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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 asymmetric cAMP/cGMP elevation and the deviation angle of growth cones during free extension.(a) Time-lapse fluorescence images of a freely extending growth cone co-expressing Epac1-camps and red cGES-DE5. The Roman numerals (i–v) indicate the time points shown in (b). (b) Time course of growth cone angle changes (see Methods). Right and left turns are defined as positive and negative values, respectively. The Roman numerals correspond those in (a). The orange horizontal line indicates the period when the maximum angle changes were calculated within 15 min after significant cAMP/cGMPright/left elevation (asterisk). A significant elevation was defined as a change that was 2.58-fold more than the derivative of cAMP/cGMPright/left and also cAMP/cGMPleft/right. The cyan line denotes the latency from a significant increase to the maximum angle change. (c) Spontaneous changes in the right/left ratios of cAMP/cGMP, cAMP, and cGMP (see Methods). (d) Cumulative distance during the observation of the growth cone in Fig. 2a. (e) Freely extending growth cones deviate toward the cAMP/cGMP elevated side. The maximum growth cone angle changes within 15 min after significant cAMP/cGMP elevation at the turning side were higher than in randomly selected cones (80 turnings from 14 growth cones). The randomly selected maximum growth cone angle changes were calculated as follows: select a time point from the overall time course and choose the maximum from within 15 min after the time point. Data are presented as means ± s.e.m. *P < 0.001.
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f2: Correlations between asymmetric cAMP/cGMP elevation and the deviation angle of growth cones during free extension.(a) Time-lapse fluorescence images of a freely extending growth cone co-expressing Epac1-camps and red cGES-DE5. The Roman numerals (i–v) indicate the time points shown in (b). (b) Time course of growth cone angle changes (see Methods). Right and left turns are defined as positive and negative values, respectively. The Roman numerals correspond those in (a). The orange horizontal line indicates the period when the maximum angle changes were calculated within 15 min after significant cAMP/cGMPright/left elevation (asterisk). A significant elevation was defined as a change that was 2.58-fold more than the derivative of cAMP/cGMPright/left and also cAMP/cGMPleft/right. The cyan line denotes the latency from a significant increase to the maximum angle change. (c) Spontaneous changes in the right/left ratios of cAMP/cGMP, cAMP, and cGMP (see Methods). (d) Cumulative distance during the observation of the growth cone in Fig. 2a. (e) Freely extending growth cones deviate toward the cAMP/cGMP elevated side. The maximum growth cone angle changes within 15 min after significant cAMP/cGMP elevation at the turning side were higher than in randomly selected cones (80 turnings from 14 growth cones). The randomly selected maximum growth cone angle changes were calculated as follows: select a time point from the overall time course and choose the maximum from within 15 min after the time point. Data are presented as means ± s.e.m. *P < 0.001.

Mentions: Spatially asymmetric cAMP and cGMP signals in freely deviating growth cones were visualized directly (Fig. 2a–d). There was no significant change in the right/left cAMP:cGMP ratio (cAMP/cGMPright/left) in freely extending growth cones. However, significant cAMP/cGMPright/left elevations were observed in the turning-side of the growth cones at 9.7 ± 0.5 min before growth cone angle change. We observed significant increases in the maximum angle change within 15 min after significant cAMP/cGMPright/left elevation (Fig. 2e). Although cytosolic cAMP/cGMP affects growth cone turning responses to netrin 19, little is known about the spontaneous dynamics of both cAMP and cGMP. Our result suggests that endogenous changes in cAMP and cGMP affect the growth cone turning angle. Consequently, our result supports a recent study that demonstrated cAMP transients driven by caged compounds steer axons7. The observed delay may reflect the signal transduction of cytoskeletal protein reorganization16.


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

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

Correlations between asymmetric cAMP/cGMP elevation and the deviation angle of growth cones during free extension.(a) Time-lapse fluorescence images of a freely extending growth cone co-expressing Epac1-camps and red cGES-DE5. The Roman numerals (i–v) indicate the time points shown in (b). (b) Time course of growth cone angle changes (see Methods). Right and left turns are defined as positive and negative values, respectively. The Roman numerals correspond those in (a). The orange horizontal line indicates the period when the maximum angle changes were calculated within 15 min after significant cAMP/cGMPright/left elevation (asterisk). A significant elevation was defined as a change that was 2.58-fold more than the derivative of cAMP/cGMPright/left and also cAMP/cGMPleft/right. The cyan line denotes the latency from a significant increase to the maximum angle change. (c) Spontaneous changes in the right/left ratios of cAMP/cGMP, cAMP, and cGMP (see Methods). (d) Cumulative distance during the observation of the growth cone in Fig. 2a. (e) Freely extending growth cones deviate toward the cAMP/cGMP elevated side. The maximum growth cone angle changes within 15 min after significant cAMP/cGMP elevation at the turning side were higher than in randomly selected cones (80 turnings from 14 growth cones). The randomly selected maximum growth cone angle changes were calculated as follows: select a time point from the overall time course and choose the maximum from within 15 min after the time point. Data are presented as means ± s.e.m. *P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Correlations between asymmetric cAMP/cGMP elevation and the deviation angle of growth cones during free extension.(a) Time-lapse fluorescence images of a freely extending growth cone co-expressing Epac1-camps and red cGES-DE5. The Roman numerals (i–v) indicate the time points shown in (b). (b) Time course of growth cone angle changes (see Methods). Right and left turns are defined as positive and negative values, respectively. The Roman numerals correspond those in (a). The orange horizontal line indicates the period when the maximum angle changes were calculated within 15 min after significant cAMP/cGMPright/left elevation (asterisk). A significant elevation was defined as a change that was 2.58-fold more than the derivative of cAMP/cGMPright/left and also cAMP/cGMPleft/right. The cyan line denotes the latency from a significant increase to the maximum angle change. (c) Spontaneous changes in the right/left ratios of cAMP/cGMP, cAMP, and cGMP (see Methods). (d) Cumulative distance during the observation of the growth cone in Fig. 2a. (e) Freely extending growth cones deviate toward the cAMP/cGMP elevated side. The maximum growth cone angle changes within 15 min after significant cAMP/cGMP elevation at the turning side were higher than in randomly selected cones (80 turnings from 14 growth cones). The randomly selected maximum growth cone angle changes were calculated as follows: select a time point from the overall time course and choose the maximum from within 15 min after the time point. Data are presented as means ± s.e.m. *P < 0.001.
Mentions: Spatially asymmetric cAMP and cGMP signals in freely deviating growth cones were visualized directly (Fig. 2a–d). There was no significant change in the right/left cAMP:cGMP ratio (cAMP/cGMPright/left) in freely extending growth cones. However, significant cAMP/cGMPright/left elevations were observed in the turning-side of the growth cones at 9.7 ± 0.5 min before growth cone angle change. We observed significant increases in the maximum angle change within 15 min after significant cAMP/cGMPright/left elevation (Fig. 2e). Although cytosolic cAMP/cGMP affects growth cone turning responses to netrin 19, little is known about the spontaneous dynamics of both cAMP and cGMP. Our result suggests that endogenous changes in cAMP and cGMP affect the growth cone turning angle. Consequently, our result supports a recent study that demonstrated cAMP transients driven by caged compounds steer axons7. The observed delay may reflect the signal transduction of cytoskeletal protein reorganization16.

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