Abstract
During cell division, cells must rearrange and separate their membrane-bound organelles as well as their plasma membrane to create equivalent daughter cells. The last stage of this process is cytokinesis, which is the physical separation of the cytoplasm into two cells. Cytokinesis involves the interplay of many proteins, including components of the ESCRT-III machinery. The ESCRT-III machinery is implicated in the final membrane abscission and is also involved in other cellular functions such as sealing of the nuclear membrane, cargo sorting on endosomes, and HIV-1 budding. Although in recent years some light has been shed on the functions and mechanisms of recruitment of this machinery, current knowledge is still limited. Here we used a small molecule, Prazosin, to investigate ESCRT-III in cytokinesis. Another aspect that is yet not clear in cell division is related to the roles played by lipids. Cells actively maintain complex and diverse lipidomes that encompass many thousands of lipids. Our lab has shown that the lipid composition of dividing cells is altered relative to non-dividing cells. However, the roles of these lipids remain largely unexplored. We developed a technique to systematically explore protein-lipid interactions in cytokinesis to address this outstanding question.Prazosin is a cytokinesis inhibitor that inhibits endosomal sorting and recycling. Recycling and sorting endosomes may be involved in delivering proteins to the midbody during cytokinesis. The mechanism by which this drug inhibits cytokinesis is not well understood. We found using fixed and live imaging that Prazosin treatment results in the aggregation of ESCRT-III components around the nuclear envelope and the intercellular bridge. This phenotype was rescued by the addition of Bafilomycin, a V-ATPase inhibitor, suggesting a possible connection with the endolysosomal system. We concluded that Prazosin may be a valid tool to manipulate the ESCRT-III machinery and to clarify its role during cell division.
Cells produce tens of thousands of different lipids, their order of magnitude is similar to that of proteins. However, their functions are still not well understood in cell division. It is our hypothesis that specific interactions between lipids and proteins might contribute to protein functions during cytokinesis. The first step to test this hypothesis is to determine if specific lipids are bound to cytokinetic proteins. We developed a method to explore protein-lipid interactions in cells. This involves detergent-free lysis to obtain small membrane fragments from cells expressing the protein of interest bound to GFP, followed by GFP trap pulldowns and identification of bound lipids by liquid chromatography-mass spectrometry (LC-MS). Many cytokinetic proteins are associated with the plasma membrane, including RACGAP1 and the ESCRT-III protein CHMP4B. We have identified which lipids specifically associate with these proteins. We then moved to investigate the functional consequences of these interactions and observed that some of the lipid species identified from CHMP4B were essential for its midbody localization. In conclusion, we developed a new way to study protein-lipid interactions in cytokinesis and this allowed us to shed light on a previously unknown aspect of cell division.
| Date of Award | 1 Jun 2022 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Ulrike Eggert (Supervisor) & Jeremy Carlton (Supervisor) |