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Behavior and regulation of centriolar appendage proteins during mitosis

Viol, Linda-Isabella

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Abstract

Summary The centrosome is a small non-membranous organelle composed of two centrioles and surrounded by the pericentriolar material. The two primary functions of the centrosome are first, to act as the main microtubule-organizing center in interphase and mitosis and second, to generate the primary cilium. The primary cilium is a microtubule-based structure that projects from the plasma membrane, where it acts as a signaling hub to transfer extracellular signals into intracellular responses. Thereby, the cilium coordinates diverse signaling pathways implicated in development, tissue homeostasis and disease. The primary cilium originates from the older centriole of the pair, called the mother centriole. This mother centriole is decorated at its distal tip with a nine-fold symmetric ring of distal and subdistal appendage proteins. Both centrioles duplicate once per cell cycle to generate one copy of themselves and hence one of the centriole contains the oldest appendages. Studies in model organisms proposed that those inherently asymmetric centrosomes potentially work as a scaffold for asymmetric distribution of cell fate determinants during mitosis and thereby acting as an intrinsic cue for asymmetric cell division. Yet, how centrosome asymmetry is established and how, if at all, influences asymmetric cell division in human stem cells remains unclear. Thus, one aim of this study was to characterize centrosome asymmetry in somatic and stem cells. I observed centrosomal asymmetry during mitosis for a subset of appendages, while others dispersed from the centrosome upon the G2/M transition. My data show that an appendage core (composed of ODF2, Cep83 and SCLT1) remained at the mother centriole from interphase to mitosis, whereas a sub-set of appendages including Ninein, Centriolin, Cep123, Cep164 and LRRC45 detached from the mother centriole during mitosis. The behavior of appendages was similar in differentiated cells and human stem cells. The second aim was to unravel whether centrosome asymmetry regulates asymmetric stem cell division. I found that ODF2 can be used as a marker for centrosome asymmetry during mitosis in human hematopoietic stem and progenitor cells (HSPCs). Here, I correlated the position of the daughter and mother centrosome, visualized by ODF2, with the asymmetric distribution of the stem cell marker CD133 in dividing HSPCs using imaging flow cytometry. Although a role for centrosomes in the asymmetric cell division of HSPCs cannot fully be excluded, I could not observe a clear correlation between centrosome age and CD133 segregation. Further, reversion of centrosome asymmetry by ODF2 depletion had no impact on the differentiation potential of HSPCs. The third aim concentrated on the regulation of appendage behavior. Distal appendages, which are required for initial steps of ciliogenesis, are released from centrosomes before mitosis by a mechanism that is currently unknown. Therefore, I aimed to elucidate the regulation of the cell cycle-dependent behavior of appendages and the consequence of perturbed appendages regulation with special regards to their function in ciliogenesis. Here, I show that the mitotic kinase Nek2 regulates the distal appendage removal at the mother centriole at the onset of mitosis. Ectopic overexpression of Nek2 but not kinase-dead Nek2 prematurely displaced those appendages in interphase, indicating a kinase-specific function. This phenotype was recapitulated in breast cancer cells with high levels of Nek2. Conversely, in Nek2 knockout (KO) cells, appendages remained associated with the older centrosome during mitosis. I could show that persistence of distal appendages on the mother centrosome in mitotic Nek2 KO cells did not allow the cells to fully disassemble their cilia before mitosis, resulting in a ciliary remnant during mitosis. This triggered asymmetric inheritance of ciliary signaling components and asynchronous cilium reassembly after cell division. Asynchronous cilium growth may have consequences for cell fate determination by allowing sister cells to differentially detect environmental signals. Therefore, a ciliary remnant during mitosis might be restricted to asymmetrically dividing stem cells, which need asymmetric cilium re-assembly as a tool for differential responding to environmental signals after cell division. Together, my data established the kinase Nek2 as a central regulator of distal appendages.

Item Type: Dissertation
Supervisor: Pereira, Prof. Dr. Gislene
Place of Publication: Heidelberg
Date of thesis defense: 13 September 2019
Date Deposited: 14 Oct 2019 10:02
Date: 2020
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
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