eGFP-tagged Wnt-3a enables functional analysis of Wnt trafficking and signaling and kinetic assessment of Wnt binding to full-length Frizzled

Wesslowski, Janine; Kozielewicz, Paweł; Wang, Xianxian; Cui, Haijun; Schihada, Hannes; Kranz, D; M, Pradhipa Karuna; Levkin, Pavel A.; Gross, Julia Christina; Boutros, Michael; Schulte, Gunnar; Davidson, Gary

doi:10.1074/jbc.ra120.012892

Cited by 32 publications

(39 citation statements)

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“…Furthermore, biophysical parameters including stability, geometry and stoichiometry of the Wnt/Fzd/LRP5/6 signalling complex are likely to infer signalling dynamics. Despite some progress in recent years [137,[159][160][161][162] studies to exploit ligand/receptor-induced Wnt signalling dynamics remain challenging owing to the complex nature of the receptors and the Wnts. To overcome these difficulties, several laboratories have developed Wnt mimetics, consisting of non-lipidated Fzd-LRP5/6 'heterodimerizers' that are structurally distinct from natural Wnts but recapitulate their activities with a tuneable activity [137,159,160,163,164].…”

Section: Feedback Mechanismsmentioning

confidence: 99%

Signalling dynamics in embryonic development

Sonnen

Janda

2021

Biochemical Journal

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In multicellular organisms, cellular behaviour is tightly regulated to allow proper embryonic development and maintenance of adult tissue. A critical component in this control is the communication between cells via signalling pathways, as errors in intercellular communication can induce developmental defects or diseases such as cancer. It has become clear over the last years that signalling is not static but varies in activity over time. Feedback mechanisms present in every signalling pathway lead to diverse dynamic phenotypes, such as transient activation, signal ramping or oscillations, occurring in a cell type- and stage-dependent manner. In cells, such dynamics can exert various functions that allow organisms to develop in a robust and reproducible way. Here, we focus on Erk, Wnt and Notch signalling pathways, which are dynamic in several tissue types and organisms, including the periodic segmentation of vertebrate embryos, and are often dysregulated in cancer. We will discuss how biochemical processes influence their dynamics and how these impact on cellular behaviour within multicellular systems.

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Section: Feedback Mechanismsmentioning

confidence: 99%

Signalling dynamics in embryonic development

Sonnen

Janda

2021

Biochemical Journal

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“…For microscopic FRET experiments, fluorescence emission time courses of both FRET donor and acceptor were routinely corrected for background and spectral bleedthrough, and the FRET ratio was calculated as described earlier 9,64 . For calculating τ, agonist-independent changes in FRET due to photobleaching were subtracted.…”

Section: Data Analysis and Statisticsmentioning

confidence: 99%

Functional modulation of PTH1R activation and signalling by RAMP2

Nemec

Schihada

Kleinau

et al. 2021

Preprint

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Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein-coupled receptors (GPCRs) including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR, and an important modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function. Using different optical biosensors to measure the activation of PTH1R and its downstream signalling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique pre-activated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signalling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signalling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered β-arrestin2 recruitment to PTH1R. Employing homology modelling we describe the putative structural molecular basis underlying our functional findings. These data uncover a critical role of RAMPs in the activation and signalling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design.

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“…We used two different cell types for the formation of spheroids: first, a cell line expressing and secreting Wnt‐3a proteins (HEK 293T, Wnt‐3a), and second, a cell line harboring a TOP‐GFP fluorescent reporter (HEK 293T, TOP‐GFP) for monitoring activation of Wnt signaling in these cells. [ 38–41 ]…”

Section: Figurementioning

confidence: 99%

“…We used two different cell types for the formation of spheroids: first, a cell line expressing and secreting Wnt-3a proteins (HEK 293T, Wnt-3a), and second, a cell line harboring a TOP-GFP fluorescent reporter (HEK 293T, TOP-GFP) for monitoring activation of Wnt signaling in these cells. [38][39][40][41] In order to allow the controlled fusion of spheroids formed from these two types of cells, an array containing Wnt producing spheroids as well as Wnt reporter spheroids was generated (see Experimental Section). As described above, we print these two cell lines (200 cells in each spot) into neighboring droplets of DMA at the same time, and culture them for 2 days to obtain spheroids using the hanging drop method.…”

mentioning

confidence: 99%

“…Wnt Signaling between Spheroids: HEK 293T Wnt-3a producing, as well as the stable HEK 293T TOP-GFP reporter cell line [41] were seeded according to the scheming program ( Figure S6, Supporting Information). The Wnt producing cells (Wnt-3a transfected HEK 293T, 24 h, 1 × 10 6 cells mL −1 , and 200 nL) were printed on column 1, 3, 5, 7, 9, 11, 13 and reporter cells (stable HEK 293T TOP-GFP, 1 × 10 6 cells mL −1 , and 200 nL) were printed on column 2, 4, 6, 8, 10, 12, and 14.…”

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confidence: 99%

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Assembly of Multi‐Spheroid Cellular Architectures by Programmable Droplet Merging

et al. 2020

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Reproduction of native tissues in vitro is important as a tool, as it both enables investigation of fundamental biological processes, and drug and toxicity screenings. In order to closely mimic complex tissues in vitro, artificial multicellular systems are created from different cell types in spatially ordered structures or welldefined geometries in a 3D microenvironment. [1-3] These systems can be built from building blocks [4-7] such as cell sheets, [8] cell-laden microgels, [5] cell spheroids, [9] and organoids. [10,11] Precise control of cellular composition and spatial distribution of building blocks within artificial multicellular systems allows for reconstitution of native tissues in their healthy and disease state in vitro. [12] There are a number of methodologies developed for fabrication of complex 3D cell systems in vitro. [3-7,13,14] Directed assembly allows manual positioning or stacking building blocks to form 3D architectures. [15,16] Birey et al. applied this method for fusion of two forebrain organoids in order to mimic the human brain development and demonstrate inter-neuronal migration. [15] The method of directed assembly is, however, manual and not compatible with high throughput. Remote assembly, such as, acoustic node, [14,17] magnetic cell levitation, [13,18] optical tweezers, [19] or laser-guided direct writing, [20] can achieve assembly of cells or spheroids against gravity or viscous forces. Chen et al. demonstrated the assembly of hepatic organoids by an acoustic node technique, and the technique was able to achieve formation of bile canaliculi networks resembling native hepatic tissue. [17] Souza et al. used magnetic cell levitation to manipulate a glioblastoma cell spheroid, and a human astrocyte spheroid in order to create a cell invasion model. [18] These methods depend on sophisticated equipment, paramagnetic media, or introduce a risk of laser-induced cell damage. Another common strategy used to fabricate multicellular architecture is assembly of cell-laden hydrogels or microgels, [5,21,22] or cell seeding on scaffolds. [7,23] However, these biomaterial-based methods failed to provide high cell packing density. The use of artificial scaffolds or gel matrices additionally lead to disadvantages for constructing 3D tissue models due to their influence on cell-cell interactions, autocrine, and paracrine signaling. 3D printing [3] is a promising method for designing and achieving multicellular architectures, but it is relatively slow, not always compatible with high throughput and relies on printable bio-inks for maintaining 3D structure and cell viability. Artificial multicellular systems are gaining importance in the field of tissue engineering and regenerative medicine. Reconstruction of complex tissue architectures in vitro is nevertheless challenging, and methods permitting controllable and high-throughput fabrication of complex multicellular architectures are needed. Here, a facile and high-throughput method is developed based on a tunable droplet-fusion technique, allowing prog...

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eGFP-tagged Wnt-3a enables functional analysis of Wnt trafficking and signaling and kinetic assessment of Wnt binding to full-length Frizzled

Cited by 32 publications

References 65 publications

Signalling dynamics in embryonic development

Signalling dynamics in embryonic development

Functional modulation of PTH1R activation and signalling by RAMP2

Assembly of Multi‐Spheroid Cellular Architectures by Programmable Droplet Merging

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