Investigating roles of a few Centrosomal proteins in Ciliogenesis and Maintenance

Abstract

Centrioles are cylindrical structures which play an instrumental role in forming centrosomes and cilium. Centrosomes are critical for the spindle pole formation during mitosis in the cell cycle. During ciliogenesis, usually, one of the two centrioles of a centrosome uses specially designed appendages at its distal end to cohere to ciliary vesicles and the cell membrane, forming cilia. Both cilia and centrosomes operate as signalling hubs and are naturally associated with cell cycle progression, cell proliferation, and differentiation. Understanding the operative linkages between centrosomes and cilia is essential because perturbations in these structures have been involved in many diseases, counting cancer and ciliopathies, e.g., Retinal degeneration, Alstrom syndrome and others. However, our knowledge of the molecular mechanisms of centrosome-to-cilium conversion and cilia maintenance has just begun to be explored. The primary challenge exists in designing applicable experimental approaches to distinguish centrosome/cilia biogenesis and maintenance. In this project, we aim to study this conversion of centrosome-to-cilium in mammalian cells through different techniques such as immunofluorescence and expansion microscopy, and genetic perturbations. We also aim to discover new novel interactors by Immunoprecipitation(IP) of important centrosomal/ciliary proteins from Drosophila non-ciliated cells vs ciliated cells, followed by Mass-Spectrometry (MS). Then we developed a strategy for examining the role of three ciliopathy-associated centrosome/ciliary proteins in cilia maintenance by conditional knockdown studies in fruit flies using excellent genetically tractable approaches. By using multifaceted techniques on long-lived ciliated cells in the fruit fly and mammalian cultured cells, my work helped to highlight the following points: a. we could follow Centrosome-to-Cilium conversion in mammalian cells (using confocal and expansion microscopy), b. IP, followed by MS studies of three proteins centrosome/cilium proteins, identified a few potential common interactors between all the three proteins, and their functions have to be further studied in future and c. we identified at least two of the centriolar proteins knocked down are critical for ciliary function maintenance in adult flies.