Single-molecule imaging analysis of Ras activation in living cells

Murakoshi, Hideji; Iino, Ryota; Kobayashi, Takashi; Fujiwara, Takeshi; Ohshima, Chika; Yoshimura, Akihiko; Kusumi, Akihiro

doi:10.1073/pnas.0401354101

Cited by 361 publications

(390 citation statements)

References 36 publications

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“…K(B)-Ras signalling microdomains are not cholesterol dependent but like lipid rafts they require actin for their structural integrity [79]. Further evidence for Ras microdomain occupancy came from single particle tracking of Ras which revealed that H-and K(B)-Ras become transiently immobile upon activation thus supporting the idea that Ras associates with signalling platforms [80]. In summary, H-and K(B)-Ras appear to operate from distinct microdomains within the cell surface and Ras activation state may be critical for directing association with particular microdomains.…”

Section: Compartmentalisation Of Rasmentioning

confidence: 98%

“…However, to date GTP-bound cytosolic Ras has not been experimentally detected. Additionally, single particle analysis studies have revealed that the time course of activation of an individual Ras molecule is typically less than a second [80]. This means that guanine nucleotide turnover and effector interactions must occur within a compartment and are not sustained whilst Ras is trafficked to other regions of the cell.…”

Section: Compartmentalised Ras Signallingmentioning

confidence: 99%

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Ras proteins: paradigms for compartmentalised and isoform-specific signalling

Omerovic

Laude

Prior

2007

Cell. Mol. Life Sci.

117

122

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Ras GTPases mediate a wide variety of cellular processes by converting a multitude of extracellular stimuli into specific biological responses including proliferation, differentiation and survival. In mammalian cells, three ras genes encode four Ras isoforms (H-Ras, K-Ras4A, KRas4B and N-Ras) that are highly homologous but functionally distinct. Differences between the isoforms, including their post-translational modifications and intracellular sorting, mean that Ras has emerged as an important model system of compartmentalised signalling and membrane biology. Ras isoforms in different subcellular locations are proposed to recruit distinct upstream and downstream accessory proteins and activate multiple signalling pathways. Here, we summarise data relating to isoform specific signalling, its role in disease and the mechanisms promoting compartmentalised signalling. Further understanding of this field will reveal the role of Ras signalling in development, cellular homeostasis and cancers and may suggest new therapeutic approaches.

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Section: Compartmentalisation Of Rasmentioning

confidence: 98%

Section: Compartmentalised Ras Signallingmentioning

confidence: 99%

Ras proteins: paradigms for compartmentalised and isoform-specific signalling

Omerovic

Laude

Prior

2007

Cell. Mol. Life Sci.

117

122

View full text Add to dashboard Cite

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“…Indeed diVerential activation of Ras between raft and non-raft domains has been observed by FRET microscopy (Fukano et al 2007). SFT experiments have shown that Raf proteins bind to immobilized Ras-GTP molecules (Murakoshi et al 2004) and EM analysis shows co-clustering of Raf and Ras-GTP (Plowman et al 2005), suggesting that Raf binds to clustered active Ras. One can speculate that conWnement and clustering of Ras and its eVector molecules facilitates interactions with further components of a relevant signaling network.…”

Section: Signaling Via Protein Clusters In the Plasma Membranementioning

confidence: 99%

“…One line of evidence comes from data acquired by electron microscopy methods (Plowman et al 2005;Prior et al 2003). The dynamics of Ras in the plasma membrane have also been measured by single Xuorophore tracking (SFT) (Lommerse et al 2003(Lommerse et al , 2005Murakoshi et al 2004) and with Xuorescence recovery after photobleaching (FRAP) (Niv et al 1999(Niv et al , 2002. According to these data, H-Ras clusters into cholesterol dependent nanoscale domains in its inactive GDP-bound state.…”

Section: Signaling Via Protein Clusters In the Plasma Membranementioning

confidence: 99%

Quantitative microscopy and systems biology: seeing the whole picture

Verveer

Bastiaens

2008

Histochem Cell Biol

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Understanding cellular function requires studying the spatially resolved dynamics of protein networks. From the isolated proteins we can only learn about their individual properties, but by investigating their behavior in their natural environment, the cell, we obtain information about the overall response properties of the network module in which they operate. Fluorescence microscopy methods provide currently the only tools to study the dynamics of molecular processes in living cells with high temporal and spatial resolution. Combined with computational approaches they allow us to obtain insights in the reactiondiVusion processes that determine biological function on the scale of cells.

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“…Full-length Ras has a protein domain, adjacent to the anchor, that is required for the formation of non-raft microdomains that constitute the actual Ras signalling platform. Therefore, it is a combination of lipid anchor and protein-based sorting mechanisms that determines the final composition of an H-Ras-signalling complex 24,43,44 .…”

Section: Determinants Of Raft Stabilitymentioning

confidence: 99%

Lipid rafts: contentious only from simplistic standpoints

Hancock

2006

Nat Rev Mol Cell Biol

751

716

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The hypothesis that lipid rafts exist in plasma membranes and have crucial biological functions remains controversial. The lateral heterogeneity of proteins in the plasma membrane is undisputed, but the contribution of cholesterol-dependent lipid assemblies to this complex, non-random organization promotes vigorous debate. In the light of recent studies with model membranes, computational modelling and innovative cell biology, I propose an updated model of lipid rafts that readily accommodates diverse views on plasma-membrane micro-organization.A widely accepted hypothesis in contemporary cell biology is that freely diffusing, stable, lateral assemblies of sphingolipids and cholesterol, which are termed lipid rafts 1-3 , constitute an important organizing principle for the plasma membrane. The basic concept is that lipid rafts can facilitate selective protein-protein interactions by selectively excluding or including proteins. This lipid-based sorting mechanism has been widely implicated in the assembly of transient signalling platforms and more permanent structures such as the immunological synapse, as well as in the sorting of proteins for entry into specific exocytic and endocytic trafficking pathways [1][2][3] . Despite the undoubted theoretical utility of lipid rafts to many cell biological processes, the basic hypothesis that stable lipid rafts exist at all in biological membranes is under intense scrutiny 4,5 . This is partly because lipid rafts, if they exist in resting cell membranes, are too small to be resolved by fluorescent microscopy and have no defined ultrastructure; therefore, proving their existence is problematic. This article will consider the biophysical properties of model membranes that underpin the lipid raft hypothesis, and the limitations of the biochemical approaches that have been used to study rafts in biological membranes that largely account for the current debate on the hypothesis mentioned above. After examining the challenges that are involved in correlating observations, which have been made in model and cellular membranes, I focus on synthesizing recent data on the size of lipid domains in model membranes with observations that have been obtained by imaging intact plasma membranes. These imaging approaches include single particle tracking (SPT), single fluorophore video tracking (SFVT), fluorescence resonance energy transfer (FRET), homo-FRET and electron microscopy (EM). On the one hand, these studies challenge the simplistic null hypothesis that lipid-based assemblies, such as lipid rafts, do not exist in biological membranes. On the other hand, it is timely to reconsider the raft hypothesis in the light of these new data because the consensus model that emerges is more complex than a simplistic notion of stable, freely diffusing lipid rafts. Some intriguing new questions aboutCompeting interests statement The author declares no competing financial interests. DATABASES

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Single-molecule imaging analysis of Ras activation in living cells

Cited by 361 publications

References 36 publications

Ras proteins: paradigms for compartmentalised and isoform-specific signalling

Ras proteins: paradigms for compartmentalised and isoform-specific signalling

Quantitative microscopy and systems biology: seeing the whole picture

Lipid rafts: contentious only from simplistic standpoints

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