Spotlight: pacFA Ceramide
February 26, 2016
Dr. Erhard Bieberich’s laboratory at Georgia Reagents University is interested in proteins that mediate the regulation of cilia and flagella by ceramide. Flagella and cilia are sensory and motor cellular organelle responsible for moving extracellular fluids and particles and propelling cells and unicellular organisms . The bioactive sphingolipid, ceramide, has been found to directly interact with atypical protein kinase Cζ (aPKCζ) to promote ciliogenesis . In order to begin to identify additional proteins that interact with ceramide, Dr. Bieberich’s laboratory used the novel bifunctional ceramide analog, pacFA Ceramide, available from Avanti Polar Lipids (catalog # 900404). In a typical protocol, living cells are incubated with this analog added to the medium. The pacFA Ceramide is then UV-crosslinked to the ceramide binding proteins. Following crosslinking, the cells are fixed, and the pacFA Ceramide-protein complex is visualized by covalently linking the pacFA Ceramide to a fluorescent dye using click chemistry. Work using this molecule is showcased in a recent publication from his laboratory in Molecular Biology of the Cell, “Regulation of Chlamydomonas flagella and ependymal cell motile cilia by ceramide-mediated translocation of GSK3”. In this study, Dr. Bieberich’s laboratory very eloquently demonstrates that Chlamydomonas flagella and ependymal cell cilia display a similar distribution of ceramides that binds to the protein glycogen synthase kinase-3 (GSK3), which is essential for the regulation of ciliogenesis by a mechanism involving the ceramide-mediated translocation of GSK3 into flagella and cilia . Co-localization data with atypical PKCζ, a known ceramide binding protein, indicates that pacFA Ceramide distributes in membranes in the same manner as endogenous ceramides . This study is a compelling example of how the bifunctional ceramide analog, pacFA Ceramide, is a useful addition to research involving the visualization and identification of ceramide binding proteins.
In addition to ceramide, Avanti offers pacFA incorporated into a variety of lipids, as well as, free pacFA, that can be used for additional binding studies.
- Kong JN, Hardin K, Dinkins M, Wang G, He Q, Mujadzic T, Zhu G, Bielawska J, Spassieva S, Bieberich E. (2015) Regulation of Chlamydomonas flagella and ependymal cell motile cilia by ceramide-meidated translocation of GSK3. Mol. Bio. Cell 26(24): 4451-4465