3 research outputs found
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(3Ă—). 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 Ca<sup>2+</sup>/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(3Ă—). 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 Ca<sup>2+</sup>/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(3Ă—). 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 Ca<sup>2+</sup>/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