{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/386987"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/386987","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Analysis of the functions of phosphoprotein phosphatase 1 catalytic subunits during cell division","abstract":"Mitotic cell division allows for the generation of two daughter cells from a mother cell. This requires the segregation of the duplicated genomes and the physical division of the cytoplasm to generate two separate daughters. Failure of any part of this process can have drastic effects and ultimately defects in cell division have been implicated in many diseases, such as cancer. Phosphorylation is extensively used during cell division to regulate the activity, function, and localisation of mitotic proteins. One of the most abundant families of phosphatases in the eukaryotic cell is the phosphoprotein phosphatase 1 (PP1) family of phosphatases. These phosphatases are holoenzymes composed of one of three catalytic subunits -α, β, and γ - in complex with one or two regulatory subunits. There is some evidence indicating that the three PP1 catalytic subunits might have distinct roles during cell division, but our knowledge of PP1-mediated dephosphorylation and the identity of their substrates is limited. To address this, I employed genome editing, targeted proteolysis, and quantitative phosphoproteomics to characterise the roles of the different PP1 catalytic subunits during cell division and to identify their potential substrates. I initially verified a method to synchronise large populations of the popular human telomerase reverse transcriptase-retinal pigment epitelium-1 (hTERT-RPE-1) cell line in mitosis. I investigated the validity of using the auxin response factor (ARF)-auxin inducible degron (AiD) system for targeted proteolysis in hTERT-RPE-1 and found that this system resulted in leaky degradation of the target protein. I next tested the AiD2 system to study PP1 phosphatases and ultimately generated human colon tumour 116 (HCT116) cell lines containing endogenously tagged PP1β and PP1γ and supervised the generation of a PP1α cell line. I found that degradation of PP1β in a late stage of cell division seems to be affecting processes required for the central spindle, whereas it contributes less to progressing cells through cell division in an earlier stage. Time-lapse imaging also revealed a few cells with defects in DNA segregation or central spindle formation when PP1β was degraded. Degradation of PP1α during both an early and late stage of division on the other hand did not seem to largely affect many processes required for progression through cell division. Additionally, I investigated the spatial regulation of PP1β during the late stages of cell division using an inducible system and found that ectopic expression of PP1β at the central spindle did not impact the levels of a common readout of cytokinesis failure. Overall, this work adds to our understanding of the functions that the different PP1 catalytic subunits may contribute to during cell division.","abstract_html":"Mitotic cell division allows for the generation of two daughter cells from a mother cell. This requires the segregation of the duplicated genomes and the physical division of the cytoplasm to generate two separate daughters. Failure of any part of this process can have drastic effects and ultimately defects in cell division have been implicated in many diseases, such as cancer. Phosphorylation is extensively used during cell division to regulate the activity, function, and localisation of mitotic proteins. One of the most abundant families of phosphatases in the eukaryotic cell is the phosphoprotein phosphatase 1 (PP1) family of phosphatases. These phosphatases are holoenzymes composed of one of three catalytic subunits -α, β, and γ - in complex with one or two regulatory subunits. There is some evidence indicating that the three PP1 catalytic subunits might have distinct roles during cell division, but our knowledge of PP1-mediated dephosphorylation and the identity of their substrates is limited. To address this, I employed genome editing, targeted proteolysis, and quantitative phosphoproteomics to characterise the roles of the different PP1 catalytic subunits during cell division and to identify their potential substrates. I initially verified a method to synchronise large populations of the popular human telomerase reverse transcriptase-retinal pigment epitelium-1 (hTERT-RPE-1) cell line in mitosis. I investigated the validity of using the auxin response factor (ARF)-auxin inducible degron (AiD) system for targeted proteolysis in hTERT-RPE-1 and found that this system resulted in leaky degradation of the target protein. I next tested the AiD2 system to study PP1 phosphatases and ultimately generated human colon tumour 116 (HCT116) cell lines containing endogenously tagged PP1β and PP1γ and supervised the generation of a PP1α cell line. I found that degradation of PP1β in a late stage of cell division seems to be affecting processes required for the central spindle, whereas it contributes less to progressing cells through cell division in an earlier stage. Time-lapse imaging also revealed a few cells with defects in DNA segregation or central spindle formation when PP1β was degraded. Degradation of PP1α during both an early and late stage of division on the other hand did not seem to largely affect many processes required for progression through cell division. Additionally, I investigated the spatial regulation of PP1β during the late stages of cell division using an inducible system and found that ectopic expression of PP1β at the central spindle did not impact the levels of a common readout of cytokinesis failure. 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