Phosphorylation of CEP83 by TTBK2 is necessary for cilia initiation

Lo, Chien-Hui; Lin, I-Hsuan; Yang, T. Tony; Huang, Yen-Chun; Tanos, B; Chou, Po-Chun; Chang, Chih-Hung; Tsay, Yeou Guang; Liao, Jung-Chi; Wang, Won Jing

doi:10.1083/jcb.201811142

Cited by 60 publications

(76 citation statements)

References 39 publications

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“…In untreated R1441C LRRK2 MEF cells, we often saw a weak signal of TTBK2 adjacent to gamma-tubulin positive centrioles which we ascribe to the established localization of TTBK2 to the plus-ends of microtubules (21,22). Lo et al (14) also report a scattered radial signal of TTBK2 around the mother centriole in cells without serum starvation. We quantified the enrichment of TTBK2 over mother centriole (marked by CEP164) using CellProfiler.…”

Section: Mechanism Of Ciliation Blockadementioning

confidence: 91%

Pathogenic LRRK2 regulates ciliation probability upstream of Tau Tubulin kinase 2

Sobu

Wawro

Dhekne

et al. 2020

Preprint

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Mutations that activate LRRK2 protein kinase cause Parkinson's disease. We have shown previously that Rab10 phosphorylation by LRRK2 enhances its binding to RILPL1 and together, these proteins block cilia formation in a variety of cell types including patient derived iPS cells. We have used live cell fluorescence microscopy to identify, more precisely, the effect of LRRK2 kinase activity on both the formation of cilia triggered by serum starvation and loss of cilia seen upon serum re-addition. LRRK2 activity decreases the overall probability of ciliation without changing the rates of cilia formation in R1441C LRRK2 MEF cells. Cilia loss in these cells is accompanied by ciliary decapitation. Kinase activity does not change the timing or frequency of decapitation or the rate of cilia loss, but increases the percent of cilia that are lost upon serum addition. LRRK2 activity, or overexpression of RILPL1 protein, blocks release of CP110 from the mother centriole, a step normally required for early ciliogenesis. In both cases, failure of CP110 uncapping was due to failure to recruit TTBK2, a kinase needed for CP110 release. In contrast, recruitment of EHD1, another step important for ciliogenesis, appears unaltered. These experiments provide critical detail to our understanding of the cellular consequences of pathogenic LRRK2 mutation, and indicate that LRRK2 blocks ciliogenesis upstream of TTBK2 and enhances the deciliation process in response to serum addition.KEY WORDS: LRRK2, Rab GTPase, primary cilia, Parkinson's disease SIGNIFICANCE STATEMENTMutations that activate LRRK2 protein kinase cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases, in particular Rab8 and Rab10. Phosphorylated Rabs bind preferentially to a distinct set of effectors and block in primary ciliation in multiple cell types. We show here that the cilia blockade is upstream of the recruitment of TTBK2 kinase to the mother centriole, a step required for the release of CP110 and subsequent cilia formation. This study provides fundamental information related to how pathogenic LRRK2 interferes with normal cell physiology.

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Section: Mechanism Of Ciliation Blockadementioning

confidence: 91%

Pathogenic LRRK2 regulates ciliation probability upstream of Tau Tubulin kinase 2

Sobu

Wawro

Dhekne

et al. 2020

Preprint

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“…1e, 1f). It is interesting to note that 24-hour serum starvation, which promotes ciliogenesis in around 60% RPE-1 cells (Lo et al, 2019), does have an effect on the completeness of ring occupancy of ciliogenesis-independent sDAP proteins. In addition, sDAPs may not always possess a nine-punctum pattern as that of DAPs in RPE-1 cells.…”

Section: Super-resolution Microscopy Reveals That Completeness Of Rinmentioning

confidence: 99%

Super-resolution microscopy reveals coupling between mammalian centriole subdistal appendages and distal appendages

Chong

Wang

et al. 2020

eLife

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Subdistal appendages (sDAPs) are centriolar elements that are observed proximal to the distal appendages (DAPs) in vertebrates. Despite the obvious presence of sDAPs, structural and functional understanding of them remains elusive. Here, by combining super-resolved localization analysis and CRISPR-Cas9 genetic perturbation, we find that although DAPs and sDAPs are primarily responsible for distinct functions in ciliogenesis and microtubule anchoring, respectively, the presence of one element actually affects the positioning of the other. Specifically, we find dual layers of both ODF2 and CEP89, where their localizations are differentially regulated by DAP and sDAP integrity. DAP depletion relaxes longitudinal occupancy of sDAP protein ninein to cover the DAP region, implying a role of DAPs in sDAP positioning. Removing sDAPs alter the distal border of centrosomal γ-tubulins, illustrating a new role of sDAPs. Together, our results provide an architectural framework for sDAPs that sheds light on functional understanding, surprisingly revealing coupling between DAPs and sDAPs.

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“…( Figure 6e). The difference between these results could be explained by the fact that TTKBs contribute to initiation of ciliogenesis (Goetz, Liem, & Anderson, 2012;Lo et al, 2019), whereas ciliogenesis can progress (to some degree) in the absence of DAW1 (Ahmed & Mitchell, 2005; Gao et al, 2010; Solomon et al, 2017; this study),…”

Section: Discussionmentioning

confidence: 53%

Dynein assembly factor with WD repeat domains 1 (DAW1) is required for the function of motile cilia in the planarian Schmidtea mediterranea

Lesko

Rouhana

2020

Dev Growth Differ

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Motile cilia propel directed cell movements and sweep fluids across the surface of tissues. Orthologs of Dynein Assembly Factor with WD Repeat Domains 1 (DAW1) support normal ciliary beating by enhancing delivery of dynein complexes to axonemal microtubules. DAW1 mutations in vertebrates result in multiple developmental abnormalities and early or prenatal lethality, complicating functional assessment of DAW1 in adult structures. Planarian flatworms maintain cellular homeostasis and regenerate through differentiation of adult pluripotent stem cells, and systemic RNA‐interference (RNAi) can be induced to analyze gene function at any point after birth. A single ortholog of DAW1 was identified in the genome of the planarian Schmidtea mediterranea (Smed‐daw1). Smed‐DAW1 is composed of eight WD repeats, which are 55% identical to the founding member of this protein family (Chlamydomonas reinhardtii ODA16) and 58% identical to human DAW1. Smed‐daw1 is expressed in the planarian epidermis, protonephridial excretory system, and testes, all of which contain cells functionally dependent on motile cilia. Smed‐daw1 RNAi resulted in locomotion defects and edema, which are phenotypes characteristic of multiciliated epidermis and protonephridial dysfunction, respectively. Changes in abundance or length of motile cilia were not observed at the onset of phenotypic manifestations upon Smed‐daw1 RNAi, corroborating with studies showing that DAW‐1 loss of function leads to aberrant movement of motile cilia in other organisms, rather than loss of cilia per se. However, extended RNAi treatments did result in shorter epidermal cilia and decreased abundance of ciliated protonephridia, suggesting that Smed‐daw1 is required for homeostatic maintenance of these structures in flatworms.

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Phosphorylation of CEP83 by TTBK2 is necessary for cilia initiation

Cited by 60 publications

References 39 publications

Pathogenic LRRK2 regulates ciliation probability upstream of Tau Tubulin kinase 2

Pathogenic LRRK2 regulates ciliation probability upstream of Tau Tubulin kinase 2

Super-resolution microscopy reveals coupling between mammalian centriole subdistal appendages and distal appendages

Dynein assembly factor with WD repeat domains 1 (DAW1) is required for the function of motile cilia in the planarian Schmidtea mediterranea

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