A protocol for generation and live-cell imaging analysis of primary cilia reporter cell lines

Binó, Lucia; Mikulenková, Erika; Štěpánek, Luděk; Bernatík, Ondřej; Vysloužil, David; Pejšková, Petra; Gorilák, Peter; Huranová, Martina; Varga, Vladimír; Čajánek, Lukáš

doi:10.1016/j.xpro.2022.101199

Cited by 2 publications

(1 citation statement)

References 31 publications

(42 reference statements)

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“…Reporter hTERT-RPE-1 Flp-In T-Rex TTBK2 KO cell lines with DOX-inducible expression of Flag-TTBK2 constructs and constitutive mNeonGreen-ARL13B reporter expression were prepared as described in 50 . For the time-lapse live imaging experiment cells were seeded in DMEM/F12 medium, 10%FBS, 1%L-glutamine and Penicilin/streptomycin, supplemented with 1 μg/mL DOX on a 10-well glass-bottom CELLVIEW CELL CULTURE SLIDE, PS, 75/25 MM (Greiner Bio-One) at a high density (∼30.000 cells per well).…”

Section: Live-cell Imagingmentioning

confidence: 99%

Tau-Tubulin Kinase 2 restrains microtubule-depolymerizer KIF2A to support primary cilia growth

Vysloužil

Bernatík

Renzova

et al. 2023

Preprint

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The initiation of assembly of primary cilia, organelles with crucial functions in development and disease, is under the control of Tau-Tubulin Kinase 2 (TTBK2). Recent work has implicated TTBK2 also in the regulation of primary cilia maintenance and function. However, the mechanisms underlying individual functions of TTBK2 in primary cilia are not fully understood. Here, to dissect the role of TTBK2 in primary cilia maintenance in human cells, we examined disease related TTBK2 truncations. We demonstrate that these truncated protein moieties show selective activity towards TTBK2 substrates. This creates a semi-permissive condition where partial TTBK2 activity suffices to support the initiation of ciliogenesis but fails to sustain primary cilia length. Subsequently, we show that the defects in primary cilia growth are linked to aberrant turnover of kinesin KIF2A at basal body. Furthermore, we demonstrate that TTBK2 regulates KIF2A by phosphorylation, which in turn restrains its levels at the ciliary base to promote primary cilia elongation and maintenance. Taken together, our data highlight the regulation of KIF2A by TTBK2 as an important mechanism governing primary cilia in human cells.

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Section: Live-cell Imagingmentioning

confidence: 99%

Tau-Tubulin Kinase 2 restrains microtubule-depolymerizer KIF2A to support primary cilia growth

Vysloužil

Bernatík

Renzova

et al. 2023

Preprint

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BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

Prasai,

Ivashchenko,

Maskova

et al. 2023

Preprint

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Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of the BBSome, a cargo adaptor essential for export of transmembrane receptors from cilia. Although actin-dependent ectocytosis has been proposed as compensatory cellular process for defective cargo retrieval, the underlying molecular mechanism is poorly understood, particularly in the context of BBS pathology. In this study, we investigated how actin polymerization and ectocytosis are regulated within the cilium. Our findings reveal that ciliary CDC42, a RHO-family GTPase triggers in situ actin polymerization, ciliary ectocytosis, and cilia shortening in BBSome-deficient cells. Furthermore, activation of the Sonic Hedgehog pathway further enhances CDC42 activity in BBSome-deficient cilia, but not in healthy cells. Inhibition of CDC42 decreased the frequency and duration of ciliary actin polymerization events and lead to the accumulation of the GPR161 receptor in bulges along the axoneme in BBSome-deficient cells during Sonic Hedgehog signaling. Overall, our study identifies CDC42 as a key trigger of ciliary ectocytosis. Moreover, the hyperactivated ciliary CDC42-actin-ectocytosis axis in BBSome-deficient cells results in cilia shortening and loss of ciliary material, potentially impacting disease severity.

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A protocol for generation and live-cell imaging analysis of primary cilia reporter cell lines

Cited by 2 publications

References 31 publications

Tau-Tubulin Kinase 2 restrains microtubule-depolymerizer KIF2A to support primary cilia growth

Tau-Tubulin Kinase 2 restrains microtubule-depolymerizer KIF2A to support primary cilia growth

BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

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