (CAD) most enhanced the Magnet system. The present CAD-Magnet system overcame a trade-off issue between the expression level and binding affinity. The CAD-converged 12 pMag photoswitches exhibited a stronger interaction with nMag after blue light irradiation compared with monomeric pMag. Additionally, the CAD played a key role in converging effector proteins as well in a single complex. Owing to these substantial improvements, the CAD-Magnet system combined with Tiam1 allowed us to robustly induce localized formation of vertical ruffles on the apical plasma membrane. The CAD-Magnet system combined with 4D imaging was instrumental in revealing the dynamics of ruffle formation

Assembly Domain-Based Optogenetic System for the Efficient Control of Cellular Signaling

We previously developed the Magnet system, which consists of two distinct Vivid protein variants, one positively and one negatively charged, designated the positive Magnet (pMag) and negative Magnet (nMag), respectively. These two proteins bind to each other through electrostatic interactions, preventing unwanted homodimerization and providing selective light-induced heterodimerization. The Magnet system enables the manipulation of cellular functions such as protein–protein interactions and genome editing, although the system could be improved further. To enhance the ability of pMagFast2 (a pMag variant with fast kinetics) to bind nMag, we introduced several pMagFast2 modules in tandem into a single construct, pMagFast2(3×). However, the expression level of this construct decreased drastically with increasing number of pMagFast2 molecules integrated into a single construct. In the present study, we applied a new approach to improve the Magnet system based on an assembly domain (AD). Among several ADs, the Ca²⁺/calmodulin-dependent protein kinase IIα association domain (CAD) most enhanced the Magnet system. The present CAD-Magnet system overcame a trade-off issue between the expression level and binding affinity. The CAD-converged 12 pMag photoswitches exhibited a stronger interaction with nMag after blue light irradiation compared with monomeric pMag. Additionally, the CAD played a key role in converging effector proteins as well in a single complex. Owing to these substantial improvements, the CAD-Magnet system combined with Tiam1 allowed us to robustly induce localized formation of vertical ruffles on the apical plasma membrane. The CAD-Magnet system combined with 4D imaging was instrumental in revealing the dynamics of ruffle formation

Assembly Domain-Based Optogenetic System for the Efficient Control of Cellular Signaling

We previously developed the Magnet system, which consists of two distinct Vivid protein variants, one positively and one negatively charged, designated the positive Magnet (pMag) and negative Magnet (nMag), respectively. These two proteins bind to each other through electrostatic interactions, preventing unwanted homodimerization and providing selective light-induced heterodimerization. The Magnet system enables the manipulation of cellular functions such as protein–protein interactions and genome editing, although the system could be improved further. To enhance the ability of pMagFast2 (a pMag variant with fast kinetics) to bind nMag, we introduced several pMagFast2 modules in tandem into a single construct, pMagFast2(3×). However, the expression level of this construct decreased drastically with increasing number of pMagFast2 molecules integrated into a single construct. In the present study, we applied a new approach to improve the Magnet system based on an assembly domain (AD). Among several ADs, the Ca²⁺/calmodulin-dependent protein kinase IIα association domain (CAD) most enhanced the Magnet system. The present CAD-Magnet system overcame a trade-off issue between the expression level and binding affinity. The CAD-converged 12 pMag photoswitches exhibited a stronger interaction with nMag after blue light irradiation compared with monomeric pMag. Additionally, the CAD played a key role in converging effector proteins as well in a single complex. Owing to these substantial improvements, the CAD-Magnet system combined with Tiam1 allowed us to robustly induce localized formation of vertical ruffles on the apical plasma membrane. The CAD-Magnet system combined with 4D imaging was instrumental in revealing the dynamics of ruffle formation

Inhibition of endogenous PLC prevented the alpha-toxin-induced release of IL-8 and formation of diacylglycerol.

<p>(A) A549 cells were pretreated with various amounts of U73122 or U73343 at 37°C for 60 min, and then incubated with or without alpha-toxin (1.0 μg/mL) at 37°C for 3 h. The concentration of IL-8 in culture supernatants was determined by ELISA. (B) A549 cells were pretreated with various amounts of U73122 or U73343 at 37°C for 60 min, and then incubated with or without alpha-toxin (1.0 μg/mL) at 37°C for 60 min and intracellular DAG levels were determined. Values represent mean ± S.E.; <i>n</i> = 4; *, <i>p</i> < 0.01.</p

Schematic model of alpha-toxin-induced membrane dynamics and accumulation of the GM1a/TrkA complex

<p>Schematic model of alpha-toxin-induced membrane dynamics and accumulation of the GM1a/TrkA complex</p

Transbilayer movement of DAG on the membrane treated with alpha-toxin.

<p>DNA transfection was used to express EYFP-C1AB in A549 cells. After 24 h, cells were incubated with 1.0 μg/mL wild-type alpha-toxin (A) or H148G alpha-toxin (B) at 37°C. The cells were visualized by confocal fluorescence microscopy. Scale bar, 10 μm.</p

Clustering of GM1a and phosphorylation of TrkA in the membrane of cells treated with alpha-toxin.

<p>(A) A549 cells stained with BODIPY-GM1a were incubated with 1.0 μg/mL wild-type alpha-toxin or H148G alpha-toxin at 37°C for 60 min. The cells were fixed in 4% paraformaldehyde and stained with Hoechst 33342. GM1a (green) and nuclei (blue) were visualized by fluorescence microscopy. Scale bar, 10 μm. (B) Bodipy-GM1a fluorescence intensity was measured as described in Materials and Methods. Values represent the mean ± SE; n = 3; *, <i>p</i> < 0.01. (C) A549 cells were incubated with 1.0 μg/mL wild-type or H148G alpha-toxin at 37°C for 60 min. The cells were fixed, permeabilized, and stained with phospho-TrkA antibody and Hoechst 33342. Phospho-TrkA (red) and nuclei (blue) were visualized by fluorescence microscopy. Scale bar, 10 μm. (D) Phospho-TrkA fluorescence intensity was measured as described in Materials and Methods. Values represent the mean ± SE; n = 5; *, <i>p</i> < 0.01.</p

Inhibition of endogenous PLC affected the clustering of GM1a and phosphorylation of TrkA.

<p>(A) A549 cells were preincubated with 40 μM U73122 (endogenous PLC inhibitor) or U73343 (U73122 analogue) at 37°C for 60 min. The treated cells were stained with BODIPY-GM1a and incubated with 1.0 μg/mL wild-type or H148G alpha-toxin at 37°C for 60 min. The cells were fixed in 4% paraformaldehyde and stained with Hoechst 33342. GM1a (green) and nuclei (blue) were visualized by fluorescence microscopy. Scale bar, 10 μm. (B) A549 cells were preincubated with 40 μM U73122 or U73343 at 37°C for 60 min. The treated cells were incubated with 1.0 μg/mL wild-type or H148G alpha-toxin at 37°C for 60 min. The cells were fixed, permeabilized, and stained with phospho-TrkA antibody and Hoechst 33342. Phospho-TrkA (red) images and nuclei (blue) were visualized by fluorescence microscopy. Scale bar, 10 μm. (C) Bodipy-GM1a fluorescence intensity was measured as described in Materials and Methods. Values represent the mean ± SE; n = 3; *, <i>p</i> < 0.01. (D) Phospho-TrkA fluorescence intensity was measured as described in Materials and Methods. Values represent the mean ± SE; n = 5; *, <i>p</i> < 0.01.</p