Systematic analysis of cilia characteristics and Hedgehog signaling in five immortal cell lines

Gómez, Arianna; Christman, Angela K; Weghe, Julie C. Van De; Finn, Malaney; Doherty, Dan

doi:10.1371/journal.pone.0266433

Cited by 4 publications

(3 citation statements)

References 36 publications

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“…Although favorable for GLI stabilization, these responses risk depleting cilia of cAMP, which can slow IFT and destabilize PC length homeostasis. Consistent with this scenario, we and others have observed that SHH stimulation of cultured murine fibroblasts or IMCD3 cells can trigger modest reductions in ciliary length that occasionally reach statistical significance (this study and Gomez et al [2022] ). However, SHH-stimulated IMCD3 PC shortening does not exceed ∼12% unless SHH-EP 4 crosstalk is blocked, whereupon length reductions are consistently significant and range from ∼25 to 40%.…”

Section: Discussionsupporting

confidence: 90%

Sonic Hedgehog activates prostaglandin signaling to stabilize primary cilium length

Ansari,

Dillard,

Zhang

et al. 2024

Journal of Cell Biology

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Sonic Hedgehog (SHH) is a driver of embryonic patterning that, when corrupted, triggers developmental disorders and cancers. SHH effector responses are organized through primary cilia (PC) that grow and retract with the cell cycle and in response to extracellular cues. Disruption of PC homeostasis corrupts SHH regulation, placing significant pressure on the pathway to maintain ciliary fitness. Mechanisms by which ciliary robustness is ensured in SHH-stimulated cells are not yet known. Herein, we reveal a crosstalk circuit induced by SHH activation of Phospholipase A2α that drives ciliary E-type prostanoid receptor 4 (EP4) signaling to ensure PC function and stabilize ciliary length. We demonstrate that blockade of SHH-EP4 crosstalk destabilizes PC cyclic AMP (cAMP) equilibrium, slows ciliary transport, reduces ciliary length, and attenuates SHH pathway induction. Accordingly, Ep4−/− mice display shortened neuroepithelial PC and altered SHH-dependent neuronal cell fate specification. Thus, SHH initiates coordination between distinct ciliary receptors to maintain PC function and length homeostasis for robust downstream signaling.

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Section: Discussionsupporting

confidence: 90%

Sonic Hedgehog activates prostaglandin signaling to stabilize primary cilium length

Ansari,

Dillard,

Zhang

et al. 2024

Journal of Cell Biology

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“…Signal intensity profiles were obtained with the Plot Profile function of Fiji/ImageJ, also as described ( Cilleros-Rodriguez et al, 2022 ). For HEK293T cells, our protocol was the same, except for two modifications based on recent work: i) coverslips were coated with poly-L-lysine and gelatin; and ii) cells were not serum-starved, as this does not affect their ciliation ( Gomez et al, 2022 ).…”

Section: Methodsmentioning

confidence: 99%

EVC-EVC2 complex stability and ciliary targeting are regulated by modification with ubiquitin and SUMO

Barbeito,

Martin-Morales,

Palencia-Campos

et al. 2023

Front. Cell Dev. Biol.

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Ellis van Creveld syndrome and Weyers acrofacial dysostosis are two rare genetic diseases affecting skeletal development. They are both ciliopathies, as they are due to malfunction of primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae and are required for Hedgehog signaling, a key pathway during skeletal morphogenesis. These ciliopathies are caused by mutations affecting the EVC-EVC2 complex, a transmembrane protein heterodimer that regulates Hedgehog signaling from inside primary cilia. Despite the importance of this complex, the mechanisms underlying its stability, targeting and function are poorly understood. To address this, we characterized the endogenous EVC protein interactome in control and Evc-null cells. This proteomic screen confirmed EVC’s main known interactors (EVC2, IQCE, EFCAB7), while revealing new ones, including USP7, a deubiquitinating enzyme involved in Hedgehog signaling. We therefore looked at EVC-EVC2 complex ubiquitination. Such ubiquitination exists but is independent of USP7 (and of USP48, also involved in Hh signaling). We did find, however, that monoubiquitination of EVC-EVC2 cytosolic tails greatly reduces their protein levels. On the other hand, modification of EVC-EVC2 cytosolic tails with the small ubiquitin-related modifier SUMO3 has a different effect, enhancing complex accumulation at the EvC zone, immediately distal to the ciliary transition zone, possibly via increased binding to the EFCAB7-IQCE complex. Lastly, we find that EvC zone targeting of EVC-EVC2 depends on two separate EFCAB7-binding motifs within EVC2’s Weyers-deleted peptide. Only one of these motifs had been characterized previously, so we have mapped the second herein. Altogether, our data shed light on EVC-EVC2 complex regulatory mechanisms, with implications for ciliopathies.

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“…To associate the ciliation phenotype observed in patient cells with the TUBB G308S variant, the effect of the mutation must be studied in a context that is different from the patient's genetic background, and isogenic between control and mutant cells. Human hTERT-RPE1 cells are a common model to study primary cilia in vitro due to efficient ciliation and the functional properties of their cilium 40 . Therefore, we assessed ciliation in the TUBB G308S cell model compared to the isogenic TUBB WT cell line.…”

Section: The Tubb G308s Variant Causes a Primary Cilia Defectmentioning

confidence: 99%

Brain development mutations in the β-tubulin TUBB result in defective ciliogenesis

Mollica,

Omer,

Evagelou

et al. 2023

Preprint

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Tubulinopathies and neurodevelopmental ciliopathies are two groups of genetic disorders characterized by abnormal brain development resulting in structural brain malformations. Tubulinopathies are caused by dominant missense mutations in genes encoding for tubulins, the building blocks of microtubules. Neurodevelopmental ciliopathies are mostly recessive disorders caused by defects in the function of the primary cilium, a sensory organelle that modulates signaling pathways important for brain development. Though more than 40 genes have been associated with neurodevelopmental ciliopathies, many patients still do not have an identified genetic etiology. Herein, we present a novelde novoheterozygous missense variant in Tubulin Beta Class I (TUBB) identified through whole-genome sequencing analysis in a patient with both ciliopathy and tubulinopathy brain features. While microtubules are fundamental to primary cilia formation and function, no association between mutations in tubulin genes and neurodevelopmental ciliopathies has been found to date. Using patient-derived cells and gene-edited isogenic cell lines, we show that the identified variant impairs the early stages of cilia formation by altering microtubule dynamics and structure. Furthermore, we demonstrate that the disease mechanism is not haploinsufficiency and that other patient mutations inTUBBaffect cilia formationin vitro, putting forward defective ciliogenesis as a contributing pathogenic factor in a subset of tubulinopathy patients.

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Systematic analysis of cilia characteristics and Hedgehog signaling in five immortal cell lines

Cited by 4 publications

References 36 publications

Sonic Hedgehog activates prostaglandin signaling to stabilize primary cilium length

Sonic Hedgehog activates prostaglandin signaling to stabilize primary cilium length

EVC-EVC2 complex stability and ciliary targeting are regulated by modification with ubiquitin and SUMO

Brain development mutations in the β-tubulin TUBB result in defective ciliogenesis

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