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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 fluors during bleb formation. The typical distribution of fluorescence intensity of cytoplasmic GFP along the axis indicated of a human neutrophil (a) and a Dictyostelium induced to bleb by ionophore and high extracellular Ca2+ (a′) (13 mM). (b) The fluorescence enhancement in the blebs, marked in the histogram “bl”, was similar to that observed with organelle-free cytoplasm at the leading edge or in phagocytic pseudopodia (marked “ps”) and was seen for both GFP and fluorescein (marked “GFP” and “fluor”, respectively [n = 17, 4, 8, and 5 for each column in order]). (c) The correlation between the increased fluorescence and the appearance of cytoplasmic blebs is shown in this time sequence in which the top panel shows fluorescein fluorescence and the bottom panel the corresponding phase-contrast images. The location of two blebs are marked by asterisks in the third image pair. Bars: (a) 8 µm; (c) 10 µm. The complete data for bleb formation and localized intensity increase are shown in Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1).
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fig6: Asymmetrical signals with cytoplasmic fluors during bleb formation. The typical distribution of fluorescence intensity of cytoplasmic GFP along the axis indicated of a human neutrophil (a) and a Dictyostelium induced to bleb by ionophore and high extracellular Ca2+ (a′) (13 mM). (b) The fluorescence enhancement in the blebs, marked in the histogram “bl”, was similar to that observed with organelle-free cytoplasm at the leading edge or in phagocytic pseudopodia (marked “ps”) and was seen for both GFP and fluorescein (marked “GFP” and “fluor”, respectively [n = 17, 4, 8, and 5 for each column in order]). (c) The correlation between the increased fluorescence and the appearance of cytoplasmic blebs is shown in this time sequence in which the top panel shows fluorescein fluorescence and the bottom panel the corresponding phase-contrast images. The location of two blebs are marked by asterisks in the third image pair. Bars: (a) 8 µm; (c) 10 µm. The complete data for bleb formation and localized intensity increase are shown in Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1).

Mentions: As a further demonstration that the intensity of GFP is related to the localized and unique optical properties of granule-free cytoplasm rather than to physiologically induced polarization or pseudopodia formation, granule-free zones were created artificially by elevating cytosolic free Ca2+ to pathologically high levels using Ca2+ ionophore and high extracellular Ca2+. Under these conditions, the normally wrinkled plasma membrane unfurls uncontrollably giving rise to “blebs” (Hallett and Dewitt, 2007). These blebs are detached from the actin cytoskeleton (Charras et al., 2006) and are devoid of organelles or granules. The mechanism by which they form is therefore dissimilar to the dynamic pseudopodia formation at the leading edge of motile cells, yet cytosolic GFP or fluorescein showed a similar increase in intensity at sites of membrane blebbing (Fig. 6, a and c). This effect is more prominent in cord blood–derived granulocytes (Fig. 6 a) and primary neutrophils (Fig. 6 c) than in Dictyostelium (Fig. 6 a'), partly because of the smaller and transient nature of the blebs that form in Dictyostelium. However, even in Dictyostelium, fluorescence intensity is increased at the granule free-cytoplasmic boundary (Fig. 6 a'). In neutrophils, the geometry of the longer-lived granule-free “blebs,” which are hemispherical, is different from the more planar pseudopodia (Fig. 6 c). An optical section can easily be found that passes through the equators of both the cell and the bleb, removing any possible sampling basis. Under these conditions, there is a similar increase in fluorescence intensity observed in the bleb as in the leading edge or phagocytic pseudopodia of neutrophils. In addition, intensity increase is independent of the size of the bleb, as predicted (Fig. 6).


Translocation or just location? Pseudopodia affect fluorescent signals.

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

Asymmetrical signals with cytoplasmic fluors during bleb formation. The typical distribution of fluorescence intensity of cytoplasmic GFP along the axis indicated of a human neutrophil (a) and a Dictyostelium induced to bleb by ionophore and high extracellular Ca2+ (a′) (13 mM). (b) The fluorescence enhancement in the blebs, marked in the histogram “bl”, was similar to that observed with organelle-free cytoplasm at the leading edge or in phagocytic pseudopodia (marked “ps”) and was seen for both GFP and fluorescein (marked “GFP” and “fluor”, respectively [n = 17, 4, 8, and 5 for each column in order]). (c) The correlation between the increased fluorescence and the appearance of cytoplasmic blebs is shown in this time sequence in which the top panel shows fluorescein fluorescence and the bottom panel the corresponding phase-contrast images. The location of two blebs are marked by asterisks in the third image pair. Bars: (a) 8 µm; (c) 10 µm. The complete data for bleb formation and localized intensity increase are shown in Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: Asymmetrical signals with cytoplasmic fluors during bleb formation. The typical distribution of fluorescence intensity of cytoplasmic GFP along the axis indicated of a human neutrophil (a) and a Dictyostelium induced to bleb by ionophore and high extracellular Ca2+ (a′) (13 mM). (b) The fluorescence enhancement in the blebs, marked in the histogram “bl”, was similar to that observed with organelle-free cytoplasm at the leading edge or in phagocytic pseudopodia (marked “ps”) and was seen for both GFP and fluorescein (marked “GFP” and “fluor”, respectively [n = 17, 4, 8, and 5 for each column in order]). (c) The correlation between the increased fluorescence and the appearance of cytoplasmic blebs is shown in this time sequence in which the top panel shows fluorescein fluorescence and the bottom panel the corresponding phase-contrast images. The location of two blebs are marked by asterisks in the third image pair. Bars: (a) 8 µm; (c) 10 µm. The complete data for bleb formation and localized intensity increase are shown in Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1).
Mentions: As a further demonstration that the intensity of GFP is related to the localized and unique optical properties of granule-free cytoplasm rather than to physiologically induced polarization or pseudopodia formation, granule-free zones were created artificially by elevating cytosolic free Ca2+ to pathologically high levels using Ca2+ ionophore and high extracellular Ca2+. Under these conditions, the normally wrinkled plasma membrane unfurls uncontrollably giving rise to “blebs” (Hallett and Dewitt, 2007). These blebs are detached from the actin cytoskeleton (Charras et al., 2006) and are devoid of organelles or granules. The mechanism by which they form is therefore dissimilar to the dynamic pseudopodia formation at the leading edge of motile cells, yet cytosolic GFP or fluorescein showed a similar increase in intensity at sites of membrane blebbing (Fig. 6, a and c). This effect is more prominent in cord blood–derived granulocytes (Fig. 6 a) and primary neutrophils (Fig. 6 c) than in Dictyostelium (Fig. 6 a'), partly because of the smaller and transient nature of the blebs that form in Dictyostelium. However, even in Dictyostelium, fluorescence intensity is increased at the granule free-cytoplasmic boundary (Fig. 6 a'). In neutrophils, the geometry of the longer-lived granule-free “blebs,” which are hemispherical, is different from the more planar pseudopodia (Fig. 6 c). An optical section can easily be found that passes through the equators of both the cell and the bleb, removing any possible sampling basis. Under these conditions, there is a similar increase in fluorescence intensity observed in the bleb as in the leading edge or phagocytic pseudopodia of neutrophils. In addition, intensity increase is independent of the size of the bleb, as predicted (Fig. 6).

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