Ras GTPases are small lipid-anchored proteins that are associated predominantly with the inner leaflet of the plasma membrane. Ras proteins must be anchored to cell membranes for biological activity. These lipid anchors are attached via a series of post-translational modifications. Ras GTPases function as molecular switches on the inner leaflet of the plasma membrane, conveying extracellular signals to the cell interior. It exists in three isoforms H-N-, and K-Ras which are ubiquitously expressed in mammalian cells.
About 40% of Ras proteins are organized into nanoscale domains called nanoclusters (1). Nanoclusters comprise of about 7 Ras proteins and have radii in the range of 6-11 nm. Ras proteins that are not in nanoclusters are randomly distributed as monomers over the plasma membrane. Different Ras isoforms drive the formation of spatially distinct nanoclusters, which have varying requirements for plasma membrane cholesterol and the actin cytoskeleton. These nanoscale domains are thought to function as dynamic and transient signaling platforms. These become sites for the scaffolding of the components of the signaling module, activating the signaling pathway via generation of a digital output which delivers high fidelity signal transmission across the plasma membrane.
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It is demonstrated by Shalom-Feuerstein et al (2) that Ras-GTP nanoclusters are the sites of Raf/MEK and ERK recruitment to the plasma membrane. Ras GTPases nanoscale domains' dynamic nature ranges over multiple length and time scales causing a non-random distribution of proteins. It has been proposed that selective concentration of signalling proteins by proteolipid-based sorting to discrete areas of the cell membrane may increase the efficiency and specificity of signalling events and prevent cross talk between different pathways (3).
Proteins that stabilizes Ras nanoclusters
Nucleophosmin regulate cell proliferation and transformation and are overexpressed in multiple cancers. However, the physiological role of NPM in carcinogenesis remains controversial because it has been described as both an oncogene and a tumor suppressor. This nucleoli-bound phosphoprotein has housekeeping function in the cells with its most significant role being a molecular chaperone shuttling proteins between nucleus and cytoplasm. In acute myeloid leukemia (AML), about a third of its occurrence is caused by a mutation that caused an aberrant localization of NPM in the cytoplasm.
It is hypothesized that these cytoplasmic NPM mutant (NPMc) interacts with Ras GTPases in the nanoclusters thus stabilizes its formation and causes an increased and sustained activation of MAPK which subsequently leads to activation of prosurvival genes. Thus elucidating what affects the stability of Ras nanoclusters together with its prosurvival outcome in cells through MAPK activation, allows for a drug-able target contributing to AML therapy.
Recently, it is shown that ectopic expression of a scaffold protein galectin-1 increases the extent of H-Ras-GTP nanoclustering and also the radius of the nanoclusters (4, 5) whereas knockdown of galectin-1 expression significantly abrogates H-Ras-GTP nanoclustering (4). Modulation of H-Ras nanoclustering by galectin-1 levels also correlates closely with changes in H-Ras signal output (6-8). Interestingly, another galectin family member, galectin-3, predominantly interacts with activated K-Ras and increases Raf-1 and phosphoinositide 3-kinase activation (9).
Challenges in the measurements of Ras nanoclusters on the plasma membrane
Plasma membrane heterogeneity forms a complex environment for the measurement of protein-protein interactions and proved to be a major challenge. It is a dynamic structure which includes several thousand species of lipid, compartmentalization by a sub-membrane actin cytoskeleton, and the lateral assembly of sphingolipids and cholesterol into transient liquid-ordered domains. Secondly, Ras protein dynamics, organization and lifetime as nanoclusters on the membrane may vary. It is shown that the lifetime of Ras-GTP nanoclusters last longer (0.5-1 s) than inactive Ras-GDP clusters (<0.1 s) (5).
Bioimaging techniques in Ras nanoclusters measurements
Electron microscopy (EM) - It was first shown that Ras isoforms are segregated into spatially distinct domains on the plasma membrane via membrane anchors using electron microscopy (EM) of intact apical plasma membrane sheets followed by statistical analysis of the immunogold point patterns. What this technique managed to elucidate is that the H-Ras and K-Ras do not show any significant overlap in nanodomains localization and it also exhibit GTP-dependent lateral segregation on the plasma membrane. These suggest that these patterning could be a platform for signaling pathways (10, 11).
Single fluorophore video tracking (SVFT) - Only 20-40% of Ras proteins are in nanoclusters, the remainder is randomly arrayed over the plasma membrane (3). Further insights into Ras surface organization have been revealed using single fluorophore video tracking (SFVT) to analyze the diffusion and mobility of individual Ras proteins.
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Fluorescence recovery after photobleaching (FRAP) - Which components of the Ras protein are necessary for nanoclustering? Analysis of a large set of hypervariable regions (HVR) deletion and alanine substitution mutants of H-Ras in the GTP- and GDP-bound states using a combination of EM and fluorescence recovery after photobleaching (FRAP) has allowed the mapping of the critical determinants of H-Ras membrane affinity and lateral segregation.
Bimolecular fluorescence complementation (BiFC) - Hu et al uses BIFC to investigate the subcellular localization of H-Ras/Gal-1 complexes in live cells (12). Here yellow fluorescent protein (YFP) is split into two non-fluorescent fragments (the N-terminal YN and the C-terminal YC) that are fused to the protein of interest (YC-H-Ras) and its suspected binding partner (YN-Gal-1). Reconstitution of fluorescence occurs when the two fragments of the split fluorophore are brought together by protein-protein interactions.
Atomic Fluorescence Microscopy (AFM) - Studies of cholesterol-based mixtures have shown the existence of a state where lipids are tightly packed and ordered, but where the lateral diffusion process is almost as fast as in the classical liquid-disordered (Ld) state. The coexistence of this cholesterol-containing state known as the liquid-ordered (Lo) phase and the (Ld) phase has strongly stimulated current thinking about the physical characteristics of cell membrane domains. AFM imaging of full length N-Ras on three component lipid bilayers identifies a significant fraction of the protein at the interface of Lo and Ld regions of the membrane (13).
Fluorescence correlation spectroscopy (FCS) - FCS provides information about the density of the fluorescent probes and contrary to FRAP, deals solely with the diffusion occurring within the observation volume and requires very low probe concentration levels. It also gives a more-detailed quantitative kinetic data. An extension of FCS which allows for a two-color cross-correlation spectroscopy have been used to analyze, sort, and detect conformational states of a few or single molecules in the excitation volume.
Single molecule measurements
The study of biological systems is complex especially when standard measurements are needed in dynamic and heterogeneous states of the system. Single molecule measurements bypass the need to synchronize an otherwise complex reaction involving ensembles of molecules that moved stochastically resulting in an averaged behavior upon normalization. Examples range from enzyme reactions with proposed multiple conformational sub states and protein folding with multiple unfolded states, pathways, intermediates, and transition regions to interactions between cell surface receptors or more downstream components of signal transduction pathways.
Single-molecule methods also permit the observation of processes at extremely low molecular concentrations; for example, conformational properties of individual prion proteins under aggregation conditions may be studied. A very intriguing application of single-molecule detection is the simultaneous observation of transitions occurring in different parts of a system, allowing the direct evaluation of synergistic effects during biopolymer structural transitions, assembly, or enzyme catalysis. Finally, these methods may lead to significant technical advances in areas such as high-throughput screening and DNA sequencing.
The detection of fluorescence from single molecules involves repeated cycling of the molecule between ground and excited states and detection of the series of emitted photons. These fluorescence photon bursts are generated through the diffusion or flow of single molecules in a liquid traversing the laser excitation volume. Such bursts can be analyzed for their duration, brightness, spectrum, and fluorescence lifetime, thereby providing molecular information on identity, size, diffusion coefficient, and concentration.
These bursts are short (i.e. typically on a millisecond time scale) and provide little information on slower fluctuations. However, they can provide invaluable information about the distributions of molecular properties of interest, undisturbed by surface effects, and about changes in these distributions under non equilibrium conditions. Because a large number of events (photon bursts) can be collected in a relatively short time, statistical analyses of these data are possible, and histograms can be constructed. Most notably, subpopulations of molecules in heterogeneous ensembles can be identified (28-30), and the properties of these subpopulations can be individually investigated.
Fluorescence resonance energy transfer (FRET)
An established single-molecule measurement is fluorescence resonance energy transfer (FRET) method. FRET has been developed to observe the activation of the small G protein Ras at the level of individual molecules and provides a powerful technique to study the signal-transduction mechanisms of various G proteins. It would also allow direct investigations of the interaction of activated Ras with its effector and scaffolding proteins and its localization in specialized domains, which would provide valuable information for understanding the signal-transduction mechanism after Ras becomes activated. This method is able to distinguish between cooperative formations of large, activated Ras-signaling complexes for signaling transduction from random simple collisional mechanisms.
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Previously, Mochizuki et al. (14) developed a method to detect Ras activation by using CFP (cyan FP) and YFP (yellow FP). Observations of FRET is usually done using total internal reflection fluorescence (TIRF) microscope where the fluorescence images are separated by a dichroic mirror and projected into two detection arms with bandpass filters. Efficiencies of energy transfer between the protein tagged donor (x-CFP) and protein tagged acceptor (y-YFP) were calculated and the size of the nanoclusters can be determined. The analyses of donor de-quenching upon acceptor photobleaching in the absence or presence of acceptor fluorophores are used to determine magnitude of FRET (15). The Stochastic optical reconstruction microscopy (STORM) or photo-activated localization microscopy (PALM) systems which incorporate TRIF can be used for fluorescence detection.
Enhancements in FRET
FRET measurements alone without any temporal resolution cannot be extended for correlation studies as they lack kinetics and dynamic measurements. These measurements are also quite susceptible to variations in expression level or molecule diffusion inherent in the sample. This also applies to external influences such as sample movements and excitation fluctuation. Fluorescence Lifetime Imaging (FLIM) is an added enhancement to FRET microscopy. It scrutinizes protein binding and estimates intermolecular distances on an Angström scale as well as allows FRET measurements to be calibrated internally. Since the emission of FRET will decay overtime, the half life can be measured using FLIM.
To determine that increased Ras nanoclusters lifetime correlates with intensity of MAPK activation.
To determine that NPMc interacts with Ras nanoclusters in its activated isoform.
To measure the binding constants (kd) and stoichiometries of NPMc interactions with Ras nanoclusters.
To determine that interaction of NPMc with Ras nanoclusters stabilizes its formation thus increased its lifetime on plasma membrane.
To determine that the stabilization of Ras nanoclusters by NPMc increased the recruitment of MAPK signaling modules to the plasma membrane and its subsequent increased activation.
FRET as a tool to answer study objectives
Using single-molecule imaging technique like FRET, the location, movement, interaction, conformational state like dimer or oligomers and biochemical reaction of single Ras molecules can be detected. Confocal microscopy can only detect co localization of two proteins but its interactions and kinetics are not elucidated. Using FRET, the temporal resolution between two fluorophores can be reduced to 1-10nm scale. FRET can be used to answer the question of how mutant cytoplasmic NPM (NPMc) interacts with Ras nanoclusters at the plasma membrane thus resulting in an increased MAPK activation through nanoclusters stabilization.
FRET in tandem with FLIM help facilitates visualization of the activation of single individual molecules of Ras and the behavior of activated Ras molecules at nanoseconds rate. Changes like binding constants (kd) and stoichiometries in NPMc and Ras nanoclusters interaction can be quantified using FRET. FLIM-FRET quantifies the proximity of two proteins by measuring changes in the fluorescence lifetime of the donor fluorophore, for example Ras-CFP, when it interacts with the acceptor fluorophore NPMc-YFP. We can first analyzed cells expressing Ras-CFP or a constitutive GTP activated state of Ras (RasG12V-CFP) in the presence or absence of NPMc-YFP. The fluorescence lifetime was measured when each protein was expressed alone or when co expressed with NPMc-YFP and its change quantified.
FRET could be used to delineate role of specific domains of NPMc or Ras GTPases involved in interaction by fluorescent labeling its deletion mutants. Interactions between native Ras proteins or its post translational states (phosphorylation, farnesylation or palmitoylation) with NPMc can also be elucidated using FLIM-FRET. Although immuno electron microscopy using gold labeled antibodies are commonly used to measure nanoclusters distributions, FRET could easily be used to replace this.
Most interestingly, FLIM-FRET can be used to track the movement of NPMc with respect to Ras nanoclusters from the plasma membrane across other organelles like Golgi complex. Fluorescence recovery after photobleaching (FRAP) could be used in tandem to test protein recovery of the system. Cells can be pretreated with cycloheximide to block protein synthesis assuring that measurements were made on recycling not newly synthesized proteins.
The state of Ras at which NPMc binds and its role in the stabilizing the Ras nanoclusters can be elucidated using FLIM-FRET. A system that measure changes in nanoseconds is indispensable to measure nanoclusters dynamics. As we hypothesizes that these nanoclusters are platform for signaling complex assemblies and activation, its lifetime correlates with intensity of signaling activation downstream.
Drawbacks of FRET in the study
FRET system has its disadvantages. The generation of fluorophore tagged proteins used in FRET should be properly folded to render it biologically active. The protein should behave similarly to its native states and fluorophores should not interfere with protein-protein interactions. To overcome this, an in vitro FRET observation experiments can be carried out to test if the system is viable before carrying it on live cells imaging. As demonstrated by Murakoshi et al (16), in vitro FRET observation is used to confirm that H-Ras could be activated even after YFP fusion. This finding was further confirmed by a pull-down assay, with use of the Ras-binding domain of Raf-1 kinase. There are several factors that could interfere with a good FRET setup such as the choice of emission and excitation wavelengths of fluorophores chosen which should overlap slightly to ensure efficient energy transfer, the local concentration of probes should be homogenous before results becomes reliable and the orientation of linker angles are important to ensure energy transfer.