When cells extend, move, or divide, a tiny organelle called the centrosome provides a cytoskeletal anchor and regulatory hub. It was long held that centrosomes are similar in composition across cells. But a spatial proteomics study on centrosomes in human neural stem cells and neurons reveals their composition differs between cell types and phases. The findings provide a rich resource for probing cell type and stage-specific etiologies of neurodevelopmental diseases, such as epilepsy and periventricular heterotopia (PH).
The study, conducted in the laboratory of Magdalena Götz, PhD, professor and chair of physiological genomics at the Ludwig-Maximilians-University in Munich, and director of the Institute of Stem Cell Research at Helmholtz Munich, was published in the journal Science, in an article titled, “Spatial centrosome proteome of human neural cells uncovers disease-relevant heterogeneity.”
“The location of a protein is crucial for a disease. With our centrosome analysis, we now have an important resource to test further associations with neuronal diseases. Our research can explain for the first time why a protein that is present in all cells, causes a phenotype only in the brain and not in other organs when mutated. This will allow further insights into disease mechanisms—and thus get one step closer to their treatment,” said Götz.
Victor Borrell, PhD, a scientist at the Institute for Neurosciences, Alicante, Spain, who was not involved in this study, said, “This study is a real tour de force that begins to uncover an outstanding question in biology in general, and in developmental neuroscience in particular, which remarkably has received little attention so far: how genetic mutations that affect a protein expressed in many or all tissues and cells, only cause defects in a very particular set of cells or developmental events? They find the answer in the centrosome.”
In an earlier study, Götz and her team had discovered a centrosome protein called Akna that was present in neural stem cells that differentiate but not in those that self-renew. This led Götz to wonder if Akna was an exception or the tip of an iceberg of as-yet-unknown proteins that localize to the centrosome in a cell-type and cell-stage specific manner.
“There is so much we don’t yet know about these cells, including how the centrosomes of neurons compare to those of neural stem cells and other cell types,” said Götz.
Conventionally, centrosomes have been purified using centrifugation but this does not isolate the organelle precisely. Fusing detectable tags to proteins in the centrosome has revealed specific protein interactions, but it is hard to scale up and does not uncover spatial information.
In the current study, the researchers used a spatial proteomics approach that enables the identification of proteins at the centrosome and their precise location. “This is done by choosing known proteins that are located at different positions at the centrosome and then determining the interactome of each of these bait proteins by immunoprecipitation followed by mass-spec analysis,” said Götz.
In collaboration with Stefanie Hauck, PhD, head of the Helmholtz Munich Proteomic Core Facility, Götz’s team found that the composition of proteins in centrosomes differs depending on the cell type. Using ten known centrosome proteins as baits, Götz’s team immunoprecipitated interacting proteins from neural stem cells derived from human induced pluripotent stem cells (iPSCs) and neurons and identified centrosome-interacting proteins using mass spectrometry. Nearly 60% of the proteins they detected had not been seen in centrosomes in other cell types, and over 50% of the proteins switched interactions and locations within the centrosome upon neuronal differentiation.
“We were surprised not only by the unexpected high degree of heterogeneity of the centrosomes, but also by the discovery of many unexpected proteins associated with them—for example, RNA-binding proteins and even proteins responsible for splicing (the processing of RNA), which normally takes place in the nucleus,” said Götz.
The researchers then overlapped the centrosome proteomes that they detected in neural stem cells and neurons with genetic variations found in patients with neurodevelopmental diseases. They found a significant overlap of gene variants found in epilepsy patients with centrosome proteomes in neurons, as well as an overlap of gene variants found in patients with PH with centrosome proteomes in neural stem cells. In PH, neurons do not migrate to their correct destinations in the fetal brain.
“This study demonstrates that the centrosome is a protein hub in cells, and that neural stem cells and neurons have a different protein composition at the centrosome. This is a completely novel mechanism of protein function selectivity, defined by the recruitment of ubiquitous proteins to the centrosome,” said Borrell. “When disease-causing mutations occur, depending on whether the mutant protein is recruited to the centrosome, they affect one cell type or another, and hence have very specific and different consequences.”
Exploring a specific PH candidate variant called PRPF6, which is a ubiquitously expressed splicing protein, the scientists found it is enriched at the centrosome in neural stem cells but not in neurons. When the researchers expressed the mutant PRPF6 in the brains of mice, they found it recapitulated the neuronal migratory defect found in PH. This highlighted the role of the PRPF6 mutant in the disease phenotype.
“That centrosomes differ hugely between cells, is of great importance in development and disease. Specific proteins are required at the centrosome for differentiating cells to leave the stem cell niche. The localization of a protein at the centrosome is key for its function and, when mutated, for its dysfunction,” said Götz. “The ubiquitously expressed splicing factor PRPF6 when mutated causes only a brain phenotype. Our work suggests that this is due to its localization at the centrosome as PRPF6 brings its splicing target RNAs to the centrosome for local translation.”
“These findings open our eyes to an entirely new way of looking at cell biology and genetic disease, taking us from the simplistic view of one gene, one function in one cell type, to one gene, many potential functions in any cell type, depending on a perfectly defined matching between otherwise ubiquitous proteins and organelles. It’ll now be exciting to understand how this cell type-specific matching between proteins and centrosome occurs, and whether similar scenarios take place for other organelles and widely expressed proteins,” Borrell added.
In future studies, Götz’s team intends to explore the mechanistic role of the novel disease candidates that they have uncovered in this study. They will also be using spatial proteomics approaches to understand the role of centrosomes in cell-type specific migration and cancer.