scholarly journals Coupling optogenetics and light-sheet microscopy, a method to study signal transduction in vivo

2017 ◽  
Author(s):  
Prameet Kaur ◽  
Timothy E. Saunders ◽  
Nicholas S. Tolwinski
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathways ◽  
Fluorescent Protein ◽  
Light Sheet ◽  
Light Illumination ◽  
Temporal Regulation ◽  
Light Sheet Microscopy ◽  
Cryptochrome 2

AbstractOptogenetics allows precise, fast and reversible intervention in biological processes. Light-sheet microscopy allows observation of the full course of embryonic development from egg to larva. Bringing the two approaches together allows unparalleled precision into the temporal regulation of signaling pathways and cellular processes in vivo. To develop this method, we investigated the regulation of canonical Wnt signaling during anterior-posterior patterning of the Drosophila embryonic epidermis. Cryptochrome 2 (CRY2) from Arabidopsis Thaliana was fused to mCherry fluorescent protein and Drosophila β–catenin to form an easy to visualize optogenetic switch. Blue light illumination caused oligomerization of the fusion protein and inhibited downstream Wnt signaling in vitro and in vivo. Temporal inactivation of β–catenin confirmed that Wnt signaling is required not only for Drosophila pattern formation, but also for maintenance later in development. We anticipate that this method will be easily extendable to other developmental signaling pathways and many other experimental systems.

scholarly journals Modulation of Calretinin Expression in Human Mesothelioma Cells Reveals the Implication of the FAK and Wnt Signaling Pathways in Conferring Chemoresistance towards Cisplatin

2019 ◽  
Vol 20 (21) ◽  
pp. 5391 ◽  
Author(s):  
Wörthmüller ◽  
Salicio ◽  
Oberson ◽  
Blum ◽  
Schwaller
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Expression System ◽  
Single Agent ◽  
Wnt Signaling Pathway ◽  
Tumor Formation ◽  
Mesenchymal Transition

Malignant mesothelioma (MM) is an aggressive asbestos-linked neoplasm, characterized by dysregulation of signaling pathways. Due to intrinsic or acquired chemoresistance, MM treatment options remain limited. Calretinin is a Ca2+-binding protein expressed during MM tumorigenesis that activates the FAK signaling pathway, promoting invasion and epithelial-to-mesenchymal transition. Constitutive calretinin downregulation decreases MM cells’ growth and survival, and impairs tumor formation in vivo. In order to evaluate early molecular events occurring during calretinin downregulation, we generated a tightly controlled IPTG-inducible expression system to modulate calretinin levels in vitro. Calretinin downregulation significantly reduced viability and proliferation of MM cells, attenuated FAK signaling and reduced the invasive phenotype of surviving cells. Importantly, surviving cells showed a higher resistance to cisplatin due to increased Wnt signaling. This resistance was abrogated by the Wnt signaling pathway inhibitor 3289-8625. In various MM cell lines and regardless of calretinin expression levels, blocking of FAK signaling activated the Wnt signaling pathway and vice versa. Thus, blocking both pathways had the strongest impact on MM cell proliferation and survival. Chemoresistance mechanisms in MM cells have resulted in a failure of single-agent therapies. Targeting of multiple components of key signaling pathways, including Wnt signaling, might be the future method-of-choice to treat MM.

scholarly journals Garcinol Regulates EMT and Wnt Signaling Pathways In Vitro and In Vivo, Leading to Anticancer Activity against Breast Cancer Cells

2012 ◽  
Vol 11 (10) ◽  
pp. 2193-2201 ◽  
Author(s):  
Aamir Ahmad ◽  
Sanila H. Sarkar ◽  
Bassam Bitar ◽  
Shadan Ali ◽  
Amro Aboukameel ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Wnt Signaling ◽  
Anticancer Activity ◽  
Cancer Cells ◽  
Signaling Pathways ◽  
Breast Cancer Cells

scholarly journals The Extracellular Domain of Lrp5/6 Inhibits Noncanonical Wnt Signaling In Vivo

2009 ◽  
Vol 20 (3) ◽  
pp. 924-936 ◽  
Author(s):  
Vitezslav Bryja ◽  
Emma R. Andersson ◽  
Alexandra Schambony ◽  
Milan Esner ◽  
Lenka Bryjová ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathways ◽  
Extracellular Domain ◽  
Convergent Extension ◽  
Mouse Development ◽  
Downstream Target ◽  
Extracellular Domains ◽  
Antisense Morpholino

Lrp5/6 are crucial coreceptors for Wnt/β-catenin signaling, a pathway biochemically distinct from noncanonical Wnt signaling pathways. Here, we examined the possible participation of Lrp5/6 in noncanonical Wnt signaling. We found that Lrp6 physically interacts with Wnt5a, but that this does not lead to phosphorylation of Lrp6 or activation of the Wnt/β-catenin pathway. Overexpression of Lrp6 blocks activation of the Wnt5a downstream target Rac1, and this effect is dependent on intact Lrp6 extracellular domains. These results suggested that the extracellular domain of Lrp6 inhibits noncanonical Wnt signaling in vitro. In vivo, Lrp6−/− mice exhibited exencephaly and a heart phenotype. Surprisingly, these defects were rescued by deletion of Wnt5a, indicating that the phenotypes resulted from noncanonical Wnt gain-of-function. Similarly, Lrp5 and Lrp6 antisense morpholino-treated Xenopus embryos exhibited convergent extension and heart phenotypes that were rescued by knockdown of noncanonical XWnt5a and XWnt11. Thus, we provide evidence that the extracellular domains of Lrp5/6 behave as physiologically relevant inhibitors of noncanonical Wnt signaling during Xenopus and mouse development in vivo.

scholarly journals Heparin-Mimicking Polymer-Based In Vitro Platform Recapitulates In Vivo Muscle Atrophy Phenotypes

2021 ◽  
Vol 22 (5) ◽  
pp. 2488
Author(s):  
Hyunbum Kim ◽  
Ji Hoon Jeong ◽  
Mona Fendereski ◽  
Hyo-Shin Lee ◽  
Da Yeon Kang ◽  
...  
Keyword(s):  
Cell Culture ◽  
Wnt Signaling ◽  
Signaling Pathways ◽  
Muscle Atrophy ◽  
Skeletal Muscle Regeneration ◽  
In Vitro Cell Culture ◽  
Gene And Protein Expression ◽  
Cell Cell

The cell–cell/cell–matrix interactions between myoblasts and their extracellular microenvironment have been shown to play a crucial role in the regulation of in vitro myogenic differentiation and in vivo skeletal muscle regeneration. In this study, by harnessing the heparin-mimicking polymer, poly(sodium-4-styrenesulfonate) (PSS), which has a negatively charged surface, we engineered an in vitro cell culture platform for the purpose of recapitulating in vivo muscle atrophy-like phenotypes. Our initial findings showed that heparin-mimicking moieties inhibited the fusion of mononucleated myoblasts into multinucleated myotubes, as indicated by the decreased gene and protein expression levels of myogenic factors, myotube fusion-related markers, and focal adhesion kinase (FAK). We further elucidated the underlying molecular mechanism via transcriptome analyses, observing that the insulin/PI3K/mTOR and Wnt signaling pathways were significantly downregulated by heparin-mimicking moieties through the inhibition of FAK/Cav3. Taken together, the easy-to-adapt heparin-mimicking polymer-based in vitro cell culture platform could be an attractive platform for potential applications in drug screening, providing clear readouts of changes in insulin/PI3K/mTOR and Wnt signaling pathways.

scholarly journals Enhanced in vivo-imaging in fish by optimized anaesthesia, fluorescent protein selection and removal of pigmentation

2018 ◽  
Author(s):  
Colin Q. Lischik ◽  
Leonie Adelmann ◽  
Joachim Wittbrodt
Keyword(s):  
Fluorescent Protein ◽  
Light Sheet ◽  
Time Lapse ◽  
Light Sheet Microscopy ◽  
Inner Organs ◽  
Traditional Approaches ◽  
Time Lapse Imaging ◽  
Targeted Inactivation ◽  
Imaging Conditions

AbstractFish are ideally suited for in vivo-imaging due to their transparency at early stages combined with a large genetic toolbox. Key challenges to further advance imaging are fluorophore selection, immobilization of the specimen and approaches to eliminate pigmentation.We addressed all three and identified the fluorophores and anaesthesia of choice by high throughput time-lapse imaging. Our results indicate that eGFP and mCherry are the best conservative choices for in vivo-fluorescence experiments, when availability of well-established antibodies and nanobodies matters. Still, mVenusNB and mGFPmut2 delivered highest absolute fluorescence intensities in vivo. Immobilization is of key importance during extended in vivo imaging. Here, traditional approaches are outperformed by mRNA injection of α-Bungarotoxin which allows a complete and reversible, transient immobilization. In combination with fully transparent juvenile and adult fish established by the targeted inactivation of both, oca2 and pnp4a via CRISPR/Cas9-mediated gene editing in medaka we could dramatically improve the state-of-the art imaging conditions in post-embryonic fish, now enabling light-sheet microscopy of the growing retina, brain, gills and inner organs in the absence of side effects caused by anaesthetic drugs or pigmentation.

scholarly journals An actin-based protrusion originating from a podosome-enriched region initiates macrophage fusion

2019 ◽  
Vol 30 (17) ◽  
pp. 2254-2267 ◽  
Author(s):  
James J. Faust ◽  
Arnat Balabiyev ◽  
John M. Heddleston ◽  
Nataly P. Podolnikova ◽  
D. Page Baluch ◽  
...  
Keyword(s):  
Phase Contrast ◽  
Cytochalasin B ◽  
Inflammatory Diseases ◽  
Leading Edge ◽  
Light Sheet ◽  
Giant Cells ◽  
Light Sheet Microscopy ◽  
Syndrome Protein

Macrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating this process is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase-contrast video microscopy demonstrated that in the majority of events, short protrusions (∼3 µm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (∼12 µm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP- and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data suggest that an actin-based protrusion formed at the leading edge initiates macrophage fusion.

scholarly journals An actin-based protrusion originating from a podosome-enriched region initiates macrophage fusion

2019 ◽  
Author(s):  
James J. Faust ◽  
Arnat Balabiyev ◽  
John M. Heddleston ◽  
Nataly P. Podolnikova ◽  
D. Page Baluch ◽  
...  
Keyword(s):  
Phase Contrast ◽  
Cytochalasin B ◽  
Inflammatory Diseases ◽  
Leading Edge ◽  
Light Sheet ◽  
Giant Cells ◽  
Light Sheet Microscopy ◽  
Syndrome Protein

AbstractMacrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating macrophage fusion is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase contrast video microscopy demonstrated that in the majority of events, short protrusions (3 ± 1 μm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (16 ± 7 μm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent actin-based protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP- and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data indicate that an actin-based protrusion formed at the leading edge initiates macrophage fusion.

scholarly journals Leptin-Induced Angiogenesis of EA.Hy926 Endothelial Cells via the Akt and Wnt Signaling Pathways In Vitro and In Vivo

2019 ◽  
Vol 10 ◽  
Author(s):  
Fei Yu ◽  
Runqing Fu ◽  
Lu Liu ◽  
Xiaoting Wang ◽  
Tingting Wu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Wnt Signaling ◽  
Signaling Pathways

scholarly journals Ex vivolive cell tracking in kidney organoids using light sheet fluorescence microscopy

2017 ◽  
Author(s):  
Marie Held ◽  
Ilaria Santeramo ◽  
Bettina Wilm ◽  
Patricia Murray ◽  
Raphaël Lévy
Keyword(s):  
Fluorescence Microscopy ◽  
Time Course ◽  
Cell Tracking ◽  
Organic Anion ◽  
Light Sheet ◽  
Differentiation Potential ◽  
Light Sheet Microscopy ◽  
Kidney Organoids

AbstractScreening cells for their differentiation potential requires a combination of tissue culture models and imaging methods that allow for long-term tracking of the location and function of cells. Embryonic kidney re-aggregationin vitroassays have been established which allow for the monitoring of organotypic cell behaviour in re-aggregated and chimeric renal organoids. However, evaluation of cell integration is hampered by the high photonic load of standard fluorescence microscopy which poses challenges for imaging three-dimensional systems in real-time over a time course. Therefore, we employed light sheet microscopy, a technique that vastly reduces photobleaching and phototoxic effects. We have also developed a new method for culturing the re-aggregates which involves immersed culture, generating organoids which more closely reflect developmentin vivo. To facilitate imaging from various angles, we embedded the organoids in a freely rotatable hydrogel cylinder. Endpoint fixing and staining were performed to provide additional biomolecular information. We succeeded in imaging labelled cells within re-aggregated kidney organoids over 15 hours and tracking their fate while simultaneously monitoring the development of organotypic morphological structures. Our results show that Wt1-expressing embryonic kidney cells obtained from transgenic mice could integrate into re-aggregated chimeric kidney organoids and contribute to developing nephrons. Furthermore, the nascent proximal tubules that formed in the re-aggregated tissues using the new culture method displayed secretory function, as evidenced by their ability to secrete an organic anion mimic into the tubular lumen.

scholarly journals Mechanism of CK2.3, a Novel Mimetic Peptide of Bone Morphogenetic Protein Receptor Type IA, Mediated Osteogenesis

2019 ◽  
Vol 20 (10) ◽  
pp. 2500 ◽  
Author(s):  
Vrathasha Vrathasha ◽  
Hilary Weidner ◽  
Anja Nohe
Keyword(s):  
Signaling Pathways ◽  
Treatment Options ◽  
Time Frame ◽  
Bmp Signaling ◽  
C2c12 Cells ◽  
Molecular Sequence ◽  
Sequence Of Events ◽  
Protein Receptor

Background: Osteoporosis is a degenerative skeletal disease with a limited number of treatment options. CK2.3, a novel peptide, may be a potential therapeutic. It induces osteogenesis and bone formation in vitro and in vivo by acting downstream of BMPRIA through releasing CK2 from the receptor. However, the detailed signaling pathways, the time frame of signaling, and genes activated remain largely unknown. Methods: Using a newly developed fluorescent CK2.3 analog, specific inhibitors for the BMP signaling pathways, Western blot, and RT-qPCR, we determined the mechanism of CK2.3 in C2C12 cells. We then confirmed the results in primary BMSCs. Results: Using these methods, we showed that CK2.3 stimulation activated OSX, ALP, and OCN. CK2.3 stimulation induced time dependent release of CK2β from BMPRIA and concurrently CK2.3 colocalized with CK2α. Furthermore, CK2.3 induced BMP signaling depends on ERK1/2 and Smad1/5/8 signaling pathways. Conclusion: CK2.3 is a novel peptide that drives osteogenesis, and we detailed the molecular sequence of events that are triggered from the stimulation of CK2.3 until the induction of mineralization. This knowledge can be applied in the development of future therapeutics for osteoporosis.

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