Biology Reference
In-Depth Information
Using Bioprobes to follow Protein
Dynamics in Living Cells
Mark R. Holt, Daniel Y. H. Soong, James Monypenny,
Ian M. Dobbie, Daniel Zicha
and
Graham A. Dunn
Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, its
designer variants and new analogues from other marine organisms, are
revolutionizing our understanding of many cell biological processes such as
cell signalling, membrane- and cytoskeletal dynamics. Contributing to this
progress are parallel developments in fluorescence microscopy enabling these
bioprobes to reveal information about molecules that goes far beyond their
mere localization, including data on protein-ligand interactions, post-
translational modification, conformational state and molecular translocation.
We discuss the application of bioprobes in the following techniques:
fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging
(FLIM), total internal reflection microscopy (TIRF), fluorescence speckle
microscopy (FSM) and the recently developed fluorescence localization after
photobleaching (FLAP). These enable the imaging of many aspects of
molecular dynamics directly in the living cell, thus complementing advances
made with genetic and biochemical methods and extending our understanding
of biological hierarchies from the molecular to the cellular level and beyond.
The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has
literally transformed the way we see cells. Extensive engineering of the native
molecule has resulted in a versatile and rapidly expanding arsenal of
multicoloured probes. Meanwhile, new living fluorophores are being
