Centrosomal Protein 55 Regulates Chromosomal Instability in Cancer Cells by Controlling Microtubule Dynamics
Abstract
:1. Introduction
2. Materials and Methods
2.1. Mammalian Cell Culture and Stable Lentiviral Knock-Down of CEP55
2.2. Stable Lentiviral Re-Expression of CEP55WT and CEP5559–428
2.3. Western Blotting
2.4. Real Time PCR
2.5. Immunofluorescence Staining
2.6. EB3 Tracking for Evaluation of MT Velocity
2.7. Cold-Induced Depolymerisation in Cellulo
2.8. Chromosome Isolation
2.9. Next-Generation Sequencing
2.10. Structure Prediction of CEP55
2.11. Recombinant Expression of His-GFP-CEP55 and CEP55 Mutants
2.12. MT-Binding
2.13. MT-Pulldown
2.14. MT-Polymerisation
2.15. Cold-Induced In Vitro MT-Depolymerisation
3. Results
3.1. Depletion of CEP55 Decreases CIN
3.2. CEP55 Depletion Reduces the Stability but Increases the Dynamics of MTs
3.3. Identification of CEP55 Peptides Required for MT-Binding
3.4. Re-Expression of a MT-Binding Deficient CEP55 Mutant Does Not Rescue Reduced CIN of CEP55 shRNA Cells
3.5. Impact of CEP55 on MT-Dynamics In Vitro
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Method | Effect of CEP55 | Extent |
---|---|---|
Cold-induced MT-depolymerization | protection | 80% |
MT-polymerization rate | slowly increasing slope | 4-fold |
conclusion | CEP55 attenuates MT depolymerization |
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Muhs, S.; Paraschiakos, T.; Schäfer, P.; Joosse, S.A.; Windhorst, S. Centrosomal Protein 55 Regulates Chromosomal Instability in Cancer Cells by Controlling Microtubule Dynamics. Cells 2024, 13, 1382. https://doi.org/10.3390/cells13161382
Muhs S, Paraschiakos T, Schäfer P, Joosse SA, Windhorst S. Centrosomal Protein 55 Regulates Chromosomal Instability in Cancer Cells by Controlling Microtubule Dynamics. Cells. 2024; 13(16):1382. https://doi.org/10.3390/cells13161382
Chicago/Turabian StyleMuhs, Stefanie, Themistoklis Paraschiakos, Paula Schäfer, Simon A. Joosse, and Sabine Windhorst. 2024. "Centrosomal Protein 55 Regulates Chromosomal Instability in Cancer Cells by Controlling Microtubule Dynamics" Cells 13, no. 16: 1382. https://doi.org/10.3390/cells13161382