Investigation of Calmodulin-like 13 and 14 as Myosin Light Chains in Arabidopsis thaliana

Abstract

Calcium ions serve as ubiquitous second messengers in the signal transduction pathways of eukaryotic cells. The evolutionarily conserved calcium sensor, calmodulin (CaM), responds to calcium signals and, through protein-protein interactions, facilitates downstream cellular responses. Plants also possess a unique family of calmodulin-like (CML) calcium sensors with 50 members in Arabidopsis thaliana. Two closely related paralogs in Arabidopsis, CML13 and CML14, were previously found to interact with the isoleucine-glutamine (IQ) domains of myosins, IQ67-domain (IQD) proteins, and CaM-activated transcription activators (CAMTAs). This thesis explored the properties and function of the interaction of Arabidopsis CML13 and CML14 with Arabidopsis myosins. Using the split-luciferase and FRET-FLIM or CAPPI assays CaM, CML13, and CML14 were found to bind to the IQ domains in the neck region of all Arabidopsis myosin VIII and XI isoforms tested, with some specificity observed among IQ-CaM/CML interactions in planta. Interestingly, this interaction was conserved in the CML13/14 orthologs in the deep-branching multicellular algae, Chara braunii, where CbCML2/4 also bound to the IQ domains of Chara myosins and CAMTAs. Furthermore, in vitro overlay and dansyl interaction experiments revealed that recombinant CaM, CML13, and CML14 exhibit specific, high-affinity, calcium-independent binding to the IQ domains of myosins. In vitro actin-motility assays using recombinant myosin VIIIs and XIs demonstrated that CaM, CML13, and CML14 function as light chains, and in most cases, either CML13 or CML14 is required for maximal motility. I also found that inducible RNA silencing of CML13 and CML14 transcripts in vivo produced a pleiotropic phenotype including reduced or terminated growth, premature senescence, and seedling mortality. Overall, my thesis indicates that CML13 and CML14 are novel myosin light chains in Arabidopsis and are important proteins throughout plant development.