Abstract
BackgroundCertain members of the Camelidae family produce a special type of antibody with only one heavy chain. The antigen binding domains are the smallest functional fragments of these heavy-chain only antibodies and as a consequence have been termed nanobodies. Discovery of these nanobodies has allowed the development of a number of therapeutic proteins and tools.In this study a class of nanobodies fused to fluorescent proteins (chromobodies), and therefore allowing antigen-binding and visualisation by fluorescence, have been used. Such chromobodies can be expressed in living cells and used as genetically encoded immunocytochemical markers.ResultsHere a modified version of the commercially available Actin-Chromobody® as a novel tool for visualising actin dynamics in tobacco leaf cells was tested. The actin-chromobody binds to actin in a specific manner. Treatment with latrunculin B, a drug which disrupts the actin cytoskeleton through inhibition of polymerisation results in loss of fluorescence after less than 30 min but this can be rapidly restored by washing out latrunculin B and thereby allowing the actin filaments to repolymerise.To test the effect of the actin-chromobody on actin dynamics and compare it to one of the conventional labelling probes, Lifeact, the effect of both probes on Golgi movement was studied as the motility of Golgi bodies is largely dependent on the actin cytoskeleton. With the actin-chromobody expressed in cells, Golgi body movement was slowed down but the manner of movement rather than speed was affected less than with Lifeact.ConclusionsThe actin-chromobody technique presented in this study provides a novel option for in vivo labelling of the actin cytoskeleton in comparison to conventionally used probes that are based on actin binding proteins.The actin-chromobody is particularly beneficial to study actin dynamics in plant cells as it does label actin without impairing dynamic movement and polymerisation of the actin filaments.
Highlights
Certain members of the Camelidae family produce a special type of antibody with only one heavy chain
This chromobody was previously used to transfect HeLa cells to show the recovery of the actin filaments after Cytochalasin D treatment (ChromoTek homepage) where it was shown that the transient binding does not influence cell viability or motility
Agrobacterium tumefaciens was transformed with these constructs and Nicotiana tabacum leaves were infiltrated with the transformed agrobacteria, either singly or with the Golgi marker consisting of the signal anchor sequence of a rat sialyl transferase fused to Green fluorescent protein (GFP) [STGFP, 31] as described in [32]
Summary
A modified version of the commercially available Actin-Chromobody® as a novel tool for visualising actin dynamics in tobacco leaf cells was tested. The actin-chromobody binds to actin in a specific manner. Treatment with latrunculin B, a drug which disrupts the actin cytoskeleton through inhibition of polymerisation results in loss of fluorescence after less than 30 min but this can be rapidly restored by washing out latrunculin B and thereby allowing the actin filaments to repolymerise. To test the effect of the actin-chromobody on actin dynamics and compare it to one of the conventional labelling probes, Lifeact, the effect of both probes on Golgi movement was studied as the motility of Golgi bodies is largely dependent on the actin cytoskeleton. With the actin-chromobody expressed in cells, Golgi body movement was slowed down but the manner of movement rather than speed was affected less than with Lifeact
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