Displaying 1 - 2 of 2
-
Uzbas, F., & O’Neill, A. (2023). Spatial Centrosome Proteomic Profiling of Human iPSC-derived Neural Cells. BIO-PROTOCOL, 13(17): e4812. doi:10.21769/BioProtoc.4812.
Abstract
The centrosome governs many pan-cellular processes including cell division, migration, and cilium formation.
However, very little is known about its cell type-specific protein composition and the sub-organellar domains where
these protein interactions take place. Here, we outline a protocol for the spatial interrogation of the centrosome
proteome in human cells, such as those differentiated from induced pluripotent stem cells (iPSCs), through co-
immunoprecipitation of protein complexes around selected baits that are known to reside at different structural parts
of the centrosome, followed by mass spectrometry. The protocol describes expansion and differentiation of human
iPSCs to dorsal forebrain neural progenitors and cortical projection neurons, harvesting and lysis of cells for protein
isolation, co-immunoprecipitation with antibodies against selected bait proteins, preparation for mass spectrometry,
processing the mass spectrometry output files using MaxQuant software, and statistical analysis using Perseus
software to identify the enriched proteins by each bait. Given the large number of cells needed for the isolation of
centrosome proteins, this protocol can be scaled up or down by modifying the number of bait proteins and can also
be carried out in batches. It can potentially be adapted for other cell types, organelles, and species as well. -
O’Neill, A. C., Uzbas, F., Antognolli, G., Merino, F., Draganova, K., Jäck, A., Zhang, S., Pedini, G., Schessner, J. P., Cramer, K., Schepers, A., Metzger, F., Esgleas, M., Smialowski, P., Guerrini, R., Falk, S., Feederle, R., Freytag, S., Wang, Z., Bahlo, M. O’Neill, A. C., Uzbas, F., Antognolli, G., Merino, F., Draganova, K., Jäck, A., Zhang, S., Pedini, G., Schessner, J. P., Cramer, K., Schepers, A., Metzger, F., Esgleas, M., Smialowski, P., Guerrini, R., Falk, S., Feederle, R., Freytag, S., Wang, Z., Bahlo, M., Jungmann, R., Bagni, C., Borner, G. H. H., Robertson, S. P., Hauck, S. M., & Götz, M. (2022). Spatial centrosome proteome of human neural cells uncovers disease-relevant heterogeneity. Science, 376(6599): eabf9088. doi:10.1126/science.abf9088.
Abstract
The centrosome provides an intracellular anchor for the cytoskeleton, regulating cell division, cell migration, and cilia formation. We used spatial proteomics to elucidate protein interaction networks at the centrosome of human induced pluripotent stem cell–derived neural stem cells (NSCs) and neurons. Centrosome-associated proteins were largely cell type–specific, with protein hubs involved in RNA dynamics. Analysis of neurodevelopmental disease cohorts identified a significant overrepresentation of NSC centrosome proteins with variants in patients with periventricular heterotopia (PH). Expressing the PH-associated mutant pre-mRNA-processing factor 6 (PRPF6) reproduced the periventricular misplacement in the developing mouse brain, highlighting missplicing of transcripts of a microtubule-associated kinase with centrosomal location as essential for the phenotype. Collectively, cell type–specific centrosome interactomes explain how genetic variants in ubiquitous proteins may convey brain-specific phenotypes.
Share this page