Reduction of meckelin leads to general loss of cilia, ciliary microtubule misalignment and distorted cell surface organization

Abstract

Abstract Background Meckelin (MKS3), a conserved protein linked to Meckel Syndrome, assists in themigration of centrioles to the cell surface for ciliogenesis. We explored foradditional functions of MKS3p using RNA interference (RNAi) and expression of FLAGepitope tagged protein in the ciliated protozoan Paramecium tetraurelia.This cell has a highly organized cell surface with thousands of cilia and basalbodies that are grouped into one or two basal body units delineated by ridges. Thehighly systematized nature of the P. tetraurelia cell surface provides aresearch model of MKS and other ciliopathies where changes in ciliary structure,subcellular organization and overall arrangement of the cell surface can be easilyobserved. We used cells reduced in IFT88 for comparison, as theinvolvement of this gene’s product with cilia maintenance and growth is wellunderstood. Results FLAG-MKS3p was found above the plane of the distal basal body in the transitionzone. Approximately 95% of those basal bodies observed had staining for FLAG-MKS3.The RNAi phenotype for MKS3 depleted cells included global shortening andloss of cilia. Basal body structure appeared unaffected. On the dorsal surface,the basal bodies and their associated rootlets appeared rotated out of alignmentfrom the normal anterior-posterior rows. Likewise, cortical units were abnormal inshape and out of alignment from normal rows. A GST pull down using the MKS3coiled-coil domain suggests previously unidentified interacting partners. Conclusions Reduction of MKS3p shows that this protein affects development and maintenance ofcilia over the entire cell surface. Reduction of MKS3p is most visible on thedorsal surface. The anterior basal body is attached to and moves along thestriated rootlet of the posterior basal body in preparation for duplication. Wepropose that with reduced MKS3p, this attachment and guidance of the basal body islost. The basal body veers off course, causing basal body rows to be misalignedand units to be misshapen. Rootlets form normally on these misaligned basal bodiesbut are rotated out of their correct orientation. Our hypothesis is furthersupported by the identification of novel interacting partners of MKS3p including akinetodesmal fiber protein, KdB2.