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Translocation or just location? Pseudopodia affect fluorescent signals.

Dewitt S, Darley RL, Hallett MB - J. Cell Biol. (2009)

Bottom Line: Localized increases in the signal from cytosolic fluorescent protein constructs, for example, are frequently used as evidence for translocation of proteins to specific sites within the cell.However, differences in optical and geometrical properties of cytoplasm can influence the recorded intensity of the probe signal.Pseudopodia are especially problematic because their cytoplasmic properties can cause abrupt increases in fluorescent signal of both GFP and fluorescein.

View Article: PubMed Central - PubMed

Affiliation: Neutrophil Signalling Group, School of Medicine, Cardiff University, Heath Park, Cardiff, Wales, UK.

ABSTRACT
The use of fluorescent probes is one of the most powerful techniques for gaining spatial and temporal knowledge of dynamic events within living cells. Localized increases in the signal from cytosolic fluorescent protein constructs, for example, are frequently used as evidence for translocation of proteins to specific sites within the cell. However, differences in optical and geometrical properties of cytoplasm can influence the recorded intensity of the probe signal. Pseudopodia are especially problematic because their cytoplasmic properties can cause abrupt increases in fluorescent signal of both GFP and fluorescein. Investigators should therefore be cautious when interpreting fluorescence changes within a cell, as these can result from either translocation of the probe or changes in the optical properties of the milieu surrounding the probe.

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Asymmetrical signals with cytoplasmic GFP. The typical distribution of fluorescence intensity of cytoplasmic GFP in human cord blood–derived neutrophils along the axis shown of a polarizing cell (a) and a cell undergoing phagocytosis (b). (c) The dynamic nature of the fluorescence asymmetry of GFP during phagocytosis is shown in a time sequence of images of a cell undergoing phagocytosis. The position of the iC3b-opsonised zymosan particle (presented with a micropipette) is indicated in the phase-contrast image and the fluorescent images below at the times indicated. The complete time course is shown in Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1). (d) The effect of localized photobleaching (30 s) within the white square on the GFP signal from the leading pseudopodia and cell body is shown and (e) quantified for the leading pseudopodia (ps) and cell body (b). The ratio of mean intensities in the two locations is also shown as the dotted line (e). Bars: (a and b) 5 µm; (c) 10 µm; (d) 10 µm. Human neutrophils expressing GFP were generated from cord blood as described previously (Omidvar et al., 2006).
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fig5: Asymmetrical signals with cytoplasmic GFP. The typical distribution of fluorescence intensity of cytoplasmic GFP in human cord blood–derived neutrophils along the axis shown of a polarizing cell (a) and a cell undergoing phagocytosis (b). (c) The dynamic nature of the fluorescence asymmetry of GFP during phagocytosis is shown in a time sequence of images of a cell undergoing phagocytosis. The position of the iC3b-opsonised zymosan particle (presented with a micropipette) is indicated in the phase-contrast image and the fluorescent images below at the times indicated. The complete time course is shown in Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1). (d) The effect of localized photobleaching (30 s) within the white square on the GFP signal from the leading pseudopodia and cell body is shown and (e) quantified for the leading pseudopodia (ps) and cell body (b). The ratio of mean intensities in the two locations is also shown as the dotted line (e). Bars: (a and b) 5 µm; (c) 10 µm; (d) 10 µm. Human neutrophils expressing GFP were generated from cord blood as described previously (Omidvar et al., 2006).

Mentions: Because GFP and its relatives are widely used as the fluorescent moiety of chimeric proteins, it is obviously important and perhaps more relevant to establish whether GFP itself can produce a similar artifact. Unless linked to a granule-targeting sequence, the larger molecular weight of GFP excludes it from diffusion across granule membranes. This guarantees that the excitation path-length available to cytosolic GFP is thus reduced by the presence of GFP-excluding granules (Fig. 2 b). It is therefore expected that, in addition to the light-scattering related phenomenon demonstrated for granule-permeant fluors such as fluorescein, the reduced light path effect would further reduce the GFP signal detected from the granular cytoplasm and thus increase its relative intensity in the granule-free cytoplasm. As with fluorescein, there was a pronounced increase in fluorescent signal at the leading edge (Fig. 5 a) and within the pseudopodia as they formed (Fig. 5 b), with an enhancement of 1.6-fold (significantly higher than fluorescein alone). The zones of increased fluorescence correlated with the organelle-free zones in the cells (Fig. 5 b) demarcated at the organelle free-granule containing boundary (Fig. 5 c). In addition, the total fluorescent signal from the cell increased during pseudopodia formation. It is conceivable that the asymmetrical distribution of GFP arose from a protein–protein interaction, such as tight binding of GFP to proteins at the leading edge. This is unlikely, as laser photobleaching at the pole opposite the zone of increased fluorescence intensity caused a decrease in the intensity of the entire GFP pool (Fig. 5 d). If GFP had accumulated at the leading edge as a result of binding to another protein, it would have been insulated from this localized photobleaching effect. However, there was no difference in the rate of decrease of GFP fluorescence in the bulk cytoplasm or the organelle-free cytoplasm, suggesting that the GFP was equally free to diffuse in both locations (Fig. 5 e). In addition, the asymmetries in GFP fluorescence were dynamic, forming and relaxing with pseudopodia formation (Fig. 5 and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1).


Translocation or just location? Pseudopodia affect fluorescent signals.

Dewitt S, Darley RL, Hallett MB - J. Cell Biol. (2009)

Asymmetrical signals with cytoplasmic GFP. The typical distribution of fluorescence intensity of cytoplasmic GFP in human cord blood–derived neutrophils along the axis shown of a polarizing cell (a) and a cell undergoing phagocytosis (b). (c) The dynamic nature of the fluorescence asymmetry of GFP during phagocytosis is shown in a time sequence of images of a cell undergoing phagocytosis. The position of the iC3b-opsonised zymosan particle (presented with a micropipette) is indicated in the phase-contrast image and the fluorescent images below at the times indicated. The complete time course is shown in Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1). (d) The effect of localized photobleaching (30 s) within the white square on the GFP signal from the leading pseudopodia and cell body is shown and (e) quantified for the leading pseudopodia (ps) and cell body (b). The ratio of mean intensities in the two locations is also shown as the dotted line (e). Bars: (a and b) 5 µm; (c) 10 µm; (d) 10 µm. Human neutrophils expressing GFP were generated from cord blood as described previously (Omidvar et al., 2006).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: Asymmetrical signals with cytoplasmic GFP. The typical distribution of fluorescence intensity of cytoplasmic GFP in human cord blood–derived neutrophils along the axis shown of a polarizing cell (a) and a cell undergoing phagocytosis (b). (c) The dynamic nature of the fluorescence asymmetry of GFP during phagocytosis is shown in a time sequence of images of a cell undergoing phagocytosis. The position of the iC3b-opsonised zymosan particle (presented with a micropipette) is indicated in the phase-contrast image and the fluorescent images below at the times indicated. The complete time course is shown in Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1). (d) The effect of localized photobleaching (30 s) within the white square on the GFP signal from the leading pseudopodia and cell body is shown and (e) quantified for the leading pseudopodia (ps) and cell body (b). The ratio of mean intensities in the two locations is also shown as the dotted line (e). Bars: (a and b) 5 µm; (c) 10 µm; (d) 10 µm. Human neutrophils expressing GFP were generated from cord blood as described previously (Omidvar et al., 2006).
Mentions: Because GFP and its relatives are widely used as the fluorescent moiety of chimeric proteins, it is obviously important and perhaps more relevant to establish whether GFP itself can produce a similar artifact. Unless linked to a granule-targeting sequence, the larger molecular weight of GFP excludes it from diffusion across granule membranes. This guarantees that the excitation path-length available to cytosolic GFP is thus reduced by the presence of GFP-excluding granules (Fig. 2 b). It is therefore expected that, in addition to the light-scattering related phenomenon demonstrated for granule-permeant fluors such as fluorescein, the reduced light path effect would further reduce the GFP signal detected from the granular cytoplasm and thus increase its relative intensity in the granule-free cytoplasm. As with fluorescein, there was a pronounced increase in fluorescent signal at the leading edge (Fig. 5 a) and within the pseudopodia as they formed (Fig. 5 b), with an enhancement of 1.6-fold (significantly higher than fluorescein alone). The zones of increased fluorescence correlated with the organelle-free zones in the cells (Fig. 5 b) demarcated at the organelle free-granule containing boundary (Fig. 5 c). In addition, the total fluorescent signal from the cell increased during pseudopodia formation. It is conceivable that the asymmetrical distribution of GFP arose from a protein–protein interaction, such as tight binding of GFP to proteins at the leading edge. This is unlikely, as laser photobleaching at the pole opposite the zone of increased fluorescence intensity caused a decrease in the intensity of the entire GFP pool (Fig. 5 d). If GFP had accumulated at the leading edge as a result of binding to another protein, it would have been insulated from this localized photobleaching effect. However, there was no difference in the rate of decrease of GFP fluorescence in the bulk cytoplasm or the organelle-free cytoplasm, suggesting that the GFP was equally free to diffuse in both locations (Fig. 5 e). In addition, the asymmetries in GFP fluorescence were dynamic, forming and relaxing with pseudopodia formation (Fig. 5 and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1).

Bottom Line: Localized increases in the signal from cytosolic fluorescent protein constructs, for example, are frequently used as evidence for translocation of proteins to specific sites within the cell.However, differences in optical and geometrical properties of cytoplasm can influence the recorded intensity of the probe signal.Pseudopodia are especially problematic because their cytoplasmic properties can cause abrupt increases in fluorescent signal of both GFP and fluorescein.

View Article: PubMed Central - PubMed

Affiliation: Neutrophil Signalling Group, School of Medicine, Cardiff University, Heath Park, Cardiff, Wales, UK.

ABSTRACT
The use of fluorescent probes is one of the most powerful techniques for gaining spatial and temporal knowledge of dynamic events within living cells. Localized increases in the signal from cytosolic fluorescent protein constructs, for example, are frequently used as evidence for translocation of proteins to specific sites within the cell. However, differences in optical and geometrical properties of cytoplasm can influence the recorded intensity of the probe signal. Pseudopodia are especially problematic because their cytoplasmic properties can cause abrupt increases in fluorescent signal of both GFP and fluorescein. Investigators should therefore be cautious when interpreting fluorescence changes within a cell, as these can result from either translocation of the probe or changes in the optical properties of the milieu surrounding the probe.

Show MeSH
Related in: MedlinePlus