Single-molecule diffusion measurements of H-Ras at the plasma membrane of live cells reveal microdomain localization upon activation

Lommerse, Piet H.M.; Snaar-Jagalska, B. Ewa; Spaink, Herman P.; Schmidt, Thomas

doi:10.1242/jcs.02300

Cited by 111 publications

(125 citation statements)

References 48 publications

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“…Association of CAAX to membrane domains between 20 and 200 nm in size has been suggested previously based on results from studies that used various indirect methods (9,12). Here, we performed superresolution experiments to directly visualize those potential domains in fixed cells.…”

Section: Caax Clusteringmentioning

confidence: 93%

“…The pcDNA3.1 vector encoding the 10 C-terminal amino acids of human HRas, including the CAAX motif fused to yellow fluorescent protein (YFP) (12), was cut using the same restriction sites. Dendra2 was subsequently ligated into the pcDNA 3.1 vector.…”

Section: Plasmidsmentioning

confidence: 99%

“…Although the first suggestions of protein clustering and membrane heterogeneity were highlighted in the 1970s in the classical article by Singer and Nicholson (2), it is only in the past decade that strong evidence of their existence has emerged. Domains ranging in size from a few tens of nanometers to~100-200 nm in diameter have been found in the past decade, both through indirect biochemical assays (3) and using sophisticated microscopy techniques including fluorescence recovery after photobleaching (4,5), fluorescence resonance energy transfer (6), fluorescence correlation spectroscopy (7-9), single-particle tracking (10)(11)(12), single-molecule microscopy (13)(14)(15), and electron microscopy (16). However, a common view on the nature and biological role of such membrane domains is still lacking.…”

Section: Introductionmentioning

confidence: 99%

“…This uncertainty in part arises from the inability of the above techniques to directly identify domains in live cells. Earlier techniques identified domains indirectly by analysis of their effect on such processes as lateral diffusion of proteins and lipids (10)(11)(12)17). To date, direct observation of domains has been achieved in membrane sheets through cryo-immunogold electron microscopy (16).…”

Section: Introductionmentioning

confidence: 99%

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Visualization of HRas Domains in the Plasma Membrane of Fibroblasts

Pezzarossa

Zosel

Schmidt

2015

Biophysical Journal

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The plasma membrane is a highly complex, organized structure where the lateral organization of signaling proteins is tightly regulated. In the case of Ras proteins, it has been suggested that the differential activity of the various isoforms is due to protein localization in separate membrane compartments. To date, direct visualization of such compartmentalization has been achieved only by electron microscopy on membrane sheets. Here, we combine photoactivated light microscopy with quantitative statistical analysis to visualize protein distribution in intact cells. In particular, we focus on the localization of HRas and its minimal anchoring domain, CAAX. We demonstrate the existence of a complex partitioning behavior, where small domains coexist with larger ones. The protein content in these domains varied from two molecules to tens of molecules. We found that 40% of CAAX and 60% of HRas were localized in domains. Subsequently, we were able to manipulate protein distributions by inducing coalescence of supposedly cholesterol-enriched domains. Clustering resulted in an increase of the localized fraction by 15%.

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Section: Caax Clusteringmentioning

confidence: 93%

Section: Plasmidsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Visualization of HRas Domains in the Plasma Membrane of Fibroblasts

Pezzarossa

Zosel

Schmidt

2015

Biophysical Journal

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“…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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The Macroscopic Effects of Microscopic Heterogeneity in Cell Signaling

Mugler¹,

Wolde²

2013

Advances in Chemical Physics

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Over the past decade, advances in super-resolution microscopy and particle-based modeling have driven an intense interest in investigating spatial heterogeneity at the level of single molecules in cells. Remarkably, it is becoming clear that spatiotemporal correlations between just a few molecules can have profound effects on the signaling behavior of the entire cell. While such correlations are often explicitly imposed by molecular structures such as rafts, clusters, or scaffolds, they also arise intrinsically, due strictly to the small numbers of molecules involved, the finite speed of diffusion, and the effects of macromolecular crowding. In this chapter we review examples of both explicitly imposed and intrinsic correlations, focusing on the mechanisms by which microscopic heterogeneity is amplified to macroscopic effect.

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Single-molecule diffusion measurements of H-Ras at the plasma membrane of live cells reveal microdomain localization upon activation

Cited by 111 publications

References 48 publications

Visualization of HRas Domains in the Plasma Membrane of Fibroblasts

Visualization of HRas Domains in the Plasma Membrane of Fibroblasts

Quantitative microscopy and systems biology: seeing the whole picture

The Macroscopic Effects of Microscopic Heterogeneity in Cell Signaling

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