Plk4 Regulates Centriole Asymmetry and Spindle Orientation in Neural Stem Cells

Defects in mitotic spindle orientation (MSO) disrupt the organization of stem cell niches impacting tissue morphogenesis and homeostasis. Mutations in centrosome genes reduce MSO fidelity, leading to tissue dysplasia and causing several diseases such as microcephaly, dwarfism, and cancer. Whether these mutations perturb spindle orientation solely by affecting astral microtubule nucleation or whether centrosome proteins have more direct functions in regulatingMSO is unknown. To investigate this question, we analyzed the consequences of deregulating Plk4 (the master centriole duplication kinase) activity in Drosophila asymmetrically dividing neural stem cells. We found that Plk4 functions upstream of MSO control, orchestrating centriole symmetry breaking and consequently centrosome positioning. Mechanistically, we show that Plk4 acts through Spd2 phosphorylation, which induces centriole release from the apical cortex. Overall, this work not only reveals a role for Plk4 in regulating centrosome function but also links the centrosome biogenesis machinery with the MSO apparatus.ERC starting grant CentroStemCancer [242598]; Institut Curie; CNRS; NCI [P30CA23074]; NIGMS [R01 GM110166, GM126035]; FRM; IC; FRM installation grant; ATIP grantOpen access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Explorer le rôle de Plk4 in vivo

Les centrosomes sont les principaux centres organisateurs des microtubules dans les cellules animales, impliqués dans la division, la motilité, la polarité cellulaire. Ils participent à l'élaboration du fuseau mitotique, qui permet la séparation des chromosomes dans les cellules filles. Dans les neuroblastes de drosophile en interphase, un des deux centrosomes maintient son activité et sa position apicale dans la cellule, alors que l'autre est inactivé et se déplace vers le pôle basal. La duplication des centrioles est initiée par la kinase Plk4 une seule fois par cycle cellulaire. Toute dérégulation des niveaux de Plk4 conduit à un défaut du nombre de centrosomes, à l'origine de pathologies comme le cancer et la microcéphalie. Pendant ma thèse, j'ai étudié les rôles et régulations de Plk4 in vivo dans les neuroblastes de drosophile. J'ai montré un nouveau rôle de Plk4 dans l'établissement de l'asymétrie des centrosomes durant l'interphase. Plk4 favorise un comportement basal des centrosomes en inhibant la nucléation des microtubules et l'ancrage au pôle apical. Plk4 régule négativement la localisation du co-activateur de l'APC/C, Fizzy-related, que j'ai identifié comme un régulateur positif de l'activation du centrosome. APC/C est une E3 ubiquitine-ligase, qui cible les protéines régulant le cycle cellulaire vers la dégradation. J'ai montré que Plk4 interagit avec ce complexe in vivo. Des mutations du motif de liaison à l'APC/C conduisent à la stabilisation de Plk4 et à une dérégulation de son accumulation au centrosome au début de l'interphase. Mon étude a donc démontré que dans les neuroblastes Plk4 coordonne la duplication des centrioles et le cycle des centrosomes.The centrosome is the main microtubule-organizing centre of animal cells with important roles in cell division, motility and polarity. In cycling cells, upon duplication, two centrosomes form the mitotic spindle, the apparatus that physically segregates the chromosomes into the daughter cells. In Drosophila neural stem cells of the larval brain, called neuroblasts, during interphase, one centrosome stays active and static at the apical side of the cell, while the other one is inactive and moves toward the basal side of the cell. Centriole duplication, which occurs only once per cell cycle, is initiated by the Polo-like kinase 4 (Plk4). Deregulation of Plk4 levels leads to alteration in centrosome number, a defect that can cause diseases such as cancer and microcephaly. During my PhD I studied the role/s and regulation of Plk4 in vivo in Drosophila neuroblasts. I found that Plk4 plays an important role in establishing centrosome asymmetry during interphase. Plk4 promotes centrosome basal-like behaviour, through inhibition of MT nucleation and centrosome apical anchorage. Plk4 negatively regulates the centrosomal localization of the APC/C co-activator Fizzy-related (Fzr) that I identified as a positive regulator of centrosome activation. The APC/C complex is an E3 ubiquitin-ligase that targets cell-cycle-related proteins to degradation. I showed that APC/C and Plk4 interact in vivo. Mutations in the APC/C binding motif lead to stabilization of Plk4 that presents unscheduled accumulation at the centrosome in early interphase neuroblasts.In conclusion, my study demonstrates that in neuroblasts, the kinase Plk4 couples centriole duplication and centrosome cycles

Isolation and Fluorescence Imaging for Single-particle Reconstruction of <em>Chlamydomonas</em> Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies

Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

Previous attempts to combine expansion microscopy (ExM) and single molecule localisation microscopy (SMLM) have proved challenging. Here the authors show that post-labelling Ex-SMLM improves labelling efficiency, reduces linkage error, and preserves ultrastructural details

Imaging cellular ultrastructures using expansion microscopy (U-ExM)

International audienc

A helical inner scaffold provides a structural basis for centriole cohesion

International audienceThe ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo-electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry

A helical inner scaffold provides a structural basis for centriole cohesion

The ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo–electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry

Imaging cellular ultrastructures using expansion microscopy (U-ExM)

Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy