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Generation of Double-Labeled Reporter Cell Lines for Studying Co-Dynamics of Endogenous Proteins in

Summary: Generation of Double-Labeled Reporter Cell Lines for
Studying Co-Dynamics of Endogenous Proteins in
Individual Human Cells
Irina Issaeva.
*, Ariel A. Cohen.
, Eran Eden, Cellina Cohen-Saidon, Tamar Danon, Lydia Cohen, Uri Alon*
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel
Understanding the dynamic relationship between components of a system or pathway at the individual cell level is a
current challenge. To address this, we developed an approach that allows simultaneous tracking of several endogenous
proteins of choice within individual living human cells. The approach is based on fluorescent tagging of proteins at their
native locus by directed gene targeting. A fluorescent tag-encoding DNA is introduced as a new exon into the intronic
region of the gene of interest, resulting in expression of a full-length fluorescently tagged protein. We used this approach to
establish human cell lines simultaneously expressing two components of a major antioxidant defense system, thioredoxin 1
(Trx) and thioredoxin reductase 1 (TrxR1), labeled with CFP and YFP, respectively. We find that the distributions of both
proteins between nuclear and cytoplasmic compartments were highly variable between cells. However, the two proteins
did not vary independently of each other: protein levels of Trx and TrxR1 in both the whole cell and the nucleus were
substantially correlated. We further find that in response to a stress-inducing drug (CPT), both Trx and TrxR1 accumulated in
the nuclei in a manner that was highly temporally correlated. This accumulation considerably reduced cell-to-cell variability
in nuclear content of both proteins, suggesting a uniform response of the thioredoxin system to stress. These results


Source: Alon, Uri - Departments of Molecular Cell Biology & Physics of Complex Systems, Weizmann Institute of Science


Collections: Biology and Medicine