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Crosstalk between second messengers predicts the motility of the growth cone.

Kobayashi T, Nagase F, Hotta K, Oka K - Sci Rep (2013)

Bottom Line: Axon guidance involves multiple second messenger signal transduction pathways.Here, we applied a simultaneous second messenger imaging method to the growth cone and demonstrated correlations between cAMP, cGMP, and Ca(2+).These results indicate that we succeed in relating second messenger crosstalk to growth cone deviation and extension, and also indicate the possibility of predicting axon guidance from this second messenger crosstalk.

View Article: PubMed Central - PubMed

Affiliation: Center for Biosciences and Informatics, School of Fundamental Sciences and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa, Japan.

ABSTRACT
Axon guidance involves multiple second messenger signal transduction pathways. Although each signal transduction pathway has been characterized, only a few studies have examined crosstalk between these cascades. Here, we applied a simultaneous second messenger imaging method to the growth cone and demonstrated correlations between cAMP, cGMP, and Ca(2+). The levels of cAMP and cGMP in non-stimulated freely extending growth cones showed a negative correlation without delay. Although there was no direct correlation between cAMP and Ca(2+), examination of cross correlations using small time windows showed frequent switching behavior from negative to positive and vice versa. Furthermore, spatially asymmetric cAMP and cGMP signals in freely deviating growth cones were visualized directly. These results indicate that we succeed in relating second messenger crosstalk to growth cone deviation and extension, and also indicate the possibility of predicting axon guidance from this second messenger crosstalk.

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Correlations between cAMP, cGMP, and Ca2+ in growth cones.(a) Representative fluorescence image of an Epac1-camps and red cGES-DE5 co-expressing growth cone. Lines drawn as cyan, green, yellow, and orange indicate the cell edge at 0, 20, 40, and 60 min after the onset of image acquisition, respectively. Scale bar represents 10 μm. (b) Spontaneous changes of ΔR/Raverage of ECFP/EYFP (cAMP) and T-Sapphire/dimer2 (cGMP) averaged within the ROIs shown in (a). (c) Negative correlation between cAMP and cGMP levels in the growth cones (correlation coefficient is −0.71, 5611 time points from 13 growth cones). (d) Cross correlation function between the spontaneous dynamics of cAMP and cGMP in the growth cones (n = 13) showed a negative correlation without delay. Data are presented as means ± s.e.m. (e) Representative fluorescence image of an Epac1-camps and SapRC2.12 co-expressing growth cone. The colored lines are the same as in (a). Scale bar represents 10 μm. (f) Representative time course changes in spontaneous ΔR/Raverage values of ECFP/EYFP (cAMP) and dimer2/T-Sapphire (Ca2+) averaged within the ROIs shown in (e). (g) No correlation between cAMP and Ca2+ levels in the growth cones (correlation coefficient is −0.19, 8529 time points from 27 growth cones). (h) Heat map showing representative temporal changes in the cross correlation function between the short-term dynamics of cAMP and Ca2+ shown in (f) (see Methods).
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f1: Correlations between cAMP, cGMP, and Ca2+ in growth cones.(a) Representative fluorescence image of an Epac1-camps and red cGES-DE5 co-expressing growth cone. Lines drawn as cyan, green, yellow, and orange indicate the cell edge at 0, 20, 40, and 60 min after the onset of image acquisition, respectively. Scale bar represents 10 μm. (b) Spontaneous changes of ΔR/Raverage of ECFP/EYFP (cAMP) and T-Sapphire/dimer2 (cGMP) averaged within the ROIs shown in (a). (c) Negative correlation between cAMP and cGMP levels in the growth cones (correlation coefficient is −0.71, 5611 time points from 13 growth cones). (d) Cross correlation function between the spontaneous dynamics of cAMP and cGMP in the growth cones (n = 13) showed a negative correlation without delay. Data are presented as means ± s.e.m. (e) Representative fluorescence image of an Epac1-camps and SapRC2.12 co-expressing growth cone. The colored lines are the same as in (a). Scale bar represents 10 μm. (f) Representative time course changes in spontaneous ΔR/Raverage values of ECFP/EYFP (cAMP) and dimer2/T-Sapphire (Ca2+) averaged within the ROIs shown in (e). (g) No correlation between cAMP and Ca2+ levels in the growth cones (correlation coefficient is −0.19, 8529 time points from 27 growth cones). (h) Heat map showing representative temporal changes in the cross correlation function between the short-term dynamics of cAMP and Ca2+ shown in (f) (see Methods).

Mentions: A negative correlation between cAMP and cGMP levels in non-stimulated freely extending growth cones was visualized directly by simultaneous imaging (Fig. 1a–c). The rate of axon extension was unaffected by the excitation laser and the expression of the FRET sensors (Supplementary Fig. 5). As expected, these results support previous studies using pharmacological stimuli89, and indicate that cAMP and cGMP are regulated reciprocally via an endogenous constitutive mechanism. Additionally, in the experiment to acquire images every 20 s, there was no delay in their correlation in freely extending growth cones (Fig. 1d). This negative correlation may be caused from ensembles of the phosphodiesterase family that have cAMP- and cGMP-selective characteristics81415. In contrast, there was no correlation between cAMP and Ca2+ (Fig. 1e–g). However, examination of cross correlations using small time windows showed frequent switching behavior from negative to positive and vice versa (Fig. 1h). The mechanism underlying this result may be positive feedback regulation of Ca2+ and cAMP67.


Crosstalk between second messengers predicts the motility of the growth cone.

Kobayashi T, Nagase F, Hotta K, Oka K - Sci Rep (2013)

Correlations between cAMP, cGMP, and Ca2+ in growth cones.(a) Representative fluorescence image of an Epac1-camps and red cGES-DE5 co-expressing growth cone. Lines drawn as cyan, green, yellow, and orange indicate the cell edge at 0, 20, 40, and 60 min after the onset of image acquisition, respectively. Scale bar represents 10 μm. (b) Spontaneous changes of ΔR/Raverage of ECFP/EYFP (cAMP) and T-Sapphire/dimer2 (cGMP) averaged within the ROIs shown in (a). (c) Negative correlation between cAMP and cGMP levels in the growth cones (correlation coefficient is −0.71, 5611 time points from 13 growth cones). (d) Cross correlation function between the spontaneous dynamics of cAMP and cGMP in the growth cones (n = 13) showed a negative correlation without delay. Data are presented as means ± s.e.m. (e) Representative fluorescence image of an Epac1-camps and SapRC2.12 co-expressing growth cone. The colored lines are the same as in (a). Scale bar represents 10 μm. (f) Representative time course changes in spontaneous ΔR/Raverage values of ECFP/EYFP (cAMP) and dimer2/T-Sapphire (Ca2+) averaged within the ROIs shown in (e). (g) No correlation between cAMP and Ca2+ levels in the growth cones (correlation coefficient is −0.19, 8529 time points from 27 growth cones). (h) Heat map showing representative temporal changes in the cross correlation function between the short-term dynamics of cAMP and Ca2+ shown in (f) (see Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814829&req=5

f1: Correlations between cAMP, cGMP, and Ca2+ in growth cones.(a) Representative fluorescence image of an Epac1-camps and red cGES-DE5 co-expressing growth cone. Lines drawn as cyan, green, yellow, and orange indicate the cell edge at 0, 20, 40, and 60 min after the onset of image acquisition, respectively. Scale bar represents 10 μm. (b) Spontaneous changes of ΔR/Raverage of ECFP/EYFP (cAMP) and T-Sapphire/dimer2 (cGMP) averaged within the ROIs shown in (a). (c) Negative correlation between cAMP and cGMP levels in the growth cones (correlation coefficient is −0.71, 5611 time points from 13 growth cones). (d) Cross correlation function between the spontaneous dynamics of cAMP and cGMP in the growth cones (n = 13) showed a negative correlation without delay. Data are presented as means ± s.e.m. (e) Representative fluorescence image of an Epac1-camps and SapRC2.12 co-expressing growth cone. The colored lines are the same as in (a). Scale bar represents 10 μm. (f) Representative time course changes in spontaneous ΔR/Raverage values of ECFP/EYFP (cAMP) and dimer2/T-Sapphire (Ca2+) averaged within the ROIs shown in (e). (g) No correlation between cAMP and Ca2+ levels in the growth cones (correlation coefficient is −0.19, 8529 time points from 27 growth cones). (h) Heat map showing representative temporal changes in the cross correlation function between the short-term dynamics of cAMP and Ca2+ shown in (f) (see Methods).
Mentions: A negative correlation between cAMP and cGMP levels in non-stimulated freely extending growth cones was visualized directly by simultaneous imaging (Fig. 1a–c). The rate of axon extension was unaffected by the excitation laser and the expression of the FRET sensors (Supplementary Fig. 5). As expected, these results support previous studies using pharmacological stimuli89, and indicate that cAMP and cGMP are regulated reciprocally via an endogenous constitutive mechanism. Additionally, in the experiment to acquire images every 20 s, there was no delay in their correlation in freely extending growth cones (Fig. 1d). This negative correlation may be caused from ensembles of the phosphodiesterase family that have cAMP- and cGMP-selective characteristics81415. In contrast, there was no correlation between cAMP and Ca2+ (Fig. 1e–g). However, examination of cross correlations using small time windows showed frequent switching behavior from negative to positive and vice versa (Fig. 1h). The mechanism underlying this result may be positive feedback regulation of Ca2+ and cAMP67.

Bottom Line: Axon guidance involves multiple second messenger signal transduction pathways.Here, we applied a simultaneous second messenger imaging method to the growth cone and demonstrated correlations between cAMP, cGMP, and Ca(2+).These results indicate that we succeed in relating second messenger crosstalk to growth cone deviation and extension, and also indicate the possibility of predicting axon guidance from this second messenger crosstalk.

View Article: PubMed Central - PubMed

Affiliation: Center for Biosciences and Informatics, School of Fundamental Sciences and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa, Japan.

ABSTRACT
Axon guidance involves multiple second messenger signal transduction pathways. Although each signal transduction pathway has been characterized, only a few studies have examined crosstalk between these cascades. Here, we applied a simultaneous second messenger imaging method to the growth cone and demonstrated correlations between cAMP, cGMP, and Ca(2+). The levels of cAMP and cGMP in non-stimulated freely extending growth cones showed a negative correlation without delay. Although there was no direct correlation between cAMP and Ca(2+), examination of cross correlations using small time windows showed frequent switching behavior from negative to positive and vice versa. Furthermore, spatially asymmetric cAMP and cGMP signals in freely deviating growth cones were visualized directly. These results indicate that we succeed in relating second messenger crosstalk to growth cone deviation and extension, and also indicate the possibility of predicting axon guidance from this second messenger crosstalk.

Show MeSH
Related in: MedlinePlus