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Downscaling the analysis of complex transmembrane signaling cascades to closed attoliter volumes.

Grasso L, Wyss R, Piguet J, Werner M, Hassaïne G, Hovius R, Vogel H - PLoS ONE (2013)

Bottom Line: Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells.Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity.They comprise parts of a cell's plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Physical Chemistry of Polymers and Membranes, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

ABSTRACT
Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity. The vesicles are derived from live mammalian cells using chemicals or optical tweezers. They comprise parts of a cell's plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells. Using fluorescence microscopies, we measured in individual vesicles the different steps of G-protein-coupled receptor mediated signaling like ligand binding to receptors, subsequent G-protein activation and finally arrestin translocation indicating receptor deactivation. Observing cellular signaling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis, and investigating receptor mediated signaling in multiarray format.

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Formation of plasma membrane vesicles from live cells.(A) Scheme: After adding cytochalasin B, cultured cells formed within a few minutes blebbing structures on their plasma membranes which can be sheared off (by shaking or by pulling with an optical tweezer) as (sub-)micrometer-sized closed plasma membrane vesicles. (B,C) Confocal micrographs showing the YFP fluorescence of HEK cells expressing A2AR-YFP before (B) and after (C) addition of cytochalasin B (typical final concentration 25 µg/ml); scale bars: 10 µm. (D) Confocal micrograph of a blebbing cell expressing a fluorescent membrane receptor (A2A-YFP, green) and a cytosolic protein (mCherry, red). Both proteins are present in the cell and in the shed vesicles; scale bar: 3 µm. (E) Array of vesicles produced from HEK cells expressing A2AR-YFP; scale bar: 10 µm.
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pone-0070929-g001: Formation of plasma membrane vesicles from live cells.(A) Scheme: After adding cytochalasin B, cultured cells formed within a few minutes blebbing structures on their plasma membranes which can be sheared off (by shaking or by pulling with an optical tweezer) as (sub-)micrometer-sized closed plasma membrane vesicles. (B,C) Confocal micrographs showing the YFP fluorescence of HEK cells expressing A2AR-YFP before (B) and after (C) addition of cytochalasin B (typical final concentration 25 µg/ml); scale bars: 10 µm. (D) Confocal micrograph of a blebbing cell expressing a fluorescent membrane receptor (A2A-YFP, green) and a cytosolic protein (mCherry, red). Both proteins are present in the cell and in the shed vesicles; scale bar: 3 µm. (E) Array of vesicles produced from HEK cells expressing A2AR-YFP; scale bar: 10 µm.

Mentions: We first report on experiments using vesicles obtained by cytochalasin B treatment. The formation of plasma membrane vesicles is depicted schematically in Figure 1A and in the micrographs of Figure 1B–E for HEK cells expressing fluorescent membrane and cytosolic proteins, demonstrating that the (sub)micrometer sized vesicles (size distribution: Figure S1) comprise portions of a cell’s membrane and cytosol. Using fluorescence correlation spectroscopy (FCS), we determined the concentration and mobility of a prototypical GPCR, the adenosine A2A receptor fused to YFP (A2AR-YFP), in the plasma membrane of both individual native vesicles and their mother cells. From the measured autocorrelation function (ACF) (Figure 2B and Figures S2A,B), we calculated receptor densities of 500±41 (n = 21) and 580±39 (n = 21) receptors/µm2 in the plasma membranes of the vesicles and the mother cells, respectively. These data show that during the formation of plasma membrane vesicles the native receptor density is maintained. The mobility of A2AR-YFP was also investigated by FCS (Figure 2B). The measured ACF curves were best described by 2D-diffusion of a single component and considering triplet state formation, yielding a typical receptor diffusion coefficient D = 0.59±0.04 µm2/s (n = 21) in vesicles, and D = 0.17±0.02 µm2/s (n = 21) in cells (Figure S2A,B). The difference of the receptor mobility in cells and vesicles might be due to the interaction of GPCRs with the cell’s cytoskeleton [21] and local roughness of the cell’s plasma membrane, which both are absent in cytochalasin-derived vesicles [3], [22]. Plasma membrane vesicles are particularly suited to measure processes on/in membranes by FCS as vesicles do not show morphological changes, which are typical for living cells [23]. Therefore the measured fluorescence traces are very stable and reproducible with considerable lower background fluorescence as compared to cells. In contrast, FCS measurements on the plasma membranes of living cells are prone to artifacts due to the intrinsic movements of a cell.


Downscaling the analysis of complex transmembrane signaling cascades to closed attoliter volumes.

Grasso L, Wyss R, Piguet J, Werner M, Hassaïne G, Hovius R, Vogel H - PLoS ONE (2013)

Formation of plasma membrane vesicles from live cells.(A) Scheme: After adding cytochalasin B, cultured cells formed within a few minutes blebbing structures on their plasma membranes which can be sheared off (by shaking or by pulling with an optical tweezer) as (sub-)micrometer-sized closed plasma membrane vesicles. (B,C) Confocal micrographs showing the YFP fluorescence of HEK cells expressing A2AR-YFP before (B) and after (C) addition of cytochalasin B (typical final concentration 25 µg/ml); scale bars: 10 µm. (D) Confocal micrograph of a blebbing cell expressing a fluorescent membrane receptor (A2A-YFP, green) and a cytosolic protein (mCherry, red). Both proteins are present in the cell and in the shed vesicles; scale bar: 3 µm. (E) Array of vesicles produced from HEK cells expressing A2AR-YFP; scale bar: 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3733713&req=5

pone-0070929-g001: Formation of plasma membrane vesicles from live cells.(A) Scheme: After adding cytochalasin B, cultured cells formed within a few minutes blebbing structures on their plasma membranes which can be sheared off (by shaking or by pulling with an optical tweezer) as (sub-)micrometer-sized closed plasma membrane vesicles. (B,C) Confocal micrographs showing the YFP fluorescence of HEK cells expressing A2AR-YFP before (B) and after (C) addition of cytochalasin B (typical final concentration 25 µg/ml); scale bars: 10 µm. (D) Confocal micrograph of a blebbing cell expressing a fluorescent membrane receptor (A2A-YFP, green) and a cytosolic protein (mCherry, red). Both proteins are present in the cell and in the shed vesicles; scale bar: 3 µm. (E) Array of vesicles produced from HEK cells expressing A2AR-YFP; scale bar: 10 µm.
Mentions: We first report on experiments using vesicles obtained by cytochalasin B treatment. The formation of plasma membrane vesicles is depicted schematically in Figure 1A and in the micrographs of Figure 1B–E for HEK cells expressing fluorescent membrane and cytosolic proteins, demonstrating that the (sub)micrometer sized vesicles (size distribution: Figure S1) comprise portions of a cell’s membrane and cytosol. Using fluorescence correlation spectroscopy (FCS), we determined the concentration and mobility of a prototypical GPCR, the adenosine A2A receptor fused to YFP (A2AR-YFP), in the plasma membrane of both individual native vesicles and their mother cells. From the measured autocorrelation function (ACF) (Figure 2B and Figures S2A,B), we calculated receptor densities of 500±41 (n = 21) and 580±39 (n = 21) receptors/µm2 in the plasma membranes of the vesicles and the mother cells, respectively. These data show that during the formation of plasma membrane vesicles the native receptor density is maintained. The mobility of A2AR-YFP was also investigated by FCS (Figure 2B). The measured ACF curves were best described by 2D-diffusion of a single component and considering triplet state formation, yielding a typical receptor diffusion coefficient D = 0.59±0.04 µm2/s (n = 21) in vesicles, and D = 0.17±0.02 µm2/s (n = 21) in cells (Figure S2A,B). The difference of the receptor mobility in cells and vesicles might be due to the interaction of GPCRs with the cell’s cytoskeleton [21] and local roughness of the cell’s plasma membrane, which both are absent in cytochalasin-derived vesicles [3], [22]. Plasma membrane vesicles are particularly suited to measure processes on/in membranes by FCS as vesicles do not show morphological changes, which are typical for living cells [23]. Therefore the measured fluorescence traces are very stable and reproducible with considerable lower background fluorescence as compared to cells. In contrast, FCS measurements on the plasma membranes of living cells are prone to artifacts due to the intrinsic movements of a cell.

Bottom Line: Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells.Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity.They comprise parts of a cell's plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Physical Chemistry of Polymers and Membranes, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

ABSTRACT
Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity. The vesicles are derived from live mammalian cells using chemicals or optical tweezers. They comprise parts of a cell's plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells. Using fluorescence microscopies, we measured in individual vesicles the different steps of G-protein-coupled receptor mediated signaling like ligand binding to receptors, subsequent G-protein activation and finally arrestin translocation indicating receptor deactivation. Observing cellular signaling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis, and investigating receptor mediated signaling in multiarray format.

Show MeSH
Related in: MedlinePlus