RESUMO
Macromolecular machines participate in almost every cell biological function. These machines can take the form of well-defined protein structures such as the kinetochore, or more loosely organized protein assemblies like the endocytic coat. The protein architecture of these machines-the arrangement of multiple copies of protein subunits at the nanoscale, is necessary for understanding their cell biological function and biophysical mechanism. Defining this architecture in vivo presents a major challenge. High density of protein molecules within macromolecular machines severely limits the effectiveness of super-resolution microscopy. However, this density is ideal for Forster Resonance Energy Transfer (FRET), which can determine the proximity between neighboring molecules. Here, we present a simple FRET quantitation scheme that calibrates a standard epifluorescence microscope for measuring donor-acceptor separations. This calibration can be used to deduce FRET efficiency fluorescence intensity measurements. This method will allow accurate determination of FRET efficiency over a wide range of values and FRET pair number. It will also allow dynamic FRET measurements with high spatiotemporal resolution under cell biological conditions. Although the poor maturation efficiency of genetically encoded fluorescent proteins presents a challenge, we show that its effects can be alleviated. To demonstrate this methodology, we probe the in vivo architecture of the γ-Tubulin Ring. Our technique can be applied to study the architecture and dynamics of a wide range of macromolecular machines.
