Live imaging of endogenous protein dynamics in zebrafish using chromobodies.
Bottom Line: We generated zebrafish lines expressing chromobodies that trace the major cytoskeletal component actin and the cell cycle marker PCNA with spatial and temporal specificity.Using these chromobodies, we captured full localization dynamics of the endogenous antigens in different cell types and at different stages of development.In combination with improved chromobody selection systems, we anticipate a rapid adaptation of this technique to new intracellular antigens and model organisms, allowing the faithful description of cellular and molecular processes in their dynamic state.
Affiliation: Max-Planck-Institut für Entwicklungsbiologie, Abteilung Genetik, Spemannstraße 35, Tübingen 72076, Germany firstname.lastname@example.org.Show MeSH
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Mentions: Cell lines that stably express chromobodies against the major component of the cellular cytoskeleton, F-actin, and against human PCNA have recently become available (Burgess et al., 2012; Rocchetti et al., 2014; Akopyan et al., 2014; Kaiser et al., 2014). In our first approach, we used HeLa cells stably expressing either chromobody to visualize the localization dynamics of the corresponding antigens in real time. To analyse their intracellular binding properties, we performed FRAP (fluorescence recovery after photobleaching) experiments. Both chromobodies show significantly faster recovery after photobleaching compared with their fluorescently labelled antigens (GFP-actin and GFP-PCNA) in cells (Fig. 1A,B). These data are indicative of a large mobile chromobody fraction composed of highly diffusible molecules. Furthermore, immediately after bleaching, we observed the relocalization of chromobodies to cellular structures that were marked in the prebleaching condition. These results suggest a transient but specific antigen-binding mode, which is characterized by a high on-rate combined with a high off-rate, for both chromobodies in living cells. We therefore hypothesized that this reversibility in binding can minimize any interference these chromobodies might exert on target protein function. In agreement with our findings, we could successfully visualize detailed cytoskeletal remodelling after incubation with F-actin-modulating compounds (Fig. 1C). Similarly, PCNA chromobodies recapitulate the dynamics of endogenous PCNA throughout the cell cycle (Fig. 1D). This is in accordance with previous findings, showing that the expression of chromobodies in eukaryotic cells does not interfere with cell cycle progression (Burgess et al., 2012) or formation of actin filaments (Plessner et al., 2015; Rocchetti et al., 2014). Based on these results, we asked whether the chromobody technology is applicable to living organisms such as zebrafish.Fig. 1.
Affiliation: Max-Planck-Institut für Entwicklungsbiologie, Abteilung Genetik, Spemannstraße 35, Tübingen 72076, Germany email@example.com.