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Live imaging of endogenous protein dynamics in zebrafish using chromobodies.

ABSTRACT: Chromobodies are intracellular nanoprobes that combine the specificity of antibodies with the convenience of live fluorescence imaging in a flexible, DNA-encoded reagent. Here, we present the first application of this technique to an intact living vertebrate organism. 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. For the first time, the chromobody technology enables live imaging of endogenous subcellular structures in an animal, with the remarkable advantage of avoiding target protein overexpression or tagging. 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.

SUBMITTER: Panza P

PROVIDER: S-EPMC4440926 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Neutrophil granules (NGs) are key components of the innate immune response and mark the development of neutrophilic granulocytes in mammals. However, there has been no specific fluorescent vital stain up to now to monitor their dynamics within a whole live organism. We rationally designed a benzochalcone fluorescent probe (HAB) featuring high tissue permeability and optimal photophysics such as elevated quantum yield, pronounced solvatochromism and target-induced fluorogenesis. Phenotypic screening identified HAB as the first cell- and organelle-specific small-molecule fluorescent tracer of NGs in live zebrafish larvae, with no labeling of other cell types or organelles. HAB staining was independent of the state of neutrophil activation, labeling NGs of both resting and phagocytically active neutrophils with equal specificity. By high-resolution live imaging, we documented the dynamics of HAB-stained NGs during phagocytosis. Upon zymosan injection, labeled NGs were rapidly recruited to the forming phagosomes. Despite being a reversible ligand, HAB could not be displaced by high concentrations of pharmacologically relevant competing chalcones, indicating that this specific labeling was the result of the HAB's precise physicochemical signature rather than a general feature of chalcones. However, one of the competitors was discovered as a promising interstitial fluorescent tracer illuminating zebrafish histology, similarly to BODIPY-ceramide. As a yellow-emitting histopermeable vital stain, HAB functionally and spectrally complements most genetically incorporated fluorescent tags commonly used in live zebrafish biology, holding promise for the study of neutrophil-dependent responses relevant to human physiopathology such as developmental defects, inflammation and infection. Furthermore, HAB intensely labeled isolated live human neutrophils at the level of granulated subcellular structures consistent with human NGs, suggesting that the labeling of NGs by HAB is not restricted to the zebrafish model but also relevant to mammalian systems.

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Live imaging of endogenous protein dynamics in zebrafish using chromobodies.

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