A RhoA-FRET Biosensor Mouse for Intravital Imaging in Normal Tissue Homeostasis and Disease Contexts

Max Nobis; David Herrmann; Sean C Warren; Shereen Kadir; Wilfred Leung; Monica Killen; Astrid Magenau; David Stevenson; Morghan C Lucas; Nadine Reischmann; Claire Vennin; James R W Conway; Alice Boulghourjian; Anaiis Zaratzian; Andrew M Law; David Gallego-Ortega; Christopher J Ormandy; Stacey N Walters; Shane T Grey; Jacqueline Bailey; Tatyana Chtanova; Julian M W Quinn; Paul A Baldock; Peter I Croucher; Juliane P Schwarz; Agata Mrowinska; Lei Zhang; Herbert Herzog; Andrius Masedunskas; Edna C Hardeman; Peter W Gunning; Gonzalo Del Monte-Nieto; Richard P Harvey; Michael S Samuel; Marina Pajic; Ewan J McGhee; Anna-Karin E Johnsson; Owen J Sansom; Heidi C E Welch; Jennifer P Morton; Douglas Strathdee; Kurt I Anderson; Paul Timpson

doi:10.1016/j.celrep.2017.09.022

A RhoA-FRET Biosensor Mouse for Intravital Imaging in Normal Tissue Homeostasis and Disease Contexts

Cell Rep. 2017 Oct 3;21(1):274-288. doi: 10.1016/j.celrep.2017.09.022.

Authors

Max Nobis¹, David Herrmann¹, Sean C Warren¹, Shereen Kadir², Wilfred Leung¹, Monica Killen¹, Astrid Magenau¹, David Stevenson², Morghan C Lucas¹, Nadine Reischmann¹, Claire Vennin¹, James R W Conway¹, Alice Boulghourjian¹, Anaiis Zaratzian¹, Andrew M Law¹, David Gallego-Ortega¹, Christopher J Ormandy¹, Stacey N Walters¹, Shane T Grey¹, Jacqueline Bailey¹, Tatyana Chtanova¹, Julian M W Quinn¹, Paul A Baldock¹, Peter I Croucher¹, Juliane P Schwarz², Agata Mrowinska², Lei Zhang¹, Herbert Herzog¹, Andrius Masedunskas³, Edna C Hardeman⁴, Peter W Gunning⁵, Gonzalo Del Monte-Nieto⁶, Richard P Harvey⁶, Michael S Samuel⁷, Marina Pajic¹, Ewan J McGhee², Anna-Karin E Johnsson⁸, Owen J Sansom², Heidi C E Welch⁸, Jennifer P Morton², Douglas Strathdee², Kurt I Anderson⁹, Paul Timpson¹⁰

Affiliations

¹ The Garvan Institute of Medical Research, St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW 2010, Australia.
² Cancer Research UK Beatson Institute, Switchback Road, Bearsden, Glasgow G611BD, UK.
³ Neuromuscular and Regenerative Medicine Unit, University of New South Wales, Sydney, NSW 2010, Australia; Oncology Research Unit, School of Medical Sciences, University of New South Wales, Sydney, NSW 2010, Australia.
⁴ Neuromuscular and Regenerative Medicine Unit, University of New South Wales, Sydney, NSW 2010, Australia.
⁵ Oncology Research Unit, School of Medical Sciences, University of New South Wales, Sydney, NSW 2010, Australia.
⁶ Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Sydney, NSW 2010, Australia; St. Vincent's Clinical School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
⁷ Centre for Cancer Biology, SA Pathology and University of South Australia School of Medicine, University of Adelaide, Adelaide, SA 5000, Australia.
⁸ Signalling Programme, Babraham Institute, Cambridge CB223AT, UK.
⁹ Francis Crick Institute, London NW11AT, UK. Electronic address: [email protected].
¹⁰ The Garvan Institute of Medical Research, St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW 2010, Australia. Electronic address: [email protected].

PMID: 28978480
DOI: 10.1016/j.celrep.2017.09.022

Abstract

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time.

Keywords: FLIM-FRET; actin; biosensors; breast cancer; cell biology; development; immunology; intravital imaging; pancreatic cancer; small GTPase RhoA.

MeSH terms

Animals
Antineoplastic Agents / pharmacology
Biosensing Techniques*
Bone and Bones / cytology
Bone and Bones / metabolism
Cell Movement / drug effects
Dasatinib / pharmacology
Erlotinib Hydrochloride / pharmacology
Female
Fluorescence Resonance Energy Transfer / instrumentation
Fluorescence Resonance Energy Transfer / methods*
Gene Expression Regulation
Intestine, Small / metabolism
Intestine, Small / ultrastructure
Intravital Microscopy / instrumentation
Intravital Microscopy / methods*
Mammary Glands, Animal / blood supply
Mammary Glands, Animal / drug effects
Mammary Glands, Animal / ultrastructure
Mammary Neoplasms, Experimental / blood supply
Mammary Neoplasms, Experimental / drug therapy
Mammary Neoplasms, Experimental / genetics
Mammary Neoplasms, Experimental / ultrastructure
Mechanotransduction, Cellular
Mice
Mice, Transgenic
Neutrophils / metabolism
Neutrophils / ultrastructure
Osteocytes / metabolism
Osteocytes / ultrastructure
Pancreatic Neoplasms / blood supply
Pancreatic Neoplasms / drug therapy
Pancreatic Neoplasms / genetics
Pancreatic Neoplasms / ultrastructure
Time-Lapse Imaging / instrumentation
Time-Lapse Imaging / methods*
rho GTP-Binding Proteins / genetics*
rho GTP-Binding Proteins / metabolism
rhoA GTP-Binding Protein

Substances

Antineoplastic Agents
Erlotinib Hydrochloride
RhoA protein, mouse
rho GTP-Binding Proteins
rhoA GTP-Binding Protein
Dasatinib

Abstract

MeSH terms

Substances

Grants and funding