Primary Cilia Are Micro Tubule Based Organelles That Stick out of the Cell Into The

Abstract

Primary cilia are micro tubule based organelles that stick out of the cell into the extracellular space, and function in signal transduction pathways. Recently the putative MAP kinase gene dyf-5 has been identified in C.elegans, which was shown to effect cilia length and IFT. The loss of function mutant had longer and misdirected cilia, while the overexpression mutant had very short cilia. Dyf-5 has three mammalian homologs, MAK, MRK and MOK. The goal of this project was to determine if MOK has a similar function in mammalian cells as dyf-5 has in C.elegans. For these experiments two cell lines were used, RPE cells (human retinal epithelial cells) and IMCD3 cells (mouse kidney epithelial cells). Both cell lines are able to form cilia in culture. The cells were transfected with shRNA’s directed against MOK, and has been overexpressed using four different MOK clones, the wildtype MOK and a mutated (kinase death) MOK, both with a C- and N-terminal GFP tag. In addition the subcellular localization pattern has been studied.

The transfection of IMCD3 cells with shRNA’s directed against MOK had no significant effect on cilium length. In the RPE cells the cilia were longer and there was a higher percentage ciliated cells. In both cases this results are not conclusive since the experiments to show a knockdown did not work properly.

The overexpression shows only little effect in the IMCD3 cells, but the effects in RPE cells are big. There was en large decrease in number cilia formed, hinting on a role of MOK in ciliogenesis.

For the RPE cells also a rescue experiment has been done, and none of the short hairpins is able to rescue the overexpression phenotype.

The localization experiments showed a difference between MOK clones with an N- and C-terminal GFP tag. The N-terminal tagged MOK localizes to the cytoplasm, while the C-terminal MOK localizes to the mitochondria.

Overall it is now too early to say that MOK has indeed the same role in mammalian cells as dyf-5 has in C.elegans, but there is evidence hinting on a role of MOK in ciliogenesis.