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Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells.

Oancea E, Teruel MN, Quest AF, Meyer T - J. Cell Biol. (1998)

Bottom Line: This selective membrane localization was lost in the presence of arachidonic acid.GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition.These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.

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Comparison of the apparent lateral membrane diffusion coefficient and apparent  plasma membrane dissociation  time of Cys1–GFP in response to  the addition of PMA, PC-PLC,  or DiC8. Fluorescence recovery  after photobleaching was used to  determine the diffusion coefficient and dissociation time of  Cys1–GFP bound to the plasma  membrane after either PMA,  PC-PLC, or DiC8 addition. A  small region of the plasma membrane was photobleached using a  short laser pulse (8 ms), and sequential images were recorded  every 330 ms for PC-PLC and  DiC8 addition and every 1.5 s for  PMA addition. (A) Example of  four images of a cell expressing  Cys1–GFP and stimulated with PC-PLC. The images shown were recorded immediately before and 0.33, 2, and 6 s after the photobleaching pulse. The plasma membrane bleach spot is indicated by the arrow. (B) Comparison of the recovery in fluorescence intensity of Cys1–GFP at the center of the bleach spot. (C) In each series of images, the one-dimensional fluorescence intensity profiles along  the plasma membrane were measured as a function of time and each profile was fit by a Gaussian function. (D) Calculated relative increase in the square radius of each Gaussian profile as a function of time for three typical cells. Data for cells stimulated with PMA, PC-PLC, and DiC8 are shown. The apparent lateral plasma membrane diffusion coefficients are proportional to the slope of each linear fit  (dy/dt = 4 × D). (E) Calculated membrane dissociation time courses for Cys1–GFP localized to the plasma membrane by PMA, PC-PLC or DiC8 addition.
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Figure 4: Comparison of the apparent lateral membrane diffusion coefficient and apparent plasma membrane dissociation time of Cys1–GFP in response to the addition of PMA, PC-PLC, or DiC8. Fluorescence recovery after photobleaching was used to determine the diffusion coefficient and dissociation time of Cys1–GFP bound to the plasma membrane after either PMA, PC-PLC, or DiC8 addition. A small region of the plasma membrane was photobleached using a short laser pulse (8 ms), and sequential images were recorded every 330 ms for PC-PLC and DiC8 addition and every 1.5 s for PMA addition. (A) Example of four images of a cell expressing Cys1–GFP and stimulated with PC-PLC. The images shown were recorded immediately before and 0.33, 2, and 6 s after the photobleaching pulse. The plasma membrane bleach spot is indicated by the arrow. (B) Comparison of the recovery in fluorescence intensity of Cys1–GFP at the center of the bleach spot. (C) In each series of images, the one-dimensional fluorescence intensity profiles along the plasma membrane were measured as a function of time and each profile was fit by a Gaussian function. (D) Calculated relative increase in the square radius of each Gaussian profile as a function of time for three typical cells. Data for cells stimulated with PMA, PC-PLC, and DiC8 are shown. The apparent lateral plasma membrane diffusion coefficients are proportional to the slope of each linear fit (dy/dt = 4 × D). (E) Calculated membrane dissociation time courses for Cys1–GFP localized to the plasma membrane by PMA, PC-PLC or DiC8 addition.

Mentions: Whereas PMA, DiC8, and bacterial PC-PLC addition all led to a similar translocation of Cys1–GFP to the plasma membrane, photobleaching recovery measurements suggested that the dissociation time and the diffusion coefficient for the membrane-associated Cys1–GFP was markedly different for the three stimuli (Fig. 4). In these experiments, a small spot of plasma membrane localized Cys1–GFP was photobleached by a short laser pulse, and the recovery of fluorescence was monitored as a function of time using sequential imaging (Fig. 4 A shows an example of cells stimulated with PC-PLC). The plasma membrane–bound Cys1– GFP had recovery times of one second in cells stimulated by addition of PC-PLC or DiC8 (see Table I). In contrast, the recovery time after PMA-induced localization was typically 10 s and a variable fraction of the membrane-associated Cys1–GFP was completely immobile (examples of the recovery curves are shown in Fig. 4 B).


Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells.

Oancea E, Teruel MN, Quest AF, Meyer T - J. Cell Biol. (1998)

Comparison of the apparent lateral membrane diffusion coefficient and apparent  plasma membrane dissociation  time of Cys1–GFP in response to  the addition of PMA, PC-PLC,  or DiC8. Fluorescence recovery  after photobleaching was used to  determine the diffusion coefficient and dissociation time of  Cys1–GFP bound to the plasma  membrane after either PMA,  PC-PLC, or DiC8 addition. A  small region of the plasma membrane was photobleached using a  short laser pulse (8 ms), and sequential images were recorded  every 330 ms for PC-PLC and  DiC8 addition and every 1.5 s for  PMA addition. (A) Example of  four images of a cell expressing  Cys1–GFP and stimulated with PC-PLC. The images shown were recorded immediately before and 0.33, 2, and 6 s after the photobleaching pulse. The plasma membrane bleach spot is indicated by the arrow. (B) Comparison of the recovery in fluorescence intensity of Cys1–GFP at the center of the bleach spot. (C) In each series of images, the one-dimensional fluorescence intensity profiles along  the plasma membrane were measured as a function of time and each profile was fit by a Gaussian function. (D) Calculated relative increase in the square radius of each Gaussian profile as a function of time for three typical cells. Data for cells stimulated with PMA, PC-PLC, and DiC8 are shown. The apparent lateral plasma membrane diffusion coefficients are proportional to the slope of each linear fit  (dy/dt = 4 × D). (E) Calculated membrane dissociation time courses for Cys1–GFP localized to the plasma membrane by PMA, PC-PLC or DiC8 addition.
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Figure 4: Comparison of the apparent lateral membrane diffusion coefficient and apparent plasma membrane dissociation time of Cys1–GFP in response to the addition of PMA, PC-PLC, or DiC8. Fluorescence recovery after photobleaching was used to determine the diffusion coefficient and dissociation time of Cys1–GFP bound to the plasma membrane after either PMA, PC-PLC, or DiC8 addition. A small region of the plasma membrane was photobleached using a short laser pulse (8 ms), and sequential images were recorded every 330 ms for PC-PLC and DiC8 addition and every 1.5 s for PMA addition. (A) Example of four images of a cell expressing Cys1–GFP and stimulated with PC-PLC. The images shown were recorded immediately before and 0.33, 2, and 6 s after the photobleaching pulse. The plasma membrane bleach spot is indicated by the arrow. (B) Comparison of the recovery in fluorescence intensity of Cys1–GFP at the center of the bleach spot. (C) In each series of images, the one-dimensional fluorescence intensity profiles along the plasma membrane were measured as a function of time and each profile was fit by a Gaussian function. (D) Calculated relative increase in the square radius of each Gaussian profile as a function of time for three typical cells. Data for cells stimulated with PMA, PC-PLC, and DiC8 are shown. The apparent lateral plasma membrane diffusion coefficients are proportional to the slope of each linear fit (dy/dt = 4 × D). (E) Calculated membrane dissociation time courses for Cys1–GFP localized to the plasma membrane by PMA, PC-PLC or DiC8 addition.
Mentions: Whereas PMA, DiC8, and bacterial PC-PLC addition all led to a similar translocation of Cys1–GFP to the plasma membrane, photobleaching recovery measurements suggested that the dissociation time and the diffusion coefficient for the membrane-associated Cys1–GFP was markedly different for the three stimuli (Fig. 4). In these experiments, a small spot of plasma membrane localized Cys1–GFP was photobleached by a short laser pulse, and the recovery of fluorescence was monitored as a function of time using sequential imaging (Fig. 4 A shows an example of cells stimulated with PC-PLC). The plasma membrane–bound Cys1– GFP had recovery times of one second in cells stimulated by addition of PC-PLC or DiC8 (see Table I). In contrast, the recovery time after PMA-induced localization was typically 10 s and a variable fraction of the membrane-associated Cys1–GFP was completely immobile (examples of the recovery curves are shown in Fig. 4 B).

Bottom Line: This selective membrane localization was lost in the presence of arachidonic acid.GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition.These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.

Show MeSH
Related in: MedlinePlus