Single-Molecule Imaging of the H-Ras Membrane-Anchor Reveals Domains in the Cytoplasmic Leaflet of the Cell Membrane

Lommerse, Piet H.M.; Blab, Gerhard A.; Cognet, Laurent; Harms, Gregory S.; Snaar-Jagalska, B. Ewa; Spaink, Herman P.; Schmidt, Thomas

doi:10.1016/s0006-3495(04)74139-9

Cited by 140 publications

(176 citation statements)

References 42 publications

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“…Data on CAAX distribution support that~40% of the molecules were organized into clusters. In our previous study, using single-molecule tracking, we observed that 40% of CAAX in live cells were undergoing confined diffusion, whereas the remaining 60% were freely diffusing in the plane of the membrane (40). We suggested that this was related to membrane confinement of the membrane anchor, CAAX.…”

Section: Discussionmentioning

confidence: 86%

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: Discussionmentioning

confidence: 86%

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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“…It is important to stress, however, that these are just examples among many applications of single-molecule methods to many areas of biology, which include probing the dynamics and folding of proteins (11-16) and nucleic acids (9,17,18), the function of molecular motors (19)(20)(21)(22), the motions of proteins in membranes (23)(24)(25)(26), and viral infection (27,28).…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

Calmodulin, Conformational States, and Calcium Signaling. A Single-Molecule Perspective

Johnson

2006

Biochemistry

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Single-molecule fluorescence measurements can provide a new perspective on the conformations, dynamics, and interactions of proteins. Recent examples are described illustrating the application of single-molecule fluorescence spectroscopy to calcium signaling proteins with an emphasis on the new information available in single-molecule fluorescence burst measurements, resonance energy transfer, and polarization modulation methods. Calcium signaling pathways are crucial in many cellular processes. The calcium binding protein calmodulin (CaM) serves as a molecular switch to regulate a network of calcium signaling pathways. Single-molecule spectroscopic methods can yield insights into conformations and dynamics of CaM and CaM-regulated proteins. Examples include studies of the conformations and dynamics of CaM, binding of target peptides, and interaction with the plasma-membrane Ca 2+ pump. Single-molecule resonance energy transfer measurements revealed conformational substates of CaM, and single-molecule polarization modulation spectroscopy was used to probe interactions between CaM and the plasma-membrane Ca 2+ -ATPase.Single-molecule fluorescence methods have the potential to resolve details about protein conformations, interactions, and dynamics that were previously hidden by ensemble averaging. As the practice of single-molecule methods expands, it is useful to examine what new information can be obtained about biomolecules and what the limitations of the methods are. This paper will examine these questions in the context of recent applications of single-molecule fluorescence techniques in the author's laboratory to the conformations, dynamics, and interactions of the calcium signaling protein calmodulin (CaM) (see Fig. 1).Proteins are dynamic molecules. Multiple conformational states are required for them to function. CaM is no exception, and appears to sample an unusually wide range of conformations (1). How can the distribution of such conformations be characterized? Conventional bulk measurements detect the average properties of many (typically > 10 10 ) molecules and therefore do not resolve the range of conformations that are actually present. One way to attack this problem is to detect and characterize molecules one at a time.Introduced in the early 1990s (2-6), single-molecule fluorescence methods can now address real problems in biology (see recent reviews (7-10)). Conformational states and conformational motions can be detected that may be missed by other methods. Dynamic processes can be probed under either equilibrium or non-equilibrium conditions by following the fluctuations of an appropriate single-molecule spectroscopic signal. In kinetic *To whom correspondence should be addressed. E-mail: ckjohnson@ku.edu. † We acknowledge support for this research from NIH R01 GM58715, the American Heart Association (99513262 and 0455487Z) and a Research Corporation Research Opportunity Award. B.D.S. and J.R.U. acknowledge support from training grant NIH GM08545, and E.S.P. acknowledges support from t...

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Single-Molecule Imaging of the H-Ras Membrane-Anchor Reveals Domains in the Cytoplasmic Leaflet of the Cell Membrane

Cited by 140 publications

References 42 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

Calmodulin, Conformational States, and Calcium Signaling. A Single-Molecule Perspective

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