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dc.contributor.authorHe, Qian
dc.date.accessioned2017-12-29T15:45:26Z
dc.date.available2017-12-29T15:45:26Z
dc.date.issued2015en
dc.identifier.urihttp://hdl.handle.net/10675.2/621660
dc.description.abstractThe primary cilium is a single, antenna-like protrusion of mammalian cells, involved in many signaling pathways important for cellular processes. In Madin-Darby Canine Kidney (MDCK) cells, an apical ceramide-enriched compartment (ACEC) was observed at the base of the primary cilia. Ceramide and Rab11a vesicles showed similar protein and lipid profiles. The lipid and protein composition suggested the presence of a ceramide associated lipid-protein complex containing atypical protein kinase C (aPKC), Cdc42, Sec8, Rab11a, and Rab8 in MDCK cells. Ceramide vesicles and Rab11a vesicles were highly enriched with C16 and C18 ceramides. Expression of a ceramide-binding but dominant-negative mutant of aPKC suppressed ciliogenesis, indicating that not only the association of ceramide with aPKC, but the activation of aPKC is critical for ciliogenesis in MDCK cells. In neural progenitors (NPs) differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), the ceramide-protein interaction underlying ciliogenesis showed parallels to that with MDCK cells, but also significant differences. Ceramide was enriched in the apical region of the cell as well as primary cilia. In addition, the ceramide level was elevated by 3-fold after neural differentiation, especially C16 and C24:1 ceramide. Immunostaining showed that C16 ceramide was mainly distributed in the apical region and primary cilium, while the C24:1 antibody showed signals at the apicolateral cell membrane in addition to the apical area and the primary cilium. Immunostaining of aPKC also showed a signal at the apicolateral membrane as well as the primary cilium. Decreasing ceramide levels led to not only reduced ciliogenesis, but also translocation of membrane-bound aPKC to the cytosol, concurrent with its activation and the phosphorylation of its substrate Aurora A kinase (AurA). Incubation of ceramide-depleted cells with C24:1 ceramide restored ciliogenesis as well as membrane distribution of aPKC and accelerated neuronal process formation. The histone deacetylase (HDAC) inhibitor trichostatin A rescued ciliogenesis in ceramide-depleted MDCK cells and NPs, indicating that ceramide promotes tubulin acetylation in cilia. In summary, we concluded that ceramide promotes ciliogenesis by inhibiting HDAC6 activity in both of these two models, but via different molecular signaling pathways.
dc.relation.urlhttps://search.proquest.com/docview/1734473101?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.en
dc.subjectCiliogenesisen
dc.subjectCeramideen
dc.subjectAtypical protein kinase C (aPKC)en
dc.subjectMDCK Cellsen
dc.subjectneural progenitors (NPs) differentiated from human embryonic stem (ES) cellsen
dc.subjectHistone deacetylase 6 (HDAC6)en
dc.titleThe role of ceramide in the regulation of ciliogenesisen
dc.typeDissertationen
dc.contributor.departmentDepartment of Physiologyen
dc.description.advisorBieberich, Erharden
dc.description.degreeDoctor of Philosophy (Ph.D.)en
dc.description.committeeBollag, Wendy; Dong, Zheng; Du, Quansheng; Schoenlein, Patriciaen
refterms.dateFOA2020-05-26T17:52:12Z
html.description.abstractThe primary cilium is a single, antenna-like protrusion of mammalian cells, involved in many signaling pathways important for cellular processes. In Madin-Darby Canine Kidney (MDCK) cells, an apical ceramide-enriched compartment (ACEC) was observed at the base of the primary cilia. Ceramide and Rab11a vesicles showed similar protein and lipid profiles. The lipid and protein composition suggested the presence of a ceramide associated lipid-protein complex containing atypical protein kinase C (aPKC), Cdc42, Sec8, Rab11a, and Rab8 in MDCK cells. Ceramide vesicles and Rab11a vesicles were highly enriched with C16 and C18 ceramides. Expression of a ceramide-binding but dominant-negative mutant of aPKC suppressed ciliogenesis, indicating that not only the association of ceramide with aPKC, but the activation of aPKC is critical for ciliogenesis in MDCK cells. In neural progenitors (NPs) differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), the ceramide-protein interaction underlying ciliogenesis showed parallels to that with MDCK cells, but also significant differences. Ceramide was enriched in the apical region of the cell as well as primary cilia. In addition, the ceramide level was elevated by 3-fold after neural differentiation, especially C16 and C24:1 ceramide. Immunostaining showed that C16 ceramide was mainly distributed in the apical region and primary cilium, while the C24:1 antibody showed signals at the apicolateral cell membrane in addition to the apical area and the primary cilium. Immunostaining of aPKC also showed a signal at the apicolateral membrane as well as the primary cilium. Decreasing ceramide levels led to not only reduced ciliogenesis, but also translocation of membrane-bound aPKC to the cytosol, concurrent with its activation and the phosphorylation of its substrate Aurora A kinase (AurA). Incubation of ceramide-depleted cells with C24:1 ceramide restored ciliogenesis as well as membrane distribution of aPKC and accelerated neuronal process formation. The histone deacetylase (HDAC) inhibitor trichostatin A rescued ciliogenesis in ceramide-depleted MDCK cells and NPs, indicating that ceramide promotes tubulin acetylation in cilia. In summary, we concluded that ceramide promotes ciliogenesis by inhibiting HDAC6 activity in both of these two models, but via different molecular signaling pathways.


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