Orbit/CLASP determines centriole length by antagonising Klp10A in <i>Drosophila</i> spermatocytes

Shoda, Tsuyoshi; Yamazoe, Kanta; Tanaka, Yuri; Asano, Yuki; Inoué, Yoshihiro

doi:10.1242/jcs.251231

Cited by 8 publications

(7 citation statements)

References 54 publications

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“…Negative regulators of cilium assembly, in particular, the CP110‐CEP97 protein complex, need to be removed from the mother centriole to initiate ciliogenesis (Spektor et al , 2007). CP110 localizes to the distal end of centrioles (Chen et al , 2002), and regulates centriole length by preventing centriolar microtubule extension (Kleylein‐Sohn et al , 2007; Schmidt et al , 2009; Shoda et al , 2021). However, CP110 does not regulate cilium length, indicating that cilium elongation is a further step (Spektor et al , 2007).…”

Section: Introductionmentioning

confidence: 99%

ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis

et al. 2022

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Despite the importance of cilia in cell signaling and motility, the molecular mechanisms regulating cilium formation remain incompletely understood. Herein, we characterize enkurin domaincontaining protein 1 (ENKD1) as a novel centrosomal protein that mediates the removal of centriolar coiled-coil protein 110 (CP110) from the mother centriole to promote ciliogenesis. We show that Enkd1 knockout mice possess ciliogenesis defects in multiple organs. Super-resolution microscopy reveals that ENKD1 is a stable component of the centrosome throughout the ciliogenesis process. Simultaneous knockdown of ENKD1 and CP110 significantly reverses the ciliogenesis defects induced by ENKD1 depletion. Protein interaction analysis shows that ENKD1 competes with centrosomal protein 97 (CEP97) in binding to CP110. Depletion of ENKD1 enhances the CP110-CEP97 interaction and detains CP110 at the mother centriole. These findings thus identify ENKD1 as a centrosomal protein and uncover a novel mechanism controlling CP110 removal from the mother centriole for the initiation of ciliogenesis.

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Section: Introductionmentioning

confidence: 99%

ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis

et al. 2022

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“…In different types of Drosophila cells and tissues, dependent on the cellular context, both elongation and shrinkage of centrioles were reported upon the loss of CP110 and CEP97 8, 17-20 . The emerging picture from these studies is that CP110 and CEP97 can counteract changes in 4 centriole length imposed by well-studied positive or negative regulators of centriolar MT growth, such as CLASP or kinesin-13, respectively 9,19,20 . CP110 and CEP97 are also required for early stages of cilia formation 18,21,22 , but the cap structure that these proteins form needs to be removed from the basal body to allow the formation of axonemal MTs 15,[23][24][25] .…”

Section: Introductionmentioning

confidence: 99%

Microtubule plus-end regulation by centriolar cap proteins

Ogunmolu

Moradi

Volkov

et al. 2021

Preprint

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Centrioles are microtubule-based organelles required for the formation of centrosomes and cilia. Centriolar microtubules, unlike their cytosolic counterparts, grow very slowly and are very stable. The complex of centriolar proteins CP110 and CEP97 forms a cap that stabilizes the distal centriole end and prevents its over-elongation. Here, we used in vitro reconstitution assays to show that whereas CEP97 does not interact with microtubules directly, CP110 specifically binds microtubule plus ends, potently blocks their growth and induces microtubule pausing. Cryo-electron tomography indicated that CP110 binds to the luminal side of microtubule plus ends and reduces protofilament peeling. Furthermore, CP110 directly interacts with another centriole biogenesis factor, CPAP/SAS-4, which tracks growing microtubule plus ends, slows down their growth and prevents catastrophes. CP110 and CPAP synergize in inhibiting plus-end growth, and this synergy depends on their direct binding. Together, our data reveal a molecular mechanism controlling centriolar microtubule plus-end dynamics and centriole biogenesis.

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“…Premeiotic spermatocytes with overexpressed Orbit and depleted Klp10A frequently show loss of centriole 1 integrity. Orbit and CP110 are closely associated with each other at the end of the centrioles in earlier-stage spermatocytes (Shoda et al, 2021).…”

mentioning

confidence: 95%

“…In mammalian cells, CP110-CEP97 cap inhibits elongation of centriolar microtubules induced by CPAP . In Drosophila, loss of CP110 or CEP97 can have different effects in different cell types, causing formation of either longer or shorter centrioles Delgehyr et al, 2012;Shoda et al, 2021). This can be explained by antagonistic relationships with either positive regulators of microtubule polymerization, such as the catastrophe-suppressing and rescue-promoting factor CLASP/Orbit (Shoda et al, 2021), or with negative regulators, such as a microtubule depolymerase of the kinesin-13 family, Klp10A (Delgehyr et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

“…In Drosophila, loss of CP110 or CEP97 can have different effects in different cell types, causing formation of either longer or shorter centrioles Delgehyr et al, 2012;Shoda et al, 2021). This can be explained by antagonistic relationships with either positive regulators of microtubule polymerization, such as the catastrophe-suppressing and rescue-promoting factor CLASP/Orbit (Shoda et al, 2021), or with negative regulators, such as a microtubule depolymerase of the kinesin-13 family, Klp10A (Delgehyr et al, 2012). When centriolar microtubules start to elongate to form the ciliary axoneme, the CP110-CEP97 cap is removed , indicating that it forms an obstacle for microtubule growth.…”

Section: Introductionmentioning

confidence: 99%

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Centrosome Formation and Function: From self-assembly of pericentriolar material to microtubule organization

Chen¹

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The centrosome is the main microtubule organizing center (MTOC) in animal cells. It consists of a pair of centrioles surrounded by pericentriolar material (PCM) that nucleates and anchors microtubules. The binding of PCM to centrioles, PCM self-assembly and PCM transport by the microtubule-based motor dynein are the prerequisites for centrosome assembly, but the mechanisms of PCM self-assembly in interphase cells are poorly understood. Moreover, the relative importance of different molecular pathways of PCM assembly varies between different cell systems and different phases of the cell cycle. Previous studies have mostly focused on mitotic cells, but systematic investigations have not been performed in interphase cells. In addition, γ-tubulin ring complex (γ-TuRC) as template for microtubule assembly largely determines the function of MTOC, but the relative importance of different centrosomal components in microtubule nucleation and maintenance of microtubule networks has not been systematically investigated. Moreover, it is unclear whether γ-TuRC can nucleate microtubules and maintain microtubule arrays in mammalian cells in the absence of γ-TuRC adaptors. In this thesis, we address these questions. In addition to the PCM, the other major component of the centrosome are centrioles, microtubule-based organelles whose formation and elongation are regulated by a large number of specific proteins. In vitro reconstitution experiments showed that two centriole biogenesis factors, CPAP and CP110, cooperatively regulate microtubule plus-end growth in vitro, but the importance of their interaction for centriole formation has not been examined on cells. In this thesis, we investigated the role of the CPAP-CP110 interaction in regulating centriole elongation. In Chapter 2, we reported that when centrioles are lost due to either depletion or inhibition of PLK4, self-clustering of PCM proteins is sufficient to form a compact acentriolar MTOC (caMTOC) and thus organize a dense radial microtubule array in interphase cells. We showed that such self-clustering of PCM is dynein-dependent and requires pericentrin, CDK5RAP2, ninein and γ-tubulin, but not CEP192, NEDD1 and CEP152. We found that the formation of caMTOC is sensitive to the non-centrosomal MTOC pathways such as AKAP450-dependent PCM recruitment to the Golgi apparatus and CAMSAP2-mediated stabilization of free microtubule minus ends. We also reproduced the findings of our cellular experiments using computer simulations. In Chapter 3, we describe tools for comprehensive and systematic functional analysis of the complex regulatory pathways of microtubule nucleation and anchoring at mammalian centrosomes using a cellular model that simultaneously lacks multiple PCM components. We found that in cells lacking the non-centrosomal microtubule minus-end stabilization pathways, different γ-TuRC adaptors exert additive effects on microtubule nucleation and attachment at the centrosome, and knocking them out in interphase cells prevents γ-TuRC from generating a dense microtubule array, even though all positive regulators of microtubule dynamics, are still present. In Chapter 4, we examined the importance of CPAP-CP110 interaction in cells by replacing wild-type CPAP with CPAP mutants that cannot bind CP110. Our data indicate that centrioles, albeit shorter ones, could still form in the presence of the CPAP mutant, demonstrating that the CPAP-CP110 interaction is not essential for centriole formation, but promotes centriole elongation.

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Orbit/CLASP determines centriole length by antagonising Klp10A in Drosophila spermatocytes

Cited by 8 publications

References 54 publications

ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis

ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis

Microtubule plus-end regulation by centriolar cap proteins

Centrosome Formation and Function: From self-assembly of pericentriolar material to microtubule organization

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