Publications

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.

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.

Abstract

Recent demands for the production of biofuels from lignocellulose led to an increased interest in engineered cellulases from Trichoderma reesei or other fungal sources. While the methods to generate such mutant cellulases on DNA level are straightforward, there is often a bottleneck in their production since a correct posttranslational processing of these enzymes is needed to obtain highly active enzymes. Their production and subsequent enzymatic analysis in the homologous host T. reesei is, however, often disturbed by the concomitant production of other endogenous cellulases. As a useful alternative, we tested the production of cellulases in T. reesei in a genetic background where cellulase formation has been impaired by deletion of the major cellulase transcriptional activator gene xyr1. Three cellulase genes (cel7a, cel7b, and cel12a) were expressed under the promoter regions of the two highly expressed genes tef1 (encoding translation elongation factor 1-alpha) or cdna1 (encoding the hypothetical protein Trire2:110879). When cultivated on d-glucose as carbon source, the Δxyr1 strain secreted all three cellulases into the medium. Related to the introduced gene copy number, the cdna1 promoter appeared to be superior to the tef1 promoter. No signs of proteolysis were detected, and the individual cellulases could be assayed over a background essentially free of other cellulases. Hence this system can be used as a vehicle for rapid and high-throughput testing of cellulase muteins in a homologous background.