In Paramecium, the ciliary basal body is connected to three rootlets (Post ciliary rootlet (PCR), Transverse rootlet (TR) and Striated rootlet (SR)). The Paramecium cell surface is divided into units each of which has one or two basal bodies. The SR projects from the basal body toward the anterior in straight rows for several units. In Tetrahymena, a full-length SR is required to resist ciliary beating forces that cause the basal bodies to move out of alignment (Galati et al., 2014). We previously found that the depletion of the transition zone protein Meckelin results in the misaligned SR and disorganized basal bodies, reminiscent of the phenotype of the Tetrahymena mutant DisA. This mutant shows shortened SR and disorganized basal body rows. We wished to study the SR in Paramecium to determine its connection to Meckelin. To do so, we identified the genes for potential SR proteins. Through the expression of epitope tagged proteins, we found 9 proteins located in the SR. All of these proteins have the predicted domains of SF-assemblin protein which form the SR of Chlamydomonas. Proteins without this domain are not found in the Paramecium SR.
Immunoprecipitation (IP) of epitope tagged SR proteins followed by LC-MS/MS showed that SR family members co-IP. We purified the intact SRs and found that the epitope tagged proteins co-purify with the intact SR. LC-MS/MS analyses of the fraction of density gradients in which the SR resides show that all SR family member (SR-D, -E, -F and –G) are found together. Other proteins that co-purify are Centrin, Actin, α-, β-, and g- tubulin, Rab/Ras small GTPase.
SR-B and SR-C family members by homology but lacking the domains of SF-assemblin proteins are not located in the SR as shown by immunofluorescence, but both of are identified in the purified SR sample by LC-MS/MS.
RNAi for one of the SR family members (SR-F) showed a phenotype similar to that of the Tetrahymena mutant DisA: shortening of the SR and misaligned basal bodies rows all over the cells from the posterior to the anterior pole as compared to control cells. However, this phenotype is not found for RNAi depletion of a second SR protein SR-D4.
Acknowledgement:
LC-MS/MS analysis was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103449.