B2 Book chapter

FLIM-FRET Analysis of Ras Nanoclustering and Membrane-Anchorage




List of Authors: Parkkola Hanna, Siddiqui Farid Ahmad, Oetken-Lindholm Christina, Abankwa Daniel

Publisher: Humana Press Inc.

Publication year: 2021

Book title *: Ras Activity and Signaling: Methods and Protocols

Journal name in source: Methods in Molecular Biology

Title of series: Methods in Molecular Biology

Volume number: 2262

ISBN: 978-1-0716-1189-0

eISBN: 978-1-0716-1190-6

ISSN: 1064-3745

DOI: http://dx.doi.org/10.1007/978-1-0716-1190-6_13


Abstract

On the plasma membrane, Ras is organized into laterally segregated proteo-lipid complexes called nanoclusters. The extent of Ras nanoclustering correlates with its signaling output, positioning nanocluster as dynamic signaling gain modulators. Recent evidence suggests that stacked dimers of Ras and Raf are elemental units at least of one type of Ras nanocluster. However, it is still incompletely understood, in which physiological contexts nanoclustering is regulated and which constituents are parts of nanocluster. Nonetheless, disruption of nanoclustering faithfully diminishes Ras activity in cells, suggesting Ras nanocluster as potential drug targets.

While there are several methods available to study Ras nanocluster, fluorescence or Förster resonance energy transfer (FRET) between fluorescently labeled, nanoclustered Ras proteins is a relatively simple readout. FRET measurements using fluorescence lifetime imaging microscopy (FLIM) have proven to be robust and sensitive to determine Ras nanoclustering changes. Loss of FRET that emerges due to nanoclustering reports on all processes upstream of Ras nanoclustering, i.e., also on proper trafficking or lipid modification of Ras. Here we report our standard FLIM-FRET protocol to measure nanoclustering-dependent FRET of Ras in mammalian cells. Importantly, nanoclustering-dependent FRET is one of the few methods that can detect differences between the Ras isoforms.


Last updated on 2021-24-06 at 09:31