Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hunan Normal University, Changsha, 410081, China.
Mol Imaging Biol. 2011 Jun;13(3):424-431. doi: 10.1007/s11307-010-0381-2.
The suboptimal features of the spectral properties of the leading fluorescence resonance energy transfer (FRET) pair, cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP), limit the full promise of FRET imaging. To overcome the drawbacks, we developed FRET-based, intra-molecular biosensors consisting of CFP/discomona sp red fluorescent protein (DsRed) or green fluorescent protein (GFP)/DsRed as donor/acceptor fluorophores.
The biosensors were expressed in NIH3T3 cells. In vitro fluorescence spectroscopy and Rho GTPase activation assays were used to confirm that Rac1 or Cdc42 was activated in serum-starved cells following stimulation with insulin or bradykinin. The transient changes of the amount, location, and translocation of activated Rac1 or Cdc42 in living cells were tracked with confocal imaging.
The increase of FRET efficiency was achieved in the cells expressing the biosensors and was proportional to the levels of activated Rac1 or Cdc42. The localized, transitional, and transient FRET signals were directly and quantitatively imaged with high spatial and temporal resolution. The biosensors were used to analyze and judge the GEF or GAP activities of putative regulatory proteins for Rac1 or Cdc42.
DsRed is a more suitable acceptor in FRET pair with CFP than with GFP in terms of the spectral overlap between the donor and acceptor. The approach can also be applied to many other types of protein behavior in living cells.
领先的荧光共振能量转移（FRET）对，即青色荧光蛋白（CFP）/黄色荧光蛋白（YFP）的光谱特性存在不理想之处，限制了 FRET 成像的充分应用。为了克服这些缺点，我们开发了基于 FRET 的分子内生物传感器，由 CFP/discomona 红色荧光蛋白（DsRed）或绿色荧光蛋白（GFP）/DsRed 作为供体/受体荧光团组成。
生物传感器在 NIH3T3 细胞中表达。体外荧光光谱和 Rho GTPase 激活测定用于确认胰岛素或缓激肽刺激后血清饥饿细胞中 Rac1 或 Cdc42 被激活。通过共焦成像跟踪活细胞中激活的 Rac1 或 Cdc42 的数量、位置和易位的瞬时变化。
表达生物传感器的细胞中实现了 FRET 效率的增加，并且与激活的 Rac1 或 Cdc42 的水平成正比。局部、过渡和瞬态的 FRET 信号被直接、定量地以高时空分辨率成像。该生物传感器可用于分析和判断 Rac1 或 Cdc42 的假定调节蛋白的 GEF 或 GAP 活性。
就供体和受体之间的光谱重叠而言，DsRed 是 FRET 对与 CFP 相比 GFP 更合适的受体。该方法还可应用于活细胞中许多其他类型的蛋白质行为。
