Limits...
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.

Show MeSH
Fluorescence intensity increases of GFP constructs in forming pseudopodia. The artifactual increases in fluorescent signal are evident in forming pseudopodia in neutrophilic HL60 cells expressing p67phox-GFP (a) and PH-Akt-GFP (b). The optical artifact is evident in the forming pseudopodia of these cells (gray arrows). However, this artifact can be distinguished from the genuine translocation of PH-Akt-GFP to the phagosomal membrane (red arrow). Bars, 5 µm
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: Fluorescence intensity increases of GFP constructs in forming pseudopodia. The artifactual increases in fluorescent signal are evident in forming pseudopodia in neutrophilic HL60 cells expressing p67phox-GFP (a) and PH-Akt-GFP (b). The optical artifact is evident in the forming pseudopodia of these cells (gray arrows). However, this artifact can be distinguished from the genuine translocation of PH-Akt-GFP to the phagosomal membrane (red arrow). Bars, 5 µm

Mentions: This should strike a note of caution, but not of despair. First, the artifact can be easily distinguished from protein translocation by monitoring total cell fluorescence (Fig. 7). Appropriate controls, such as an irrelevant or nonsignaling GFP chimeric protein, should also be used to indicate the magnitude of the artifact. Second, not all cells are equally prone to the problem, and imaging cells with granular cytoplasms has an increased risk for flawed interpretation of localization data. Finally, once the problem has been recognized, there are also solutions. For example, taking the ratio of fluorescent images from signaling and “control” probes is often used when investigating pseudopodia formation or phagocytosis. There are examples of this good practice in the papers from Swanson's group (Kamen et al., 2007, 2008). Also, the depth attenuation effect shown in Fig. 3 makes the problem worse at optical sections deeper within the cell. Therefore, another simple precaution is to compare images taken at different optical sections. In our hands, the translocation of PH-Akt-GFP to forming phagocytic cups does not correlate temporally or spatially with optically clear pseudopodia (Dewitt et al., 2006). Instead, the increased fluorescent signal is associated with the plasma membrane, as expected from a probe for PIP3. To exclude the possibility that the increased intensity was due to submicroscopic convolution of the membrane, DiIC16(3) was used as an appropriate control marker of the plasma membrane (Dewitt et al., 2006). Ratio images of the control fluorescence and the probe confirms that the increase in intensity of the probes could not be explicable by a membrane convolution effect or other “nonspecific” effect (Fig. 8).


Translocation or just location? Pseudopodia affect fluorescent signals.

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

Fluorescence intensity increases of GFP constructs in forming pseudopodia. The artifactual increases in fluorescent signal are evident in forming pseudopodia in neutrophilic HL60 cells expressing p67phox-GFP (a) and PH-Akt-GFP (b). The optical artifact is evident in the forming pseudopodia of these cells (gray arrows). However, this artifact can be distinguished from the genuine translocation of PH-Akt-GFP to the phagosomal membrane (red arrow). Bars, 5 µm
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: Fluorescence intensity increases of GFP constructs in forming pseudopodia. The artifactual increases in fluorescent signal are evident in forming pseudopodia in neutrophilic HL60 cells expressing p67phox-GFP (a) and PH-Akt-GFP (b). The optical artifact is evident in the forming pseudopodia of these cells (gray arrows). However, this artifact can be distinguished from the genuine translocation of PH-Akt-GFP to the phagosomal membrane (red arrow). Bars, 5 µm
Mentions: This should strike a note of caution, but not of despair. First, the artifact can be easily distinguished from protein translocation by monitoring total cell fluorescence (Fig. 7). Appropriate controls, such as an irrelevant or nonsignaling GFP chimeric protein, should also be used to indicate the magnitude of the artifact. Second, not all cells are equally prone to the problem, and imaging cells with granular cytoplasms has an increased risk for flawed interpretation of localization data. Finally, once the problem has been recognized, there are also solutions. For example, taking the ratio of fluorescent images from signaling and “control” probes is often used when investigating pseudopodia formation or phagocytosis. There are examples of this good practice in the papers from Swanson's group (Kamen et al., 2007, 2008). Also, the depth attenuation effect shown in Fig. 3 makes the problem worse at optical sections deeper within the cell. Therefore, another simple precaution is to compare images taken at different optical sections. In our hands, the translocation of PH-Akt-GFP to forming phagocytic cups does not correlate temporally or spatially with optically clear pseudopodia (Dewitt et al., 2006). Instead, the increased fluorescent signal is associated with the plasma membrane, as expected from a probe for PIP3. To exclude the possibility that the increased intensity was due to submicroscopic convolution of the membrane, DiIC16(3) was used as an appropriate control marker of the plasma membrane (Dewitt et al., 2006). Ratio images of the control fluorescence and the probe confirms that the increase in intensity of the probes could not be explicable by a membrane convolution effect or other “nonspecific” effect (Fig. 8).

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