Number and brightness analysis of sFRP4 domains in live cells demonstrates vesicle association signal of the NLD domain and dynamic intracellular responses to Wnt3a |
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| Institution: | 1. Molecular Pharmacology Laboratory, School of Pharmacy, CHIRI Biosciences Research Precinct, Curtin University, Western Australia, Australia;2. School of Biomedical Sciences, CHIRI Biosciences Research Precinct, Curtin University, Western Australia, Australia;3. Centre for Environmental Risk Assessment and Remediation, University of South Australia, South Australia, Australia;4. Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, USA;1. Department of Biochemistry and Molecular Medicine, University of California at Davis, School of Medicine, Sacramento, CA 95817, USA;2. Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children-Northern California, UC Davis School of Medicine, Sacramento, CA 95817, USA;1. Department of Infectious Diseases, The Second Affiliated Hospital of Medical School of Xi''an Jiaotong University, Xi''an, Shaanxi 710004, China;2. Department of Infectious Diseases, Tangdu Hospital, The Fourth Military Medical University, Xi''an, Shaanxi 710038, China;3. Department of Gastroenterology Diseases, Xijing Hospital, The Fourth Military Medical University, Xi''an, Shaanxi 710032, China;4. Department of Respiratory Diseases, The Second Affiliated Hospital of Medical School of Xi''an Jiaotong University, Xi''an, Shaanxi 710004, China;1. Medical College of Xi''an Jiao Tong University, Xi''an 710061, China;2. Department of Urology, Shaanxi Provincial People''s Hospital, The Third Affiliated Hospital of Xi''an Jiaotong University, Xi''an 710068, China;3. Department of Ophthalmology, Shaanxi Provincial People''s Hospital, The Third Affiliated Hospital of Xi''an Jiaotong University, Xi''an 710068, China;4. Department of Urology, The First Affiliated Hospital of Xi''an Jiaotong University, Xi''an 710061, China;1. Department of Biotechnology, Sri Ramachandra Institute of Higher Education & Research (Deemed to be University), Chennai, India;2. Department of Integrative Biology, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore, India;3. Department of Pathology, Sri Ramachandra Institute of Higher Education & Research (Deemed to be University), Chennai, India |
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| Abstract: | The Wnts are secreted, lipidated glycoproteins that play a role in cellular processes of differentiation, proliferation, migration, survival, polarity and stem cell self-renewal. The majority of Wnts biological effects are through binding to specific frizzled (Fzd) receptor complexes leading to activation of downstream pathways. Secreted frizzled-related proteins (sFRPs) were first identified as antagonists of Wnt signalling by binding directly to Wnts. They comprise two domains, a Fzd-like cysteine rich domain (CRD) and a netrin-like domain (NLD). Subsequently sFRPs have been shown to also interact with Fzd receptors and more diverse functions have been identified, including potentiation of Wnt signalling. Many aspects of the biology of this family remain to be elucidated. We used the number and brightness (N&B) method, a technique based on fluorescence fluctuation analysis, to characterise the intracellular aggregation and trafficking of sFRP4 domains. We expressed sFRP4 and its’ domains as EGFP fusions and then characterised the effect of endogenous Wnt3a by fluorescence confocal imaging. We observed vesicular trafficking of sFRP4 and that the NLD domain has a vesicular association signal. We found that sFRP4 and the CRD formed oligomeric aggregates in the perinuclear region while the NLD was distributed evenly throughout the cell with a larger proportion of aggregates. Most significantly we observed intracellular redistribution of sFRP4 in response to Wnt3a suggesting that Wnt3a can modulate intracellular localisation and secretion of sFRP4. Our results reveal a number of novel findings regarding sFRP4 which are likely to have relevance to this wider family. |
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| Keywords: | Wnt sFRP Cysteine-rich domain Netrin-like domain Protein aggregates Live cell imaging |
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