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Live imaging of endogenous protein dynamics in zebrafish using chromobodies.

Panza P, Maier J, Schmees C, Rothbauer U, Söllner C - Development (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Entwicklungsbiologie, Abteilung Genetik, Spemannstraße 35, Tübingen 72076, Germany paolo.panza@tuebingen.mpg.de.

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Localization dynamics of actin-CB and PCNA-CB in HeLa cells. (A) FRAP of actin-CB (upper row) or GFP-actin (lower row) transiently expressed in HeLa cells. Photobleaching of a small region (yellow box) shows a significantly faster recovery (t1/2: 3.83 s) of actin-CB compared with GFP-actin, indicating transient antigen binding. The plot shows average values of total fluorescence at consecutive time points, n=10. Error bars indicate s.d. (B) Photobleaching of replication foci during S phase (yellow box) shows fast recovery of the PCNA-CB signal (t1/2: 1.81 s), whereas almost no recovery of GFP-PCNA can be detected. The plot shows average values of total fluorescence at consecutive time points, n=10. Error bars indicate s.d. (C) HeLa cells stably expressing actin-CB imaged upon treatment with 2 μM cytochalasin D (an actin polymerization inhibitor) for 40 min and during subsequent recovery (180 min). Time-lapse imaging reveals drug-induced actin reorganization. (D) Time-lapse analysis of a HeLa cell stably expressing PCNA-CB. During G1, the chromobody signal is evenly distributed throughout the nucleus. Over time, granular foci redistribute at sites of DNA replication, indicating the progression of S phase (3-7.5 h), until foci disappear in G2 (8.5 h) and the cell divides (10 h). Time-stamps: min:s (A,B), h:min:s (C,D). Scale bars: 10 μm in A,B,D; 50 μm in C.
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DEV118943F1: Localization dynamics of actin-CB and PCNA-CB in HeLa cells. (A) FRAP of actin-CB (upper row) or GFP-actin (lower row) transiently expressed in HeLa cells. Photobleaching of a small region (yellow box) shows a significantly faster recovery (t1/2: 3.83 s) of actin-CB compared with GFP-actin, indicating transient antigen binding. The plot shows average values of total fluorescence at consecutive time points, n=10. Error bars indicate s.d. (B) Photobleaching of replication foci during S phase (yellow box) shows fast recovery of the PCNA-CB signal (t1/2: 1.81 s), whereas almost no recovery of GFP-PCNA can be detected. The plot shows average values of total fluorescence at consecutive time points, n=10. Error bars indicate s.d. (C) HeLa cells stably expressing actin-CB imaged upon treatment with 2 μM cytochalasin D (an actin polymerization inhibitor) for 40 min and during subsequent recovery (180 min). Time-lapse imaging reveals drug-induced actin reorganization. (D) Time-lapse analysis of a HeLa cell stably expressing PCNA-CB. During G1, the chromobody signal is evenly distributed throughout the nucleus. Over time, granular foci redistribute at sites of DNA replication, indicating the progression of S phase (3-7.5 h), until foci disappear in G2 (8.5 h) and the cell divides (10 h). Time-stamps: min:s (A,B), h:min:s (C,D). Scale bars: 10 μm in A,B,D; 50 μm in C.

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.


Live imaging of endogenous protein dynamics in zebrafish using chromobodies.

Panza P, Maier J, Schmees C, Rothbauer U, Söllner C - Development (2015)

Localization dynamics of actin-CB and PCNA-CB in HeLa cells. (A) FRAP of actin-CB (upper row) or GFP-actin (lower row) transiently expressed in HeLa cells. Photobleaching of a small region (yellow box) shows a significantly faster recovery (t1/2: 3.83 s) of actin-CB compared with GFP-actin, indicating transient antigen binding. The plot shows average values of total fluorescence at consecutive time points, n=10. Error bars indicate s.d. (B) Photobleaching of replication foci during S phase (yellow box) shows fast recovery of the PCNA-CB signal (t1/2: 1.81 s), whereas almost no recovery of GFP-PCNA can be detected. The plot shows average values of total fluorescence at consecutive time points, n=10. Error bars indicate s.d. (C) HeLa cells stably expressing actin-CB imaged upon treatment with 2 μM cytochalasin D (an actin polymerization inhibitor) for 40 min and during subsequent recovery (180 min). Time-lapse imaging reveals drug-induced actin reorganization. (D) Time-lapse analysis of a HeLa cell stably expressing PCNA-CB. During G1, the chromobody signal is evenly distributed throughout the nucleus. Over time, granular foci redistribute at sites of DNA replication, indicating the progression of S phase (3-7.5 h), until foci disappear in G2 (8.5 h) and the cell divides (10 h). Time-stamps: min:s (A,B), h:min:s (C,D). Scale bars: 10 μm in A,B,D; 50 μm in C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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DEV118943F1: Localization dynamics of actin-CB and PCNA-CB in HeLa cells. (A) FRAP of actin-CB (upper row) or GFP-actin (lower row) transiently expressed in HeLa cells. Photobleaching of a small region (yellow box) shows a significantly faster recovery (t1/2: 3.83 s) of actin-CB compared with GFP-actin, indicating transient antigen binding. The plot shows average values of total fluorescence at consecutive time points, n=10. Error bars indicate s.d. (B) Photobleaching of replication foci during S phase (yellow box) shows fast recovery of the PCNA-CB signal (t1/2: 1.81 s), whereas almost no recovery of GFP-PCNA can be detected. The plot shows average values of total fluorescence at consecutive time points, n=10. Error bars indicate s.d. (C) HeLa cells stably expressing actin-CB imaged upon treatment with 2 μM cytochalasin D (an actin polymerization inhibitor) for 40 min and during subsequent recovery (180 min). Time-lapse imaging reveals drug-induced actin reorganization. (D) Time-lapse analysis of a HeLa cell stably expressing PCNA-CB. During G1, the chromobody signal is evenly distributed throughout the nucleus. Over time, granular foci redistribute at sites of DNA replication, indicating the progression of S phase (3-7.5 h), until foci disappear in G2 (8.5 h) and the cell divides (10 h). Time-stamps: min:s (A,B), h:min:s (C,D). Scale bars: 10 μm in A,B,D; 50 μm in C.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Entwicklungsbiologie, Abteilung Genetik, Spemannstraße 35, Tübingen 72076, Germany paolo.panza@tuebingen.mpg.de.

Show MeSH
Related in: MedlinePlus