Primary cilia are hair-like sensory organelles protruding from the surface of most human cells. As cilia are dynamic, several aspects of their biology can only be revealed by real-time analysis in living cells. Here we describe the generation of primary cilia reporter cell lines. Furthermore, we provide a detailed protocol of how to use the reporter cell lines for live-cell imaging microscopy analysis of primary cilia to study their growth as well as intraciliary transport. For complete details on the use and execution of this protocol, please refer to Bernatik et al. (2020) and Pejskova et al. (2020).

• MeSH
• buněčné linie MeSH
• cilie * metabolismus   MeSH
• lidé MeSH
• mikroskopie metody   MeSH
• počítačové zpracování obrazu * metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Primary cilia play critical roles in development and disease. Their assembly and disassembly are tightly coupled to cell cycle progression. Here, we present data identifying KIF14 as a regulator of cilia formation and Hedgehog (HH) signaling. We show that RNAi depletion of KIF14 specifically leads to defects in ciliogenesis and basal body (BB) biogenesis, as its absence hampers the efficiency of primary cilium formation and the dynamics of primary cilium elongation, and disrupts the localization of the distal appendage proteins SCLT1 and FBF1 and components of the IFT-B complex. We identify deregulated Aurora A activity as a mechanism contributing to the primary cilium and BB formation defects seen after KIF14 depletion. In addition, we show that primary cilia in KIF14-depleted cells are defective in response to HH pathway activation, independently of the effects of Aurora A. In sum, our data point to KIF14 as a critical node connecting cell cycle machinery, effective ciliogenesis, and HH signaling.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• aurora kinasa A antagonisté a inhibitory   genetika   metabolismus   MeSH
• bazální tělíska metabolismus   MeSH
• buněčný cyklus genetika   MeSH
• chromatografie kapalinová MeSH
• cilie genetika   metabolismus   patologie   MeSH
• HEK293 buňky MeSH
• interfáze fyziologie   MeSH
• intracelulární signální peptidy a proteiny genetika   metabolismus   MeSH
• kineziny genetika   metabolismus   MeSH
• lidé MeSH
• mitóza genetika   MeSH
• onkogenní proteiny genetika   metabolismus   MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• proteiny hedgehog metabolismus   MeSH
• RNA interference MeSH
• signální transdukce genetika   MeSH
• sodíkové kanály metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Wnt and BMP signaling pathways are two key molecular machineries regulating development and homeostasis. The efficient coordination of Wnt and BMP is essential in many developmental processes such as establishment of antero-posterior and dorso-ventral body axis, regulation of convergent extension, or development of various organ systems. SMAD ubiquitination regulatory factor (Smurf) family of E3 ubiquitin ligases are important and evolutionary conserved regulators of TGF-β/BMP signaling pathways. Smurf2 has been previously shown to regulate Wnt/planar cell polarity (PCP) signaling pathway by ubiquitinating Prickle1, one of the key components of PCP. We explored the role of Smurf2 in Wnt pathways in further detail and identified that Smurf2 is also a ubiquitin ligase of Dishevelled (DVL), the key cytoplasmic signal transducer in the Wnt pathway. Interestingly, the Smurf2 and DVL relationship expands beyond substrate-E3 ligase. We can show that DVL activates Smurf2, which allows Smurf2 to ubiquitinate its substrates from Wnt/PCP (Prickle1) as well as TGF-β/BMP (Smad2) pathways more efficiently. Using SMAD7 as an example of Smurf2 activator we show that DVL and SMAD7 both activates Smurf2 activity. In HEK293 cells the deficiency of DVL phenocopies absence of Smurf2 and leads to the increased phosphorylation of R-Smads. Smurf2-DVL connection provides a novel and intriguing point of crosstalk for Wnt and BMP pathways.

• MeSH
• biologické modely MeSH
• HEK293 buňky MeSH
• kostní morfogenetické proteiny metabolismus   MeSH
• lidé MeSH
• nádorové supresorové proteiny metabolismus   MeSH
• protein dishevelled metabolismus   MeSH
• proteiny s doménou LIM metabolismus   MeSH
• proteolýza MeSH
• signální dráha Wnt * MeSH
• signální transdukce MeSH
• transformující růstový faktor beta metabolismus   MeSH
• ubikvitinace MeSH
• ubikvitinligasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Primary cilia are organelles necessary for proper implementation of developmental and homeostasis processes. To initiate their assembly, coordinated actions of multiple proteins are needed. Tau tubulin kinase 2 (TTBK2) is a key player in the cilium assembly pathway, controlling the final step of cilia initiation. The function of TTBK2 in ciliogenesis is critically dependent on its kinase activity; however, the precise mechanism of TTBK2 action has so far not been fully understood due to the very limited information about its relevant substrates. In this study, we demonstrate that CEP83, CEP89, CCDC92, Rabin8, and DVL3 are substrates of TTBK2 kinase activity. Further, we characterize a set of phosphosites of those substrates and CEP164 induced by TTBK2 in vitro and in vivo. Intriguingly, we further show that identified TTBK2 phosphosites and consensus sequence delineated from those are distinct from motifs previously assigned to TTBK2. Finally, we show that TTBK2 is also required for efficient phosphorylation of many S/T sites in CEP164 and provide evidence that TTBK2-induced phosphorylations of CEP164 modulate its function, which in turn seems relevant for the process of cilia formation. In summary, our work provides important insight into the substrates-TTBK2 kinase relationship and suggests that phosphorylation of substrates on multiple sites by TTBK2 is probably involved in the control of ciliogenesis in human cells.

• MeSH
• aminokyselinové motivy MeSH
• cilie metabolismus   MeSH
• fosforylace MeSH
• fosfoserin metabolismus   MeSH
• fosfothreonin metabolismus   MeSH
• HEK293 buňky MeSH
• kasein kinasa I metabolismus   MeSH
• lidé MeSH
• multiproteinové komplexy metabolismus   MeSH
• organogeneze * MeSH
• protein-serin-threoninkinasy chemie   metabolismus   MeSH
• substrátová specifita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Dishevelled (DVL) is an essential component of the Wnt signaling cascades. Function of DVL is controlled by phosphorylation but the molecular details are missing. DVL3 contains 131 serines and threonines whose phosphorylation generates complex barcodes underlying diverse DVL3 functions. In order to dissect the role of DVL phosphorylation we analyzed the phosphorylation of human DVL3 induced by previously reported (CK1ε, NEK2, PLK1, CK2α, RIPK4, PKCδ) and newly identified (TTBK2, Aurora A) DVL kinases. METHODS: Shotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on immunoprecipitates from HEK293T cells was used to identify and quantify phosphorylation of DVL3 protein induced by 8 kinases. Functional characterization was performed by in-cell analysis of phospho-mimicking/non-phosphorylatable DVL3 mutants and supported by FRET assays and NMR spectroscopy. RESULTS: We used quantitative mass spectrometry and calculated site occupancies and quantified phosphorylation of > 80 residues. Functional validation demonstrated the importance of CK1ε-induced phosphorylation of S268 and S311 for Wnt-3a-induced β-catenin activation. S630-643 cluster phosphorylation by CK1, NEK2 or TTBK2 is essential for even subcellular distribution of DVL3 when induced by CK1 and TTBK2 but not by NEK2. Further investigation showed that NEK2 utilizes a different mechanism to promote even localization of DVL3. NEK2 triggered phosphorylation of PDZ domain at S263 and S280 prevents binding of DVL C-terminus to PDZ and promotes an open conformation of DVL3 that is more prone to even subcellular localization. CONCLUSIONS: We identify unique phosphorylation barcodes associated with DVL function. Our data provide an example of functional synergy between phosphorylation in structured domains and unstructured IDRs that together dictate the biological outcome. Video Abtract.

• MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• hmotnostní spektrometrie MeSH
• kinasy NEK metabolismus   MeSH
• konformace proteinů MeSH
• kultivované buňky MeSH
• lidé MeSH
• protein dishevelled chemie   metabolismus   MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Development of neural tube has been extensively modeled in vitro using human pluripotent stem cells (hPSCs) that are able to form radially organized cellular structures called neural rosettes. While a great amount of research has been done using neural rosettes, studies have only inadequately addressed how rosettes are formed and what the molecular mechanisms and pathways involved in their formation are. Here we address this question by detailed analysis of the expression of pluripotency and differentiation-associated proteins during the early onset of differentiation of hPSCs towards neural rosettes. Additionally, we show that the BMP signaling is likely contributing to the formation of the complex cluster of neural rosettes and its inhibition leads to the altered expression of PAX6, SOX2 and SOX1 proteins and the rosette morphology. Finally, we provide evidence that the mechanism of neural rosettes formation in vitro is reminiscent of the process of secondary neurulation rather than that of primary neurulation in vivo. Since secondary neurulation is a largely unexplored process, its understanding will ultimately assist the development of methods to prevent caudal neural tube defects in humans.

• MeSH
• buněčná diferenciace * MeSH
• COUP transkripční faktor II genetika   metabolismus   MeSH
• faktory domény POU genetika   metabolismus   MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• nervové kmenové buňky cytologie   metabolismus   MeSH
• neurální trubice cytologie   embryologie   metabolismus   MeSH
• neurulace * MeSH
• pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• transkripční faktor PAX6 genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.

• MeSH
• analýza jednotlivých buněk metody   MeSH
• biosenzitivní techniky MeSH
• enzymatické testy metody   MeSH
• fluorescenční mikroskopie metody   MeSH
• fosforylace fyziologie   MeSH
• frizzled receptory metabolismus   MeSH
• genový knockout MeSH
• HEK293 buňky MeSH
• kasein kinasa 1 epsilon genetika   metabolismus   MeSH
• lidé MeSH
• mutageneze cílená MeSH
• oocyty MeSH
• PDZ domény fyziologie   MeSH
• protein dishevelled genetika   metabolismus   MeSH
• rezonanční přenos fluorescenční energie MeSH
• signální dráha Wnt fyziologie   MeSH
• simulace molekulární dynamiky MeSH
• Xenopus laevis MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Frizzleds (FZDs) are receptors for secreted lipoglycoproteins of the Wingless/Int-1 (WNT) family, initiating an important signal transduction network in multicellular organisms. FZDs are G protein-coupled receptors (GPCRs), which are well known to be regulated by phosphorylation, leading to specific downstream signaling or receptor desensitization. The role and underlying mechanisms of FZD phosphorylation remain largely unexplored. Here, we investigated the phosphorylation of human FZD6 Using MS analysis and a phospho-state- and -site-specific antibody, we found that Ser-648, located in the FZD6 C terminus, is efficiently phosphorylated by casein kinase 1 ϵ (CK1ϵ) and that this phosphorylation requires the scaffolding protein Dishevelled (DVL). In an overexpression system, DVL1, -2, and -3 promoted CK1ϵ-mediated FZD6 phosphorylation on Ser-648. This DVL activity required an intact DEP domain and FZD-mediated recruitment of this domain to the cell membrane. Substitution of the CK1ϵ-targeted phosphomotif reduced FZD6 surface expression, suggesting that Ser-648 phosphorylation controls membrane trafficking of FZD6 Phospho-Ser-648 FZD6 immunoreactivity in human fallopian tube epithelium was predominantly apical, associated with cilia in a subset of epithelial cells, compared with the total FZD6 protein expression, suggesting that FZD6 phosphorylation contributes to asymmetric localization of receptor function within the cell and to epithelial polarity. Given the key role of FZD6 in planar cell polarity, our results raise the possibility that asymmetric phosphorylation of FZD6 rather than asymmetric protein distribution accounts for polarized receptor signaling.

• MeSH
• buněčná membrána metabolismus   MeSH
• epitel metabolismus   MeSH
• fosfoproteiny imunologie   MeSH
• fosforylace MeSH
• frizzled receptory chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• hmotnostní spektrometrie MeSH
• kasein kinasa I metabolismus   MeSH
• lidé MeSH
• protein dishevelled chemie   fyziologie   MeSH
• protilátky imunologie   MeSH
• sekvence aminokyselin MeSH
• serin metabolismus   MeSH
• signální transdukce MeSH
• vejcovody metabolismus   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mammalian limb development is driven by the integrative input from several signaling pathways; a failure to receive or a misinterpretation of these signals results in skeletal defects. The brachydactylies, a group of overlapping inherited human hand malformation syndromes, are mainly caused by mutations in BMP signaling pathway components. Two closely related forms, Brachydactyly type B2 (BDB2) and BDB1 are caused by mutations in the BMP antagonist Noggin (NOG) and the atypical receptor tyrosine kinase ROR2 that acts as a receptor in the non-canonical Wnt pathway. Genetic analysis of Nog and Ror2 functional interaction via crossing Noggin and Ror2 mutant mice revealed a widening of skeletal elements in compound but not in any of the single mutants, thus indicating genetic interaction. Since ROR2 is a non-canonical Wnt co-receptor specific for Wnt-5a we speculated that this phenotype might be a result of deregulated Wnt-5a signaling activation, which is known to be essential for limb skeletal elements growth and patterning. We show that Noggin potentiates activation of the Wnt-5a-Ror2-Disheveled (Dvl) pathway in mouse embryonic fibroblast (MEF) cells in a Ror2-dependent fashion. Rat chondrosarcoma chondrocytes (RCS), however, are not able to respond to Noggin in this fashion unless growth arrest is induced by FGF2. In summary, our data demonstrate genetic interaction between Noggin and Ror2 and show that Noggin can sensitize cells to Wnt-5a/Ror2-mediated non-canonical Wnt signaling, a feature that in cartilage may depend on the presence of active FGF signaling. These findings indicate an unappreciated function of Noggin that will help to understand BMP and Wnt/PCP signaling pathway interactions.

• MeSH
• brachydaktylie genetika   MeSH
• genetické testování MeSH
• kostní morfogenetické proteiny antagonisté a inhibitory   MeSH
• lidé MeSH
• nervové dráhy MeSH
• protein Wnt 5a MeSH
• signální dráha Wnt MeSH
• signální transdukce * MeSH
• sirotčí receptory podobné receptoru tyrosinkinasy MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Frizzleds (FZDs) are unconventional G protein-coupled receptors, which activate diverse intracellular signaling pathways via the phosphoprotein Disheveled (DVL) and heterotrimeric G proteins. The interaction interplay of FZDs with DVL and G proteins is complex, involves different regions of FZD and the potential dynamics are poorly understood. In the present study, we aimed to characterize the function of a highly conserved tyrosine (Y2502.39) in the intracellular loop 1 (IL1) of human FZD4. We have found Y2502.39 to be crucial for DVL2 interaction and DVL2 translocation to the plasma membrane. Mutant FZD4-Y2502.39F, impaired in DVL2 binding, was defective in both β-catenin-dependent and β-catenin-independent WNT signaling induced in Xenopus laevis embryos. The same mutant maintained interaction with the heterotrimeric G proteins Gα12 and Gα13 and was able to mediate WNT-induced G protein dissociation and G protein-dependent YAP/TAZ signaling. We conclude from modeling and dynamics simulation efforts that Y2502.39 is important for the structural integrity of the FZD-DVL, but not for the FZD-G protein interface and hypothesize that the interaction network of Y2502.39 and H3484.46 plays a role in specifying downstream signaling pathways induced by the receptor.

• MeSH
• aminokyselinové motivy MeSH
• embryo nesavčí metabolismus   MeSH
• frizzled receptory chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• heterotrimerní G-proteiny metabolismus   MeSH
• konzervovaná sekvence * MeSH
• lidé MeSH
• mutační analýza DNA MeSH
• nádory metabolismus   patologie   MeSH
• polymerizace MeSH
• protein dishevelled chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální dráha Wnt MeSH
• signální transdukce MeSH
• simulace molekulární dynamiky MeSH
• strukturní homologie proteinů MeSH
• tyrosin metabolismus   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Xenopus laevis embryologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH