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.

Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation

“…Of note, no significant difference could be observed between control parasites and parasites expressing Cb. Together, these data confirm that expression of Cb in parasites allows detection of F-actin and that Cb expression has only minor effects on F-actin dynamics, as previously shown in other eukaryotes (Panza et al, 2015).

10.7554/eLife.24119.014Figure 5.Chromobodies are specific for parasite F-actin and do not influence total amount of F-actin in the parasite.( A ) Coomassie stained gel showing recombinant Cb purified from bacteria.

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“…In summary, expression of Cb is well tolerated by the parasite indicating that it does not significantly adversely affect normal actin functions within the cell, in agreement to data obtained in other cellular systems (Belin et al, 2014; Panza et al, 2015). However, it cannot be ruled out that actin dynamics are locally affected within the cell due to expression of Cb.…”

“…While individual actin filament kinetics could not be easily defined, due to their highly dynamic nature within the parasite cytosol (Videos 5–7), a relatively stable F-actin network was clearly visible that linked individual parasites within the vacuole. Chromobodies have been used successfully in diverse eukaryotes to analyse actin dynamics (Panza et al, 2015; Plessner et al, 2015; Rocchetti et al, 2014) and appear to have less toxicity than other F-actin sensors. However, we were concerned that Cb expression in T. gondii would perturb actin polymerisation, especially given the proposed dynamic and unstable nature of T. gondii actin.…”

“…GFP-actin shows a high signal to noise ratio in parasites causing the inability to differentiate actin filaments from the monomeric form Angrisano et al (2012a). Thus we sought a new approach to image F-actin in intracellular parasites by expressing actin-chromobody, a single chain anti-actin camel antibody that has been successfully employed in diverse eukaryotes, including plants (Rocchetti et al, 2014) and animal cells (Plessner et al, 2015; Panza et al, 2015). Chromobodies (Cb) were found to have several advantages compared to other actin probes, such as lower toxicity, less influence on F-actin dynamics and a high signal to noise ratio (Panza et al, 2015).…”

Actin visualization at a glance

“…However, in contrast to LifeAct, the Actin-Chromobody does not seem to influence apparent actin dynamics, even when it is expressed at high levels. So far, the ActinChromobody has been successfully applied to visualize actin in mammalian nuclei , in tobacco leaf cells (Rocchetti et al, 2014) and zebrafish (Panza et al, 2015). Nevertheless, at present, it remains a novel and less-well explored tool.…”