Primary Cilia Formation Does Not Rely on WNT/β-Catenin Signaling

Abstract

Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-β. However, gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/β-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/β-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast, second model proposes a positive role of WNT/β-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/β-catenin pathway, β-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/β-catenin pathway in cell lines (RPE-1, NIH3T3, and HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6, or AXIN1/2 we show that neither activation nor deactivation of the WNT/β-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/β-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/β-catenin pathway in some systems seems to moderately suppress ciliogenesis.

MAPK15 Controls Hedgehog Signaling in Medulloblastoma Cells by Regulating Primary Ciliogenesis

“…MAPK15 was reported to localize to the basal bodies of motile [ 8 ] and primary cilia [ 7 ] in vertebrates and to regulate their formation. Indeed, interfering with MAPK15 expression in both RPE1 ( Figure 1 a and Figure S1a ) and IMCD3 ( Figure 1 b and Figure S1b ) cells, two established models for studying primary ciliogenesis [ 27 , 28 ], respectively in human and murine cells, strongly inhibited their ability of assembling primary cilia. Interestingly, Kazatskaya et al recently showed that a MAPK15 kinase-dead mutant was not able to rescue siRNA-dependent inhibition of primary cilia formation in RPE1 cells [ 7 ], suggesting a key role for MAPK15 kinase activity in regulating this process.…”

“…Based on the role of MAPK15 in primary cilia biogenesis, we, therefore, decided to investigate a potential role for this kinase in HH signaling. At this scope, we first tested MAPK15-dependent primary cilia formation in NIH3T3 cells, which is a well-established model for the study of both the HH pathway [ 25 ] and of ciliogenesis [ 27 ]. To modulate MAPK15 expression in these cells, we decided to use the stable expression of shRNAs for this kinase ( Figure S2 ) and demonstrated that this approach strongly reduced starvation-dependent primary ciliogenesis in these cells ( Figure 2 a,b).…”

WNT stimulation induced conformational dynamics in the Frizzled-Dishevelled interaction

“…For all experiments, ΔFZD 1-10 HEK293T cells were used if nothing else was stated. ΔFZD 1-10 HEK293T cells ( 43 ) and ΔLRP5/6 HEK293 T-Rex cells ( 44 ) were cultured in DMEM (HyClone SH30081) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher Scientific 10270106 and 15140) in a humidified incubator at 37 °C and 5% CO 2 . All cell culture plastics were from Sarstedt unless stated otherwise.…”

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

“…In the former case, use healthy cells, consider thawing a low passage aliquot of the cells, and check routinely for Mycoplasma. In addition, to promote ciliogenesis, try to optimize the cell density, length of serum starvation period, or consider using small molecules promoting cilia formation (e.g., Cytochalasin D ( Bernatik et al., 2021 ; Kim et al., 2010 )). In case the cilia presence is DOX-inducible (i.e., in the case of rescue experiment), we observed that adding DOX already 1–2 days prior to seeding the cells for imaging experiment results in a better rescue effect.…”