UVM Theses and Dissertations
Format:
Print
Author:
Valentine, Megan Smith
Dept./Program:
Biology
Year:
2009
Degree:
MS
Abstract:
Meckelin (MKS3 or TMEM67) is a low abundance, highly conserved, seven transmembrane protein located in ciliated cells. In humans, mutations in MKS3 are most commonly associated with the ciliopathy Meckel-Gruber syndrome. This disease is characterized by occipital encephalocele, renal cystic dysplasia, and polydactyly. In a developing embryo, a null mutation in the MKS3 gene leads to the abortion of the fetus around 20 weeks. One orthologue of this gene has been found in the Annotated Paramecium Genome (GSPATG00015939001). We used RNA interference (RNAi) to study the role of this gene in Paramecium. As a comparison for MKS3 we have targeted IFT88, a vital trafficking protein to intraflagellar transport (IFT). We anticipate the RNAi phenotype of the IFT88 RNAi-fed cells will display disruption in their ciliary lengths (they should be short or missing), but should show no change in the positioning or organization of the basal bodies and the cell surface.
Cells fed the IFT88 and MKS3 RNAi bacteria, when compared to cells fed empty vector (L4440) bacteria (controls), demonstrated slow growth, abnormal cell shape, and very slow swimming. The IFT88 RNAi-fed cells showed longer backward swimming in solutions containing TEA, indicating that these cells have a diminished IK(Ca). They also demonstrated shorter backward swimming in 30 mM KC1 and mixed swimming results in 8 mM BaC1₂, indicating a diminished ICa(V). The MKS3 RNAi-fed cells showed long backward swimming in 20 mM NaC1 and shorter backward swimming in 30 mM KC1 and 8 mM BaC1₂, when compared to the controls.
The results from these cells are hard to interpret but do indicate a diminished ICa(V), most likely due to the short and missing cilia (also the case with the IFT88 RNAi-fed cells). Immunostaining studies showed dramatically shorter cilia in both RNAi-fed cell types when compared to the control cells and also showed disrupted rows of basal bodies in the MKS3 RNAifed cells. Scanning electron microscope images showed bald cells for IFT88 RNAi cells with a well organized cell surface, but a very folded and disrupted cell surface (as well as misshapen and short cilia) for the MKS3 RNAi-fed cells.
Using a 3x Flag-sequence in the vector pPXV, the genomic sequence for Paramecium MKS3 was marked at the N- or C- terminus, linearized, and injected into the macronucleus of the paramecia. Injecting C-terminal tagged MKS3 caused cell death within 3 days. N-terminal tagged MKS3 produced two separate staining results, but not within the same population of cells. In the first result, the protein clearly localizes to the cell surface, in a pattern consistent with the parasomal sacs (sites of endo- and exo- cytosis). The second pattern indicates partial co-localization with basal bodies and possibly ciliary staining. We demonstrate here the cellular localization of the Mks3 protein in Paramecium and show that this protein is involved with the maintenance of the cell and ciliary membrane, and is most likely involved in basal body positioning or anchoring.
Cells fed the IFT88 and MKS3 RNAi bacteria, when compared to cells fed empty vector (L4440) bacteria (controls), demonstrated slow growth, abnormal cell shape, and very slow swimming. The IFT88 RNAi-fed cells showed longer backward swimming in solutions containing TEA, indicating that these cells have a diminished IK(Ca). They also demonstrated shorter backward swimming in 30 mM KC1 and mixed swimming results in 8 mM BaC1₂, indicating a diminished ICa(V). The MKS3 RNAi-fed cells showed long backward swimming in 20 mM NaC1 and shorter backward swimming in 30 mM KC1 and 8 mM BaC1₂, when compared to the controls.
The results from these cells are hard to interpret but do indicate a diminished ICa(V), most likely due to the short and missing cilia (also the case with the IFT88 RNAi-fed cells). Immunostaining studies showed dramatically shorter cilia in both RNAi-fed cell types when compared to the control cells and also showed disrupted rows of basal bodies in the MKS3 RNAifed cells. Scanning electron microscope images showed bald cells for IFT88 RNAi cells with a well organized cell surface, but a very folded and disrupted cell surface (as well as misshapen and short cilia) for the MKS3 RNAi-fed cells.
Using a 3x Flag-sequence in the vector pPXV, the genomic sequence for Paramecium MKS3 was marked at the N- or C- terminus, linearized, and injected into the macronucleus of the paramecia. Injecting C-terminal tagged MKS3 caused cell death within 3 days. N-terminal tagged MKS3 produced two separate staining results, but not within the same population of cells. In the first result, the protein clearly localizes to the cell surface, in a pattern consistent with the parasomal sacs (sites of endo- and exo- cytosis). The second pattern indicates partial co-localization with basal bodies and possibly ciliary staining. We demonstrate here the cellular localization of the Mks3 protein in Paramecium and show that this protein is involved with the maintenance of the cell and ciliary membrane, and is most likely involved in basal body positioning or anchoring.