Living markers for actin block myosin-dependent motility of plant organelles and auxin

Expression-based techniques using recombinant actin-binding proteins (ABPs) have been developed as advantageous means of visualising actin filaments. As actin function is linked to the movement of cellular cargoes, and overexpression of ABPs may compete with endogenous cytoskeletal proteins, such as myosins, secondary effects on cellular motility might be observed during actin visualisation. Cytoplasmic streaming and auxin transport were chosen as examples of cargo movement and investigated in two Arabidopsis thaliana lines stably transformed with fluorescently labelled talin (GFP-mTn) or fimbrin (GFP-FABD2). In both lines, the maximal streaming velocity of organelles was reduced to 80% in hypocotyl epidermal cells, where actin was broadly equally labelled by both ABPs. In contrast, observations of streaming and actin organisation during treatments with cytochalasin D (CD) suggested GFP-mTn-labelled actin to remain more stable. Furthermore, basipetal auxin transport was undisturbed in the GFP-FABD2 line but reduced by GFP-mTn. Remarkably, treatments with CD and 2,3-butanedione monoxime, which immobilizes myosin by impairing its ATPase, produced not only failures in organelle movement but also in basipetal auxin transport in the wild-type. These observations suggest that myosin is involved in processes of auxin translocation. In parallel, reduced motility in transgenic plants may be explained by a disturbed acto-myosin interplay, if overexpressed ABPs block the processive movement of myosin along actin filaments. This report shows that the use of live markers for actin visualisation may affect motility of cellular compounds and underlines the general need for critical investigation of actin-related processes in wild-type as well as transgenic plants prior to further interpretation.