Ciliopathies are a group of disorders that arise from ciliary dysfunction. Meckelin (MKS3 or TMEM67) is a conserved transmembrane protein found at the transition zone of ciliated cells. In humans MKS3 is one of 3 genes linked to the ciliopathy Meckel Syndrome. This disease is characterized by occipital meningioencephalocoele, polycystic kidneys, fibrotic changes to the liver, postnatal polydactyly and situs inversus. Paramecium tetraurelia is a single celled ciliated eukaryote. Its surface is organized of a meshwork of cortical units that run the length of the cell. At the center of the cortical units are either one or two basal bodies. In two basal body units only the posterior basal body is ciliated. From the ciliated basal body, three rootlets project in stereotypical orientations: the post-ciliary rootlet projects posteriorly, the transverse microtubule projects toward the adjacent basal body row and the striated rootlet projects anteriorly. Both the post-ciliary rootlet and transverse microtubule are microtubule-based structures. The striated rootlet is composed of multiple subunits that are predicted to have conserved segmented coiled coil domains known as SF-Assemblin domains. In Picariello at al., 2014, we showed that MKS3 is present in the transition zone of Paramecium tetraurelia and that RNAi for MKS3 leads to global ciliary loss. Additionally, RNAi for MKS3 results in the disorganization of the basal body rows. Within the areas of disorganization, the basal bodies along with their striated rootlets, post-ciliary rootlets and transverse microtubules are rotated away from their expected orientation. Interestingly, the post-ciliary rootlet and transverse microtubule are still attached at the expected angles relative to each other within the areas of disorganization. Initial GST pull-down experiments using the coiled coil domain of MKS3 suggest a potential interaction between MKS3 and the striated rootlet family members KdC1 and KdB2. To test potential interactions between MKS3 and the striated rootlet we identified 27 potential striated rootlet family members in Paramecium. Full-length sequences for 13 of these genes were marked at their N-terminus with a 3x FLAG sequence. Components with a conserved SF-Assemblin domain were distributed uniformly within the striated rootlet. Components lacking the SF-Assemblin domain were found in various cellular locations, but not within the striated rootlet. GST pull-down experiments utilizing the MKS3 C-terminus as bait were performed using cells expressing the FLAG-tagged striated rootlet family members. Unfortunately a clear interaction between MKS3 and the striated rootlet remains elusive. The organized nature of the surface of Paramecium has allowed us to identify a previously unrealized function for MKS3. Our immunofluorescence data suggest that MKS3 functions outside the transition zone to maintain basal body row organization by potentially contributing to a link between the basal body and the striated rootlet. Without the link, the migrating basal bodies are free to rotate and project their rootlets in the wrong directions. Although the nature of the link remains elusive, the identification of disorganized basal body rows upon MKS3 reduction suggests that, in addition to ciliary dysfunction, basal body polarity defects may contribute to the development of MKS.

The Anterior Gradient 2 (AGR2) protein has been implicated in a variety of biological systems linked to cancer and metastasis, tamoxifen-induced drug resistance, pro-inflammatory diseases like IBD and asthma, and limb regeneration. The molecular mechanisms by which AGR2 mediates these various phenotypes in disease progression in both cancer and IBD are poorly understood, as is the biological function(s) of AGR2 under non-disease conditions. Here, we use a combination of biochemical techniques, organ culture, cell biology and mouse genetics to investigate the biological significance of AGR2 both in cell lines and in vivo. We present data based on phage-peptide inter-actomics screens suggesting a role for AGR2 in mediating the maturation and trafficking of a class of membrane and secretory proteins, and investigate a putative interaction between AGR2 and one member of this class of proteins. We also describe the construction of a universal vector for use in making a variety of transgenic animals, and then present data showing its use as a promoter reporter, and attempt to investigate the temporal and spatial expression of AGR2 in the developing and adult mouse. Further, we present data describing the localisation pattern of AGR2 in the developing murine kidney using a combination of organ culture and antibody staining, and suggest a role for AGR2 in the developing kidney based on this data that is in agreement with a chaperone function for membrane and secretory proteins. Together, these data suggest that AGR2 has an intrinsic consensus docking site for a subset of its client proteins, that AGR2 plays a role in protein maturation in ciliated cell types, and provides a novel biological model to dissect the role of AGR2 in ER-trafficking.

Indiana University-Purdue University Indianapolis (IUPUI) / Ciliopathies with multiorgan pathology include renal cysts and eye pathology. Previous studies showed meckelin (MKS3 protein product) are crucial to cilia function and its absence in Wpk rats (with mutated rMks3 gene) causes Leber's congenital amaurosis. Retinal photoreceptors have connecting cilium that joins the inner to the outer segment and plays a role in the transport of molecules necessary for morphological and molecular development and maintenance of the outer segment process. The present study evaluated meckelin expression during normal postnatal retinal development and the consequences of mutant meckelin on photoreceptor development and survival in Wistar-Wpk/Wpk rat. Meckelin was co-expressed in photoreceptors, amacrine, Muller glia and ganglion cells in postnatal day 10 (P10) and P21 wild type retinae. Meckelin was detected in both inner and outer segments of photoreceptors. By P10, both wild type and homozygous Wpk mutant retina had all retinal cell types. In contrast, by P21, cells expressing photoreceptor-specific markers in the Wpk mutant were fewer in number with abnormal expression patterns. Cell death assays confirmed a significant amount of cells undergoing apoptosis in the outer nuclear layer of the mutant rat retina. By electron microscopy, mutant photoreceptors did not develop an outer segment process beyond a connecting cilium and rudimentary outer segment. We conclude that MKS3 is not important for formation of connecting cilium and rudimentary outer segments, but is critical for the elongation and/or maintenance of mature outer segment processes.