PgmNr C55: Using Chlamydomonas to understand cilia assembly.

Authors:
S. K. Dutcher; Huawen Lin; Suyang Guo; Robyn Roth


Institutes
Washington Univ Sch Med, St Louis, MO.


Abstract:

The eukaryotic cilium is a complex machine with over 600 polypeptides. Motile cilia move cells or fluids. Motile and primary cilia play roles in signaling. Cilia both send and receive signals from the environment. Defects in these organelles cause a wide range of human diseases that include cancer, obesity, retinal degeneration, kidney and bone diseases as well as neurocognitive defects. We use Chlamydomonas reinhardtii, to understand the assembly and function of cilia. Ciliary proteins are highly conserved throughout the eukaryotic lineage and model organisms have been key in identifying and understanding human ciliopathies. We have been addressing three key questions using electron tomographic, biochemical, and genetic approaches. First, how are the large protein assemblies of motile cilia assembled in the cytoplasm? Second, how are cytoplasmic proteins kept out of cilium? Third, how are membrane vesicles used for ciliary signaling? Primary ciliary dyskinesia (PCD) is a disease in which the motility of cilia on the multi-ciliated epithelia of the respiratory tract is compromised. We are examining the roles of several proteins (HEATR2, DYX1C1, and IDA3) in the cytoplasmic assembly of the dynein arms.  Proteins that localize to the transition zone, which spans from the triplet microtubules of the basal body, a modified centriole, to the doublet microtubules of the ciliary axoneme cause a variety of ciliopathies characterized by kidney defects, polydactyly, and retinal degeneration. We have found that some of these transition zone proteins (NPHP4, CEP290, RPGRIP1L) as well as proteins needed for intraflagellar transport (IFT172, IFT144, IFT52) are needed for the assembly of highly organized arrays of transmembrane proteins in the ciliary necklace, which is in the ciliary membrane surrounding the transition zone. It has been postulated that the transition zone and/or the ciliary necklace provide the barrier to the entry of cytoplasmic proteins into cilia. Using mass spectroscopy of isolated flagella, we find that that the ciliary necklace does not provide barrier function. The absence of transition zone proteins affects the barrier. Loss of RPGRIP1L has the strongest effect on the barrier; the 26S proteasome and the TRiC/CCT chaperonin complex enter the mutant cilia. Understanding the consequences of the entry of these proteins is underway. Studies in Chlamydomonas and C. elegans showed that membrane vesicles referred to as ectosomes are released from the ciliary membrane (Wang et al., 2015; Wood et al., 2013; Cai et al., 2015). These ectosomes carry signaling activity. We have found that the transition zone protein, RPGRIP1L, plays a role in the production of signal-containing ectosomes. The identification of new mutants is being pursued to understand the role of the transition zone in ectosome production and signaling.