Counterregulation of cAMP-directed kinase activities controls ciliogenesis

Porpora, Monia; Sauchella, Simona; Rinaldi, Laura; Donne, Rossella Delle; Sepe, Maria; Torres‐Quesada, Omar; Intartaglia, Daniela; Garbi, Corrado; Insabato, Luigi; Santoriello, Margherita; Bachmann, Verena; Synofzik, Matthis; Lindner, Herbert; Conte, Iván; Stefan, Eduard; Feliciello, Antonio

doi:10.1038/s41467-018-03643-9

Cited by 42 publications

(57 citation statements)

References 69 publications

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“…Close-up views and copy number changes for significant paclitaxel loci are shown in Figure 7 . Consistent with the activity of paclitaxel as an antitubulin drug, there were two genes among the paclitaxel loci known to play a role in microtubule function; NEK10 (Porpora et al 2018; Yi et al 2018) and PDE4DIP (Bouguenina et al 2017; Yang, Wu, et al 2017). The role of PDE4DIP may explain the clinical utility of phosphodiesterase 4 inhibitors as antiproliferative and immunosuppressive agents (Keating 2017).…”

Section: Resultsmentioning

confidence: 75%

Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells

Lin

Wang

et al. 2019

Preprint

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Genetic screens in mammalian cells commonly focus on loss-of-function approaches. To evaluate the phenotypic consequences of extra gene copies, we used bulk segregant analysis (BSA) of radiation hybrid (RH) cells. We constructed six pools of RH cells, each consisting of ∼2500 independent clones, and placed the pools under selection in media with or without paclitaxel. Low pass sequencing identified 859 growth loci, 38 paclitaxel loci, 62 interaction loci and 3 loci for mitochondrial abundance at genome-wide significance. Resolution was measured as ∼30 kb, close to single-gene. Divergent properties were displayed by the RH-BSA growth genes compared to those from loss-of-function screens, refuting the balance hypothesis. In addition, enhanced retention of human centromeres in the RH pools suggests a new approach to functional dissection of these chromosomal elements. Pooled analysis of RH cells showed high power and resolution and should be a useful addition to the mammalian genetic toolkit.

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Section: Resultsmentioning

confidence: 75%

Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells

Lin

Wang

et al. 2019

Preprint

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“…6L). Indeed, it has been demonstrated in HEK293 cells that an increase in cellular cAMP levels and concomitant PKA-dependent protein phosphorylation at centriolar satellites induces ubiquitination and proteolysis of NEK10 by the co-assembled E3 ligase CHIP and promotes cilia resorption (Porpora et al, 2018). This is supported by elegant studies of cilia length control in Chlamydomonas (Cao, Meng et al, 2013, Hendel, Thomson et al, 2018, Hu, Liang et al, 2015, Ishikawa & Marshall, 2017, Liang, Zhu et al, 2018, Ludington, Ishikawa et al, 2015, Luo, Cao et al, 2011, Meng & Pan, 2016, Pan & Snell, 2005, Wang et al, 2019.…”

Section: Discussionmentioning

confidence: 93%

“…cAMP-dependent signaling pathways have been shown to regulate cilia length (Avasthi, Marley et al, 2012, Besschetnova et al, 2010, Kwon, Temiyasathit et al, 2010, Porpora et al, 2018. However, it has been controversially discussed whether an increase in the intracellular cAMP concentration, evoked by pharmacological stimulation, results in an increase or decrease in cilia length (Besschetnova et al, 2010, Porpora et al, 2018. In fact, we also performed pharmacological stimulation of cAMP synthesis in mIMCD-3 cells using forskolin and analyzed the change in cilia length.…”

Section: Investigating the Spatial Contribution Of Camp Signaling Tomentioning

confidence: 99%

“…Furthermore, the most prominent primary cilia signaling pathway, the Sonic hedgehog (Shh) pathway, utilizes cAMP as a second messenger in the cilium to transduce stimulation by Shh into a change in gene expression (Moore, Stepanchick et al, 2016, Mukhopadhyay, Wen et al, 2013. Finally, the dynamic modulation of primary cilia length seems to be controlled by cAMP (Besschetnova, Kolpakova-Hart et al, 2010, Porpora, Sauchella et al, 2018. However, as of yet, it has been impossible to manipulate cAMP dynamics in primary cilia independently from the cell body.…”

Section: Introductionmentioning

confidence: 99%

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Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

Hansen

Kaiser

Klausen

et al. 2020

Preprint

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Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. Primary cilia constitute a subcellular compartment that orchestrates signal transduction independent from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling and function has been challenging due to the lack of tools to manipulate and analyze ciliary signaling in living cells. Here, we describe a nanobodybased targeting approach for optogenetic tools that allows to specifically analyze ciliary signaling and function, and that is applicable in vitro and in vivo. We overcome the loss of protein function observed after direct fusion to a ciliary targeting sequence, and functionally localize the photo-activated adenylate cyclase bPAC, the light-activated phosphodiesterase LAPD, and the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we unravel the contribution of spatial cAMP signaling in controlling cilia length. Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.

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“…In humans, TTLL5 mutation causes recessive retinal dystrophy [23,24]. C. elegans NEKL-4 is homologous to human NEK10, which is a kinase specific to ciliated cells and contributes to ciliogenesis in cultured human kidney cells [25]. In humans, NEK10 mutation causes bronchiectasis, a disorder of mucociliary transport in the airway due to defective motile cilia [26].…”

Section: Introductionmentioning

confidence: 99%

Mutation of NEKL-4/NEK10 and TTLL genes opposes loss of the CCPP-1 deglutamylase and prevents neuronal ciliary degeneration

Power¹,

Akella²,

Gu³

et al. 2020

Preprint

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Ciliary microtubules are subject to post-translational modifications that act as a "Tubulin Code" to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C. elegans, mutations in ccpp-1, the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in the tubulin tyrosine ligase-like glutamylase gene. We show that mutation in ttll-4, ttll-5, or ttll-11 gene suppressed the hyperglutamylation-induced loss of microtubules and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C. elegans NEKL-4 does not localize to cilia yet plays a role in regulating axonemal microtubule stability. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C. elegans genes important for neuronal and ciliary stability, our work may inform research into human ciliopathies and neurodegenerative diseases.

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Counterregulation of cAMP-directed kinase activities controls ciliogenesis

Cited by 42 publications

References 69 publications

Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells

Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells

Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

Mutation of NEKL-4/NEK10 and TTLL genes opposes loss of the CCPP-1 deglutamylase and prevents neuronal ciliary degeneration

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