Calmodulin is Required for Paraflagellar Rod Assembly and Flagellum-Cell Body Attachment in Trypanosomes

In the flagellum of the African sleeping sickness parasite Trypanosoma brucei calmodulin (CaM) is found within the paraflagellar rod (PFR), an elaborate extra-axonemal structure, and the axoneme. In dissecting mechanisms of motility regulation we analysed CaM function using RNAi. Unexpectedly CaM depletion resulted in total and catastrophic failure in PFR assembly; even connections linking axoneme to PFR failed to form following CaM depletion. This provides an intriguing parallel with the role in the green alga Chlamydomonas of a CaM-related protein in docking outer-dynein arms to axoneme outer-doublet microtubules. Absence of CaM had no discernible effect on axoneme assembly, but the failure in PFR assembly was further compounded by loss of the normal linkage between PFR and axoneme to the flagellum attachment zone of the cell body. Thus, flagellum detachment was a secondary, time-dependent consequence of CaM RNAi, and coincided with the loss of normal trypomastigote morphology, thereby linking the presence of PFR architecture with maintenance of cell form, as well as cell motility. Finally, wider comparison between the flagellum detachment phenotypes of RNAi mutants for CaM and the FLA1 glycoprotein potentially provides new perspective into the function of the latter into establishing and maintaining flagellum-cell body attachment.