A New RhoA Biosensor
Date Issued
2011
Date
2011
Author(s)
Chen, Chen-Cheng
Abstract
RhoA, a member of the Rho family of small GTPase, regulates multiple cellular behaviors including cell migration, differentiation, cell cycle progression, and gene transcription. Like most of GTPases, RhoA cycles between the active form (GTP-bound) and the inactive form (GDP-bound). Only the active form of RhoA can interact with downstream effectors. Due to the dynamic control of RhoA activity by a variety of regulating mechanisms, it is important to determine the spatiotemporal dynamics of RhoA activity in living cell and the total steady-state level of RhoA activity in cell lysates.
In this study, a new RhoA biosensor was successfully constructed by incorporating an environment-sensitive fluorophore onto the polypeptide that can only interact with the active form RhoA. Upon binding to GTPγS-RhoA in vitro, the biosensor showed a 7.5-fold increase in fluorescence intensity and with Kd value of 200 nM. The biosensor was applied to the detection of RhoA activity in cell lysates. Furthermore, the biosensor has been conjugated to the trans-activator of transcription (TAT) domain for cell delivery, so it might be used to determine the spatiotemporal activity of endogenous RhoA in living cells.
Subjects
RhoA
Biosensor
Type
thesis
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