MOLECULAR BIOLOGY AND GENETICS MS THESIS DEFENSE BY DİLA GÜLENSOY

Title: Elucidation of the mechanisms that underlie centriolar satellite biogenesis and dynamics

 Speaker: Dila Gülensoy

 Time: 11 August, 2022, 11.00 AM

Thesis Committee Members:

Assoc. Prof. Dr. Elif Nur Fırat Karalar (Advisor, Koç University)

Assist. Prof. Dr. Hasan Demirci (Koç University)

Prof. Dr. Arzu Çelik (Boğaziçi University)

Abstract:

Centriolar satellites are the third novel component of the mammalian centrosome/cilium complex. They are membrane-less electron-dense granules clustered around the main microtubule organizing center. These granules are heterogeneous in terms of composition, size, and shape and have a variety of roles in different cellular processes such as cilium assembly/disassembly, centriole duplication, stress response, and autophagy. Therefore understanding the centriolar satellite composition, dynamics and behavior are essential to elucidate their implications for health and diseases such as cancer and ciliopathies.  The main scaffolding protein of the centriolar satellites is Pericentriolar Material 1 (PCM1), which maintains the satellite integrity since depletion of PCM1 leads to loss of centriolar satellites. Therefore understanding the biochemical and biophysical properties of the PCM1 is an essential first step to uncover the biochemistry of centriolar satellites. I found that  PCM1 is a highly disordered protein, suggesting that it might undergo liquid-liquid phase separation (LLPS) to form centriolar satellite granules. Even though they are spherical and dynamic, which aligns with the membrane-less granule properties that go under the LLPS, mature satellite granules are in a gel-like state or become gelified over time. The global RNA depletion leads to the dispersal of the PCM1 granules showing that they have a stable RNA core that helps with the granule integrity. Structure-function analysis of PCM1 showed that three different fragments within the PCM1 N-terminus form granules, whereas the fragments from the C-terminal act cytoplasmic. This result shows that N-terminal has a role in the granule formation, whereas the C-terminal is more likely to have a regulatory role. The PCM1 fragments that form granules exhibit dynamic events including fusion, fission, kiss, and split events. Lastly, the de novo PCM1 disassembly experiment shows that with decreasing protein concentration, the granules are broken into smaller granules until no visible granule is seen. Our findings show that PCM1 goes under the LLPS and shows liquid-like behavior however there is an equilibrium between both the liquid and gel-like state.