Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang , are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time scale as the diffusion of the labeled complexes through the detection volume (1– 100 ms). Since the origin of this quenching process is currently unclear, care has to be taken when the Dreiklang label is intended to be used in FCS applications.
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