Functional Analysis Of Calmodulin In Cilia-related Activities

Cilium or flagellum?exchangeable terms?,a vital and conserved organelle from the cell surface,functions in cell motility,fluid flow,signal perception and transduction,and participates in variety of cell processes including cell cycle,cell differentiation and organ development.A range of human disorders known as ciliopathies result from defects in ciliary structure or function.Further study is warranted in all sorts of physiological processes as the precise structure,complex components and delicately regulated signals of cilium.Calcium,the paramount cation for living organisms,has been involved in series of important physiological processes such as ciliary motility,assembly,disassembly,deflagellation and signal transduction.Calmodulin?CaM?,the most extensive calcium signal transducer,is critical for flagellar motility as component of ciliary radial spoke and central pair.However,whether and how CaM is implicated in other ciliary activities is unclear,therefore it is of great value to investigate the function of CaM in relation to ciliary activities.To study the function of CaM,we first analyzed the features of CaM in C.reinhardtii,and confirmed its electrophoretic mobility as the relative mobility of CaM would shift in the presence and absence of Ca²⁺.We generated an insertional mutant of CaM by transforming wild type cells with DNA fragments.The cam mutant has shorter cilia compared to the wild type and most of the mutant cells were paralyzed,with few cells twitching or slow moving,reflecting the intervention of CaM in regulation of ciliary motility.As a ciliary protein,CaM is located to the transition zone and basal body of cilia,and enriched within the nucleus-basal body connector?NBBC?to regulate its length.The initial time of ciliary assembly delayed and the assembly rate,not disassembly rate,was reduced in cam mutant,suggesting CaM is required for ciliary assembly but not involved in ciliary disassembly.Additionally,the function of CaM in ciliary assembly is independent of protein synthesis or intraflagellar transport?IFT?.Further more,the cam mutant has defects in acid-induced deflagellation instead of deflagellation upon calcium shock,mechanical shearing or mastoparan treatment,indicating the cam mutant is not defective in deflagellation machinery and CaM functions in cytosolic calcium elevations induced by pH shock.With the in-depth study,the increase of inositol 1,4,5-trisphosphate?IP₃?induced by pH shock is inhibited in the cam mutant.No interaction between PLC and CaM has been detected and PLC activity is not affected by CaM in vitro,proving CaM mediates PLC activation indirectly.Moreover,the cam mutants also have defects in calcium entry and CaM interacts directly with ADF1,a transient receptor potential?TRP?channel,revealing CaM cooperates with ADF1 to regulate calcium entry.In summary,CaM is a key regulation factor in ciliary activities which plays crucial role in calcium homeostasis upon acidic stress.