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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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Cell cycle analysis of transgenic zebrafish embryos expressing PCNA-CB. (A) Overview of the dorsal midbrain of a wnt1:gal4,UAS:GFP (green); UAS:PCNA-CB (magenta) double transgenic embryo at 38 hpf. (B) Unaltered cell cycle progression in PCNA-CB-expressing, wnt1-positive neural progenitors. PCNA-CB signal transitions from a speckled configuration (S phase) to an evenly distributed one (G2), which precedes mitosis (M). Arrowheads indicate a cell progressing through its last cycle before terminally differentiating into two daughter neurons. Time-stamps: h:min:s. Scale bars: 50 μm in A; 20 μm in B.
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DEV118943F3: Cell cycle analysis of transgenic zebrafish embryos expressing PCNA-CB. (A) Overview of the dorsal midbrain of a wnt1:gal4,UAS:GFP (green); UAS:PCNA-CB (magenta) double transgenic embryo at 38 hpf. (B) Unaltered cell cycle progression in PCNA-CB-expressing, wnt1-positive neural progenitors. PCNA-CB signal transitions from a speckled configuration (S phase) to an evenly distributed one (G2), which precedes mitosis (M). Arrowheads indicate a cell progressing through its last cycle before terminally differentiating into two daughter neurons. Time-stamps: h:min:s. Scale bars: 50 μm in A; 20 μm in B.

Mentions: To reveal the subcellular localization of PCNA in actively dividing cells, we crossed UAS:PCNA-CB founder fish to a wnt1 driver line (Volkmann et al., 2010). In these transgenic embryos, the dorsal midbrain is sparsely but strongly labelled during the first few days of development (Fig. 3A). The high level of transactivation induced by the wnt1 promoter fragment was instrumental in testing for possible cellular toxicity caused by the expression of chromobodies. We did not detect obvious signs of cellular stress or macroscopic cell death in these embryos. We reasoned that if PCNA-directed chromobodies can interfere with constitutive PCNA-dependent DNA replication, delayed neurogenesis and consequent morphological defects would be observed. By contrast, embryos that express PCNA-CB from 16 hpf were healthy and could be raised to adulthood. Live imaging conducted from 30 hpf to ∼42 hpf revealed that neighbouring cells were undergoing asynchronous cell cycles, as they showed distinct PCNA-CB localization patterns. We focused further analysis on cells displaying finely speckled chromobody signal in their nuclei, a mark of S phase. Over time, foci gradually decreased in number and increased in size, indicating the progression from early to late S phase, during which heterochromatin is replicated (Burgess et al., 2012). Immediately afterwards, a dramatic localization shift from speckles to uniform nuclear labelling was recorded shortly before the cells underwent mitosis (arrowheads in Fig. 3B; supplementary material Movies 3,6). These observations specifically mimic the reported cell cycle-dependent localization pattern of PCNA in mammalian cells (Leonhardt et al., 2000; Essers et al., 2005). After cell division, the chromobody relocalizes to the cytoplasm, from where PCNA is actively transported back into the nucleus during G1.Fig. 3.


Live imaging of endogenous protein dynamics in zebrafish using chromobodies.

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

Cell cycle analysis of transgenic zebrafish embryos expressing PCNA-CB. (A) Overview of the dorsal midbrain of a wnt1:gal4,UAS:GFP (green); UAS:PCNA-CB (magenta) double transgenic embryo at 38 hpf. (B) Unaltered cell cycle progression in PCNA-CB-expressing, wnt1-positive neural progenitors. PCNA-CB signal transitions from a speckled configuration (S phase) to an evenly distributed one (G2), which precedes mitosis (M). Arrowheads indicate a cell progressing through its last cycle before terminally differentiating into two daughter neurons. Time-stamps: h:min:s. Scale bars: 50 μm in A; 20 μm in B.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440926&req=5

DEV118943F3: Cell cycle analysis of transgenic zebrafish embryos expressing PCNA-CB. (A) Overview of the dorsal midbrain of a wnt1:gal4,UAS:GFP (green); UAS:PCNA-CB (magenta) double transgenic embryo at 38 hpf. (B) Unaltered cell cycle progression in PCNA-CB-expressing, wnt1-positive neural progenitors. PCNA-CB signal transitions from a speckled configuration (S phase) to an evenly distributed one (G2), which precedes mitosis (M). Arrowheads indicate a cell progressing through its last cycle before terminally differentiating into two daughter neurons. Time-stamps: h:min:s. Scale bars: 50 μm in A; 20 μm in B.
Mentions: To reveal the subcellular localization of PCNA in actively dividing cells, we crossed UAS:PCNA-CB founder fish to a wnt1 driver line (Volkmann et al., 2010). In these transgenic embryos, the dorsal midbrain is sparsely but strongly labelled during the first few days of development (Fig. 3A). The high level of transactivation induced by the wnt1 promoter fragment was instrumental in testing for possible cellular toxicity caused by the expression of chromobodies. We did not detect obvious signs of cellular stress or macroscopic cell death in these embryos. We reasoned that if PCNA-directed chromobodies can interfere with constitutive PCNA-dependent DNA replication, delayed neurogenesis and consequent morphological defects would be observed. By contrast, embryos that express PCNA-CB from 16 hpf were healthy and could be raised to adulthood. Live imaging conducted from 30 hpf to ∼42 hpf revealed that neighbouring cells were undergoing asynchronous cell cycles, as they showed distinct PCNA-CB localization patterns. We focused further analysis on cells displaying finely speckled chromobody signal in their nuclei, a mark of S phase. Over time, foci gradually decreased in number and increased in size, indicating the progression from early to late S phase, during which heterochromatin is replicated (Burgess et al., 2012). Immediately afterwards, a dramatic localization shift from speckles to uniform nuclear labelling was recorded shortly before the cells underwent mitosis (arrowheads in Fig. 3B; supplementary material Movies 3,6). These observations specifically mimic the reported cell cycle-dependent localization pattern of PCNA in mammalian cells (Leonhardt et al., 2000; Essers et al., 2005). After cell division, the chromobody relocalizes to the cytoplasm, from where PCNA is actively transported back into the nucleus during G1.Fig. 3.

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