Protein Expression in Kidney Epithelial Cell Primary Cilium
Collaboration
M. Chamlali and L. B. Pedersen, Department of Biology, University of Copenhagen
Research Background
This research investigates the role of DLG1, a Scribble polarity complex protein, in regulating ciliary protein trafficking in kidney epithelial cells. As a part of the research, PC2 and SDCCAG3 intensity levels were measured in spinning disk fluorescence microscopy 3D image stacks of transwells-cultured cells acquired from WT, Dlg1−/−, and rescue mCCD cell lines. The IACF created a fully automated MATLAB script to report the mean fluorescence intensity of the different proteins of interest inside subregions of the identified primary cilia.
Method
Nucleus regions were automatically identified (DAPI channel, Gaussian filtering, background subtraction and global thresholding)
Primary cilia were segmented (cilia marker channel, Gaussian filtering, and local thresholding) as the brightest 3D objects overlapping a nucleus region.
Primary cilium bases were identified as the closest cilium voxel to the center of mass of the corresponding nucleus region (assuming an outward growth of the cilia)
Primary cilium base regions were defined as the set of cilium voxels within a maximum (user-defined) geodesic distance to the corresponding base. F
SDCCAG3 and PC2 channels mean intensities were individually measured and reported inside each primary cilium, primary cilium base region, and primary cilium body (whole cilium excluding the base region) after background intensity correction (3D median filtered image subtraction).
For more details, please see the full publication and code linked below.
Publication
Rezi, C. K., Aslanyan, M. G., Diwan, G. D., Cheng, T., Chamlali, M., Junger, K., Anvarian, Z., Lorentzen, E., Pauly, K. B., Afshar-Bahadori, Y., Fernandes, E. F., Qian, F., Tosi, S., Christensen, S. T., Pedersen, S. F., Strømgaard, K., Russell, R. B., Miner, J. H., Mahjoub, M. R., & Boldt, K. (2024). DLG1 functions upstream of SDCCAG3 and IFT20 to control ciliary targeting of polycystin-2. EMBO Reports, 25(7), 3040–3063. https://doi.org/10.1038/s44319-024-00170-1