Single-Molecule Tracking Reveals Altered Dynamics Of A Transcription Regulator Expressed At Similar Levels From Different Gene Expression Systems

The development of fluorescent proteins and their application in live-cell imaging have not only validated previously known molecular processes but have also unraveled new biological mechanisms. Though the functionality and stability of fusions to fluorescent proteins are routinely characterized, there is no strict agreement in the community on how to express these fusion proteins in cells. Here, we measure differences in the single-molecule trajectories of a transcription regulator protein, TcpP, depending on its expression method in Vibrio cholerae. TcpP was fluorescently labeled with the photoactivatable fluorescent protein PAmCherry at its C-terminus and was either expressed off of the native TcpP promoter in the chromosome or from an arabinose-inducible promoter PBAD. Even though we detected similar levels of tcpP-pamcherry mRNA transcripts and TcpP-PAmCherry protein concentrations in vitro with both expression methods, single-molecule tracking of the protein in live V. cholerae reveals differences which can obfuscate biological interpretation of the role of TcpP in toxin production. In the plasmid-induced strain, we observed (1) more slowly diffusing TcpP-PAmCherry molecules, (2) an at least a five-fold increase in copies of TcpP-PAmCherry, contrary to the in vitro western results, and (3) the absence of a very rapidly diffusing population that is observed upon endogenous expression. In addition, we compared the dynamics of TcpP-PAmCherry in V. cholerae to TcpP-PAmCherry in wildtype Escherichia coli, in which background TcpP should not have any relevant biological activity. Our findings suggest that single-molecule tracking of proteins provides a very sensitive assay to detect subtle differences in protein dynamics—and thus activities—that are hidden in vitro.