MSc Student, Snedden Lab Arabidopsis CML13 and CML14 Interact with Myosins and Function as Plant-Specific Myosin Light Chains Calcium ions (Ca2+) are widely present as secondary messengers in eukaryotes. Ca2+-signals are interpreted by Ca2+-binding proteins called sensors, which then regulate various responses. Apart from the highly conserved calmodulin (CaM), plants possess a distinct family of CaM-like proteins (CMLs) that function as Ca2+-sensors. CMLs primarily consist of Ca2+-binding EF-hands and lack any other functional domains. They are believed to act as sensor-relays by undergoing conformational changes induced by Ca2+ and interacting with target proteins. Among the 50 CMLs found in Arabidopsis, AtCML13/14 are particularly intriguing due to their unique biochemical properties and high expression levels in vivo. To investigate the function of CML13/14, we screened a yeast two-hybrid library to identify potential interacting proteins. Our screen led to the discovery of three unrelated families of putative targets: IQ67 domain proteins (IQDs; microtubule scaffolds), CAMTAs (transcription factors), and myosins (motor proteins). These proteins possess a structural characteristic known as tandem IQ-motifs, which are a special type of CaM-binding domain. Through in vitro and in vivo protein-interaction assays, we found evidence suggesting that CML13, CML14, and CaM are the primary interactors of these targets via their IQ domains within the CML family. Focusing on myosins as representative targets of CML13/14, we utilized confocal microscopy, in vitro kinetic assays, and in vitro binding tests to demonstrate that these CMLs act as novel myosin light chains. To gain further insights into their functions in vivo, we employed an inducible RNAi system to specifically silence either CML13 or CML14 in Arabidopsis. The resulting phenotypes were pleiotropic, indicating that these CMLs play crucial roles in development by regulating cytoskeletal function through their interactions with myosins and IQDs. In summary, our data suggests that CML13/14 are important regulators in various biological processes. They modulate cytoskeletal activity via their association with myosins and IQDs, while potentially influencing gene expression through interactions with CAMTAs.
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