Identification and isolation of primary cilia in the HAT-7 cell line

Thesis (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2015 (Department of Molecular and Cell Biology).Includes bibliographic references: leaves 44-48.Primary cilia (PC) are 9+0 arranged microtubule based cellular organelles that are present on the surface of most eukaryotic cells. Their functions in various locations are not fully understood but current data is suggestive of important mechano- and chemo-sensory roles. More specifically PC have been linked to anterior-posterior limb patterning, endochondral bone formation, maintenance of bone, craniofacial development and tooth development. The electron microscopic presence of PC on odontoblasts and their dynamic expression on differentiating ameloblasts suggests possible non-elucidated signaling pathways. In the rat incisor tooth model, development and eruption occur continuously most likely from the cervical loop which has been labeled as the stem cell niche . The labial surface of the developing rat incisor contains a gradation of different stage cells from the progenitor cells of the cervical loop at the apex to the maturation stage ameloblasts below completely mineralized enamel. In this experiment attempts were made to identify and isolate samples of PC without non-ciliary contaminants from HAT-7 cell cultures . Protein isolates of the cytosol, membrane, nuclear and cytoskeleton of HAT-7 and IMCD3 cells were formed using fractionation techniques. All samples were examined using biochemical characterization via SOS-PAGE ... [TRUNCATED