Centrosome Regulation in Development and Dysregulation in Disease

National Heart, Lung, And Blood Institute

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Abstract

Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering mechanisms driving animal development, physiology and disease. Elucidating these mechanisms has been enhanced through the study of model organisms and the use of sophisticated genetic, biochemical and imaging tools. In the past fiscal year, we have continued our longtime work aimed at understanding the underlying causes of Microcephaly a developmental disorder where animals fail to acheive proper brain size and neuron number. Mutations in diverse genes are linked to microcephaly, including several with DNA damage repair (DDR) functions; however, it is not well understood how these DDR gene mutations limit brain size. We have focused our work on the protein TRAIP, which has multiple functions in DDR. We characterized the Drosophila TRAIP homolog nopo, hereafter traip, and found that traip mutants (traip-) have a brain-specific defect in the mushroom body (MB). traip- MBs were smaller and contained fewer neurons, but no neurodegeneration, consistent with human primary microcephaly. Reduced neuron numbers in traip- were explained by premature loss of MB neuroblasts (MB-NBs), in part via caspase-dependent cell death. Many traip- MB-NBs had prominent chromosome bridges in anaphase, along with polyploidy, aneuploidy or micronuclei. Traip localization during mitosis is sufficient for MB development, suggesting that Traip can repair chromosome bridges during mitosis if necessary. Our results suggest that proper brain size is ensured by the recently described role for TRAIP in unloading stalled replication forks in mitosis, which suppresses DNA bridges and premature neural stem cell loss to promote proper neuron number. We also continue to investigate the role of centrosomes in proper neural stem cell division and preventing microcephaly. Toward this end, we have explored the mechanism by which centrosomes are positioned in neural stem cells to ensure proper stem cell number. While we know centrosome positioning is essential for their function, not much is known about the mechanism of transport. Typically, centrosomes are transported to various cellular locations through the interaction of centrosomal microtubules (MTs) with motor proteins anchored at the cortex or the nuclear surface. However, it remains unknown how centrioles migrate in cellular contexts in which they do not nucleate MTs. Here, we demonstrate that during interphase, inactive centrioles move directly along the interphase MT network as Kinesin-1 cargo. We identify Pericentrin-Like-Protein (PLP) as a novel Kinesin-1 interacting molecule essential for centriole motility. In vitro assays show that PLP directly interacts with the cargo binding domain of Kinesin-1, allowing PLP to migrate on MTs. Binding assays using purified proteins revealed that relief of Kinesin-1 autoinhibition is critical for its interaction with PLP. Finally, our studies of neural stem cell asymmetric divisions in the Drosophila brain show that the PLP-Kinesin-1 interaction is essential for the timely separation of centrioles, the asymmetry of centrosome activity, and the age-dependent centrosome inheritance.