Dit proefschrift bevat onderzoek over verschillende processen binnen het intracellulaire domein. Als eerst wordt er onderzocht hoe een cel zijn oplosbare eiwitten in de correcte compartimenten... Show moreDit proefschrift bevat onderzoek over verschillende processen binnen het intracellulaire domein. Als eerst wordt er onderzocht hoe een cel zijn oplosbare eiwitten in de correcte compartimenten krijgt na mitosis. Het tweede omschrijft een mechanisme dat verantwoordelijk is voor het structureren van het endoplasmatisch reticulum. Als derde werd onderzocht wat voorn rol het eiwit MOSPD2 vervult binnen contacten tussen het ER, Golgi apparatus en lysosomen. Show less
Koedoot, E.; Steijn, E. van; Vermeer, M.; González-Prieto, R.; Vertegaal , A.C.O.; Martens, J.W.M.; ... ; Water, B. van de 2021
BackgroundTriple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic opportunities. Recently, splicing factors have gained attention as potential... Show moreBackgroundTriple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic opportunities. Recently, splicing factors have gained attention as potential targets for cancer treatment. Here we systematically evaluated the role of RNA splicing factors in TNBC cell proliferation.MethodsIn this study, we performed an RNAi screen targeting 244 individual splicing factors to systematically evaluate their role in TNBC cell proliferation. For top candidates, mechanistic insight was gained using amongst others western blot, PCR, FACS, molecular imaging and cloning. Pulldown followed by mass spectrometry were used to determine protein-protein interactions and patient-derived RNA sequencing data was used relate splicing factor expression levels to proliferation markers.ResultsWe identified nine splicing factors, including SNRPD2, SNRPD3 and NHP2L1, of which depletion inhibited proliferation in two TNBC cell lines by deregulation of sister chromatid cohesion (SCC) via increased sororin intron 1 retention and down-regulation of SMC1, MAU2 and ESPL1. Protein-protein interaction analysis of SNRPD2, SNRPD3 and NHP2L1 identified that seven out of the nine identified splicing factors belong to the same spliceosome complex including novel component SUN2 that was also critical for efficient sororin splicing. Finally, sororin transcript levels are highly correlated to various proliferation markers in BC patients.ConclusionWe systematically determined splicing factors that control proliferation of breast cancer cells through a mechanism that involves effective sororin splicing and thereby appropriate sister chromatid cohesion. Moreover, we identified SUN2 as an important new spliceosome complex interacting protein that is critical in this process. We anticipate that deregulating sororin levels through targeting of the relevant splicing factors might be a potential strategy to treat TNBC. Show less
Koedoot, E.; Steijn, E. van; Vermeer, M.; Gonzalez Prieto, R.; Vertegaal, A.C.O.; Martens, J.W.M.; ... ; Water, B. van de 2021
BackgroundTriple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic opportunities. Recently, splicing factors have gained attention as potential... Show moreBackgroundTriple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic opportunities. Recently, splicing factors have gained attention as potential targets for cancer treatment. Here we systematically evaluated the role of RNA splicing factors in TNBC cell proliferation.MethodsIn this study, we performed an RNAi screen targeting 244 individual splicing factors to systematically evaluate their role in TNBC cell proliferation. For top candidates, mechanistic insight was gained using amongst others western blot, PCR, FACS, molecular imaging and cloning. Pulldown followed by mass spectrometry were used to determine protein-protein interactions and patient-derived RNA sequencing data was used relate splicing factor expression levels to proliferation markers.ResultsWe identified nine splicing factors, including SNRPD2, SNRPD3 and NHP2L1, of which depletion inhibited proliferation in two TNBC cell lines by deregulation of sister chromatid cohesion (SCC) via increased sororin intron 1 retention and down-regulation of SMC1, MAU2 and ESPL1. Protein-protein interaction analysis of SNRPD2, SNRPD3 and NHP2L1 identified that seven out of the nine identified splicing factors belong to the same spliceosome complex including novel component SUN2 that was also critical for efficient sororin splicing. Finally, sororin transcript levels are highly correlated to various proliferation markers in BC patients.ConclusionWe systematically determined splicing factors that control proliferation of breast cancer cells through a mechanism that involves effective sororin splicing and thereby appropriate sister chromatid cohesion. Moreover, we identified SUN2 as an important new spliceosome complex interacting protein that is critical in this process. We anticipate that deregulating sororin levels through targeting of the relevant splicing factors might be a potential strategy to treat TNBC. Show less
Cell division ends when two daughter cells physically separate via abscission, the cleavage of the intercellular bridge. It is not clear how the anti-parallel microtubule bundles bridging daughter... Show moreCell division ends when two daughter cells physically separate via abscission, the cleavage of the intercellular bridge. It is not clear how the anti-parallel microtubule bundles bridging daughter cells are severed. Here, we present a novel abscission mechanism. We identified chromokinesin KIF4A, which is adjacent to the midbody during cytokinesis, as being required for efficient abscission. KIF4A is regulated by post-translational modifications. We evaluated modification of KIF4A by the ubiquitin-like protein SUMO. We mapped lysine 460 in KIF4A as the SUMO acceptor site and employed CRISPR-Cas9-mediated genome editing to block SUMO conjugation of endogenous KIF4A. Failure to SUMOylate this site in KIF4A delayed cytokinesis. SUMOylation of KIF4A enhanced the affinity for the microtubule destabilizer stathmin 1 (STMN1). We here present a new level of abscission regulation through the dynamic interactions between KIF4A and STMN1 as controlled by SUMO modification of KIF4A. Show less
Spits, M.; Janssen, L.J.; Voortman, L.M.; Kooij, R.; Neefjes, A.C.M.; Ovaa, H.; Neefjes, J. 2019
The human genome comprises of more than 22.000 genes. These genes encode for proteins playing a role in many general or tissue-specific biological processes, but for many of them the function is... Show moreThe human genome comprises of more than 22.000 genes. These genes encode for proteins playing a role in many general or tissue-specific biological processes, but for many of them the function is not yet unraveled yet. A way to study all the different genes at once in a certain biological process is by genome-wide screening. By considering all genes in a pathway, new players can be identified that would otherwise not have been linked to the studied process. In this thesis, I describe a siRNA-based genome-wide screen used to identify new proteins involved in MHC class II antigen presentation, expression and transport. The data obtained from this screen not only led to the identification of proteins involved in the MHC class II pathway specifically, but we also identified a regulator of a more general process namely intracellular endosome localization. This shows the importance of genome-wide screening in the identification of new players and regulators of certain biological pathways which may be considered as new drug targets to cure diseases. Show less
