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

Author

Nobis, M, Herrmann, D, Warren, SC, Kadir, S, Leung, W, Killen, M, Magenau, A, Stevenson, D, Lucas, MC, Reischmann, N, Vennin, C, Conway, JRW, Boulghourjian, A, Zaratzian, A, Law, AM, Gallego-Ortega, D, Ormandy, CJ, Walters, SN, Grey, ST, Bailey, J, Chtanova, T, Quinn, JMW, Baldock, PA, Croucher, PI, Schwarz, JP, Mrowinska, A, Zhang, L, Herzog, H, Masedunskas, A, Hardeman, EC, Gunning, PW, Del Monte-Nieto, G, Harvey, RP, Samuel, MS, Pajic, M, McGhee, EJ, Johnsson, A-KE, Sansom, OJ, Welch, HCE, Morton, JP, Strathdee, D, Anderson, KI, Timpson, P, Nobis, M, Herrmann, D, Warren, SC, Kadir, S, Leung, W, Killen, M, Magenau, A, Stevenson, D, Lucas, MC, Reischmann, N, Vennin, C, Conway, JRW, Boulghourjian, A, Zaratzian, A, Law, AM, Gallego-Ortega, D, Ormandy, CJ, Walters, SN, Grey, ST, Bailey, J, Chtanova, T, Quinn, JMW, Baldock, PA, Croucher, PI, Schwarz, JP, Mrowinska, A, Zhang, L, Herzog, H, Masedunskas, A, Hardeman, EC, Gunning, PW, Del Monte-Nieto, G, Harvey, RP, Samuel, MS, Pajic, M, McGhee, EJ, Johnsson, A-KE, Sansom, OJ, Welch, HCE, Morton, JP, Strathdee, D, Anderson, KI, and Timpson, P

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.

Published
2017

2. Intravital FRAP Imaging using an E-cadherin-GFP Mouse Reveals Disease- and Drug-Dependent Dynamic Regulation of Cell-Cell Junctions in Live Tissue.

Author

Erami, Z, Herrmann, D, Warren, SC, Nobis, M, McGhee, EJ, Lucas, MC, Leung, W, Reischmann, N, Mrowinska, A, Schwarz, JP, Kadir, S, Conway, JRW, Vennin, C, Karim, SA, Campbell, AD, Gallego-Ortega, D, Magenau, A, Murphy, KJ, Ridgway, RA, Law, AM, Walters, SN, Grey, ST, Croucher, DR, Zhang, L, Herzog, H, Hardeman, EC, Gunning, PW, Ormandy, CJ, Evans, TRJ, Strathdee, D, Sansom, OJ, Morton, JP, Anderson, KI, Timpson, P, Erami, Z, Herrmann, D, Warren, SC, Nobis, M, McGhee, EJ, Lucas, MC, Leung, W, Reischmann, N, Mrowinska, A, Schwarz, JP, Kadir, S, Conway, JRW, Vennin, C, Karim, SA, Campbell, AD, Gallego-Ortega, D, Magenau, A, Murphy, KJ, Ridgway, RA, Law, AM, Walters, SN, Grey, ST, Croucher, DR, Zhang, L, Herzog, H, Hardeman, EC, Gunning, PW, Ormandy, CJ, Evans, TRJ, Strathdee, D, Sansom, OJ, Morton, JP, Anderson, KI, and Timpson, P

Abstract

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments.

Published
2016

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

Author

Nobis M, Herrmann D, Warren SC, Kadir S, Leung W, Killen M, Magenau A, Stevenson D, Lucas MC, Reischmann N, Vennin C, Conway JRW, Boulghourjian A, Zaratzian A, Law AM, Gallego-Ortega D, Ormandy CJ, Walters SN, Grey ST, Bailey J, Chtanova T, Quinn JMW, Baldock PA, Croucher PI, Schwarz JP, Mrowinska A, Zhang L, Herzog H, Masedunskas A, Hardeman EC, Gunning PW, Del Monte-Nieto G, Harvey RP, Samuel MS, Pajic M, McGhee EJ, Johnsson AE, Sansom OJ, Welch HCE, Morton JP, Strathdee D, Anderson KI, and Timpson P

Subjects
Animals, Antineoplastic Agents pharmacology, Bone and Bones cytology, Bone and Bones metabolism, Cell Movement drug effects, Dasatinib pharmacology, Erlotinib Hydrochloride pharmacology, Female, Fluorescence Resonance Energy Transfer instrumentation, Gene Expression Regulation, Intestine, Small metabolism, Intestine, Small ultrastructure, Intravital Microscopy instrumentation, 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, rho GTP-Binding Proteins metabolism, rhoA GTP-Binding Protein, Biosensing Techniques, Fluorescence Resonance Energy Transfer methods, Intravital Microscopy methods, Time-Lapse Imaging methods, rho GTP-Binding Proteins genetics
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., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Published
2017
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4. Intravital FRAP Imaging using an E-cadherin-GFP Mouse Reveals Disease- and Drug-Dependent Dynamic Regulation of Cell-Cell Junctions in Live Tissue.

Author

Erami Z, Herrmann D, Warren SC, Nobis M, McGhee EJ, Lucas MC, Leung W, Reischmann N, Mrowinska A, Schwarz JP, Kadir S, Conway JRW, Vennin C, Karim SA, Campbell AD, Gallego-Ortega D, Magenau A, Murphy KJ, Ridgway RA, Law AM, Walters SN, Grey ST, Croucher DR, Zhang L, Herzog H, Hardeman EC, Gunning PW, Ormandy CJ, Evans TRJ, Strathdee D, Sansom OJ, Morton JP, Anderson KI, and Timpson P

Subjects
Animals, Cadherins genetics, Green Fluorescent Proteins genetics, Mice, Mice, Transgenic, Neoplasms, Experimental genetics, Organ Specificity, Proto-Oncogene Proteins p21(ras) genetics, Proto-Oncogene Proteins p21(ras) metabolism, Tumor Suppressor Protein p53 genetics, Tumor Suppressor Protein p53 metabolism, Cadherins metabolism, Green Fluorescent Proteins metabolism, Neoplasms, Experimental metabolism, Neoplasms, Experimental pathology, Optical Imaging methods, Tumor Microenvironment
Abstract

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Published
2016
Full Text
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