TY - JOUR
T1 - Detection of Ras nanoclustering-dependent homo-FRET using fluorescence anisotropy measurements
AU - babu Manoharan, Ganesh
AU - Guzmán, Camilo
AU - Najumudeen, Arafath Kaja
AU - Abankwa, Daniel
N1 - Funding Information:
The study was supported by internal funds of the University of Luxembourg , Luxembourg, the Academy of Finland and the Sigrid Juselius Foundation , Finland. We are thankful to Dominic Esposito and Vanessa Wall (Frederick National Laboratory for Cancer Research, Frederick, MD, United States) for providing the entry clones and destination vectors for multisite gateway cloning. We thank Mrs. Nesrine Ben Fredj and Mrs. Christina Laurini for the technical assistance.
Funding Information:
The study was supported by internal funds of the University of Luxembourg, Luxembourg, the Academy of Finland and the Sigrid Juselius Foundation, Finland. We are thankful to Dominic Esposito and Vanessa Wall (Frederick National Laboratory for Cancer Research, Frederick, MD, United States) for providing the entry clones and destination vectors for multisite gateway cloning. We thank Mrs. Nesrine Ben Fredj and Mrs. Christina Laurini for the technical assistance.
Publisher Copyright:
© 2023 The Authors
PY - 2023/6
Y1 - 2023/6
N2 - The small GTPase Ras is frequently mutated in cancer and a driver of tumorigenesis. The recent years have shown great progress in drug-targeting Ras and understanding how it operates on the plasma membrane. We now know that Ras is non-randomly organized into proteo-lipid complexes on the membrane, called nanoclusters. Nanoclusters contain only a few Ras proteins and are necessary for the recruitment of downstream effectors, such as Raf. If tagged with fluorescent proteins, the dense packing of Ras in nanoclusters can be analyzed by Förster/ fluorescence resonance energy transfer (FRET). Loss of FRET can therefore report on decreased nanoclustering and any process upstream of it, such as Ras lipid modifications and correct trafficking. Thus, cellular FRET screens employing Ras-derived fluorescence biosensors are potentially powerful tools to discover chemical or genetic modulators of functional Ras membrane organization. Here we implement fluorescence anisotropy-based homo-FRET measurements of Ras-derived constructs labelled with only one fluorescent protein on a confocal microscope and a fluorescence plate reader. We show that homo-FRET of both H-Ras- and K-Ras-derived constructs can sensitively report on Ras-lipidation and -trafficking inhibitors, as well as on genetic perturbations of proteins regulating membrane anchorage. By exploiting the switch I/II-binding Ras-dimerizing compound BI-2852, this assay is also suitable to report on the engagement of the K-Ras switch II pocket by small molecules such as AMG 510. Given that homo-FRET only requires one fluorescent protein tagged Ras construct, this approach has significant advantages to create Ras-nanoclustering FRET-biosensor reporter cell lines, as compared to the more common hetero-FRET approaches.
AB - The small GTPase Ras is frequently mutated in cancer and a driver of tumorigenesis. The recent years have shown great progress in drug-targeting Ras and understanding how it operates on the plasma membrane. We now know that Ras is non-randomly organized into proteo-lipid complexes on the membrane, called nanoclusters. Nanoclusters contain only a few Ras proteins and are necessary for the recruitment of downstream effectors, such as Raf. If tagged with fluorescent proteins, the dense packing of Ras in nanoclusters can be analyzed by Förster/ fluorescence resonance energy transfer (FRET). Loss of FRET can therefore report on decreased nanoclustering and any process upstream of it, such as Ras lipid modifications and correct trafficking. Thus, cellular FRET screens employing Ras-derived fluorescence biosensors are potentially powerful tools to discover chemical or genetic modulators of functional Ras membrane organization. Here we implement fluorescence anisotropy-based homo-FRET measurements of Ras-derived constructs labelled with only one fluorescent protein on a confocal microscope and a fluorescence plate reader. We show that homo-FRET of both H-Ras- and K-Ras-derived constructs can sensitively report on Ras-lipidation and -trafficking inhibitors, as well as on genetic perturbations of proteins regulating membrane anchorage. By exploiting the switch I/II-binding Ras-dimerizing compound BI-2852, this assay is also suitable to report on the engagement of the K-Ras switch II pocket by small molecules such as AMG 510. Given that homo-FRET only requires one fluorescent protein tagged Ras construct, this approach has significant advantages to create Ras-nanoclustering FRET-biosensor reporter cell lines, as compared to the more common hetero-FRET approaches.
KW - Anisotropy
KW - Drug discovery
KW - Homo-FRET
KW - Nanoclustering
KW - Ras
UR - http://www.scopus.com/inward/record.url?scp=85152128455&partnerID=8YFLogxK
UR - https://pubmed.ncbi.nlm.nih.gov/37058825
U2 - 10.1016/j.ejcb.2023.151314
DO - 10.1016/j.ejcb.2023.151314
M3 - Article
C2 - 37058825
AN - SCOPUS:85152128455
SN - 0171-9335
VL - 102
JO - European Journal of Cell Biology
JF - European Journal of Cell Biology
IS - 2
M1 - 151314
ER -