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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 fluorescein. The dynamic nature of the fluorescence asymmetry of fluorescein is shown (a) in a time sequence of images and (b) as a graph of total cellular and cytosolic fluorescent signals. The complete time sequence is shown in Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1). (c) The correlation between the increased fluorescence at the leading edge and the appearance of organelle-free cytoplasm is shown in the time sequence in which the top panel shows fluorescein fluorescence and the bottom panel the corresponding phase-contrast images. Images are shown at 10-s intervals. The maximum projection of organelle-free cytoplasm is marked by an asterisk in the third image pair. (d) The typical distribution of fluorescence intensity of cytoplasmic fluorescein along the axis of a polarized human neutrophil is shown. Bars: (a) 10 µm; (c) 8 µm; (d) 5 µm.
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fig4: Asymmetrical signals with cytoplasmic fluorescein. The dynamic nature of the fluorescence asymmetry of fluorescein is shown (a) in a time sequence of images and (b) as a graph of total cellular and cytosolic fluorescent signals. The complete time sequence is shown in Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1). (c) The correlation between the increased fluorescence at the leading edge and the appearance of organelle-free cytoplasm is shown in the time sequence in which the top panel shows fluorescein fluorescence and the bottom panel the corresponding phase-contrast images. Images are shown at 10-s intervals. The maximum projection of organelle-free cytoplasm is marked by an asterisk in the third image pair. (d) The typical distribution of fluorescence intensity of cytoplasmic fluorescein along the axis of a polarized human neutrophil is shown. Bars: (a) 10 µm; (c) 8 µm; (d) 5 µm.

Mentions: The magnitude of this problem can be easily demonstrated in human neutrophils loaded with the diacetate ester form of fluorescein. As the cells polarize and extend pseudopodia, areas of increased fluorescent signal (∼1.4-fold) become apparent. These areas correlate with sites of granule-free zones in the cell and move dynamically (Fig. 4). If this effect were the result of fluor redistribution within the cell, the total fluorescent signal would remain constant (i.e., the fluorescent signal would decrease in some sub-cellular locations and increase in others). However, in this example, the total fluorescent signal from the cell rises and falls as pseudopodia form (Fig. 4, a and b; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1). These observations are diagnostic of the spatial-optical artifact and are consistent with the formation of zones within the cell which have improved properties for excitation, not redistribution, of fluorescein. However, fluorescein is not ideal for this demonstration as its intensity is also dependent on pH (Tsien, 1989), yet the sharp boundary between high and low fluorescent zones (Fig. 4 d) makes it unlikely that fluorescein is reporting a pH gradient here. Furthermore, the same effects are also observed with other small molecular probes, such as eosin, and more importantly, by pH-insensitive GFP (Shaner et al., 2005).


Translocation or just location? Pseudopodia affect fluorescent signals.

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

Asymmetrical signals with cytoplasmic fluorescein. The dynamic nature of the fluorescence asymmetry of fluorescein is shown (a) in a time sequence of images and (b) as a graph of total cellular and cytosolic fluorescent signals. The complete time sequence is shown in Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1). (c) The correlation between the increased fluorescence at the leading edge and the appearance of organelle-free cytoplasm is shown in the time sequence in which the top panel shows fluorescein fluorescence and the bottom panel the corresponding phase-contrast images. Images are shown at 10-s intervals. The maximum projection of organelle-free cytoplasm is marked by an asterisk in the third image pair. (d) The typical distribution of fluorescence intensity of cytoplasmic fluorescein along the axis of a polarized human neutrophil is shown. Bars: (a) 10 µm; (c) 8 µm; (d) 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654297&req=5

fig4: Asymmetrical signals with cytoplasmic fluorescein. The dynamic nature of the fluorescence asymmetry of fluorescein is shown (a) in a time sequence of images and (b) as a graph of total cellular and cytosolic fluorescent signals. The complete time sequence is shown in Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1). (c) The correlation between the increased fluorescence at the leading edge and the appearance of organelle-free cytoplasm is shown in the time sequence in which the top panel shows fluorescein fluorescence and the bottom panel the corresponding phase-contrast images. Images are shown at 10-s intervals. The maximum projection of organelle-free cytoplasm is marked by an asterisk in the third image pair. (d) The typical distribution of fluorescence intensity of cytoplasmic fluorescein along the axis of a polarized human neutrophil is shown. Bars: (a) 10 µm; (c) 8 µm; (d) 5 µm.
Mentions: The magnitude of this problem can be easily demonstrated in human neutrophils loaded with the diacetate ester form of fluorescein. As the cells polarize and extend pseudopodia, areas of increased fluorescent signal (∼1.4-fold) become apparent. These areas correlate with sites of granule-free zones in the cell and move dynamically (Fig. 4). If this effect were the result of fluor redistribution within the cell, the total fluorescent signal would remain constant (i.e., the fluorescent signal would decrease in some sub-cellular locations and increase in others). However, in this example, the total fluorescent signal from the cell rises and falls as pseudopodia form (Fig. 4, a and b; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1). These observations are diagnostic of the spatial-optical artifact and are consistent with the formation of zones within the cell which have improved properties for excitation, not redistribution, of fluorescein. However, fluorescein is not ideal for this demonstration as its intensity is also dependent on pH (Tsien, 1989), yet the sharp boundary between high and low fluorescent zones (Fig. 4 d) makes it unlikely that fluorescein is reporting a pH gradient here. Furthermore, the same effects are also observed with other small molecular probes, such as eosin, and more importantly, by pH-insensitive GFP (Shaner et al., 2005).

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