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Neurotrophin regulation of beta-actin mRNA and protein localization within growth cones.

Zhang HL, Singer RH, Bassell GJ - J. Cell Biol. (1999)

Bottom Line: NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA.Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of beta-actin mRNA.These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

ABSTRACT
Neurotrophins play an essential role in the regulation of actin-dependent changes in growth cone shape and motility. We have studied whether neurotrophin signaling can promote the localization of beta-actin mRNA and protein within growth cones. The regulated localization of specific mRNAs within neuronal processes and growth cones could provide a mechanism to modulate cytoskeletal composition and growth cone dynamics during neuronal development. We have previously shown that beta-actin mRNA is localized in granules that were distributed throughout processes and growth cones of cultured neurons. In this study, we demonstrate that the localization of beta-actin mRNA and protein to growth cones of forebrain neurons is stimulated by neurotrophin-3 (NT-3). A similar response was observed when neurons were exposed to forskolin or db-cAMP, suggesting an involvement of a cAMP signaling pathway. NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA. Localization of beta-actin mRNA was blocked by prior treatment of cells with Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A. Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of beta-actin mRNA. These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones.

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Time course of β-actin mRNA and protein localization to growth cones after NT-3 treatment. Neurons were cultured for 4 d and starved in MEM for 6 h. NT-3 (25 ng/ml) was added to the medium for indicated time, and cells were fixed for in situ hybridization and immunofluorescence analysis. A rapid increase in β-actin protein localization (2 min) was observed before maximal localization of β-actin mRNA within growth cones (10 min).
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Figure 6: Time course of β-actin mRNA and protein localization to growth cones after NT-3 treatment. Neurons were cultured for 4 d and starved in MEM for 6 h. NT-3 (25 ng/ml) was added to the medium for indicated time, and cells were fixed for in situ hybridization and immunofluorescence analysis. A rapid increase in β-actin protein localization (2 min) was observed before maximal localization of β-actin mRNA within growth cones (10 min).

Mentions: The above results using cytochalasin suggest a specific mechanism to localize β-actin mRNA that is independent of any signals that NT-3 may directly impart on new actin synthesis and polymerization. We propose that the first neuronal response to NT-3 is to rapidly promote actin polymerization within the peripheral margin of the growth cone that is then followed by the microtubule-dependent targeting of β-actin mRNAs to the growth cone. We performed a time course experiment to determine whether β-actin protein localization may occur before and perhaps independent of mRNA localization. After starvation in MEM, 62% of the cells were scored as localized for β-actin protein (Fig. 6). NT-3 treatment for 2 min resulted in >85% of the cells being scored as localized for β-actin protein (Fig. 6). β-actin protein localization in response to NT-3 was initially concentrated in one or two foci within the peripheral margin, suggesting local accumulation and/or polymerization (Fig. 7 B). After several additional minutes, β-actin protein staining was observed throughout the entire peripheral margin (Fig. 7 C). An increase in β-actin mRNA localization was also observed after 2 min, although it took longer than β-actin protein to reach maximal levels, peaking at 10 min (Fig. 6). Therefore, the relocalization of β-actin mRNA was delayed relative to the protein localization. These results support the model that NT-3 signals may also promote the localization of β-actin protein by a mechanism independent of mRNA localization.


Neurotrophin regulation of beta-actin mRNA and protein localization within growth cones.

Zhang HL, Singer RH, Bassell GJ - J. Cell Biol. (1999)

Time course of β-actin mRNA and protein localization to growth cones after NT-3 treatment. Neurons were cultured for 4 d and starved in MEM for 6 h. NT-3 (25 ng/ml) was added to the medium for indicated time, and cells were fixed for in situ hybridization and immunofluorescence analysis. A rapid increase in β-actin protein localization (2 min) was observed before maximal localization of β-actin mRNA within growth cones (10 min).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164987&req=5

Figure 6: Time course of β-actin mRNA and protein localization to growth cones after NT-3 treatment. Neurons were cultured for 4 d and starved in MEM for 6 h. NT-3 (25 ng/ml) was added to the medium for indicated time, and cells were fixed for in situ hybridization and immunofluorescence analysis. A rapid increase in β-actin protein localization (2 min) was observed before maximal localization of β-actin mRNA within growth cones (10 min).
Mentions: The above results using cytochalasin suggest a specific mechanism to localize β-actin mRNA that is independent of any signals that NT-3 may directly impart on new actin synthesis and polymerization. We propose that the first neuronal response to NT-3 is to rapidly promote actin polymerization within the peripheral margin of the growth cone that is then followed by the microtubule-dependent targeting of β-actin mRNAs to the growth cone. We performed a time course experiment to determine whether β-actin protein localization may occur before and perhaps independent of mRNA localization. After starvation in MEM, 62% of the cells were scored as localized for β-actin protein (Fig. 6). NT-3 treatment for 2 min resulted in >85% of the cells being scored as localized for β-actin protein (Fig. 6). β-actin protein localization in response to NT-3 was initially concentrated in one or two foci within the peripheral margin, suggesting local accumulation and/or polymerization (Fig. 7 B). After several additional minutes, β-actin protein staining was observed throughout the entire peripheral margin (Fig. 7 C). An increase in β-actin mRNA localization was also observed after 2 min, although it took longer than β-actin protein to reach maximal levels, peaking at 10 min (Fig. 6). Therefore, the relocalization of β-actin mRNA was delayed relative to the protein localization. These results support the model that NT-3 signals may also promote the localization of β-actin protein by a mechanism independent of mRNA localization.

Bottom Line: NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA.Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of beta-actin mRNA.These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

ABSTRACT
Neurotrophins play an essential role in the regulation of actin-dependent changes in growth cone shape and motility. We have studied whether neurotrophin signaling can promote the localization of beta-actin mRNA and protein within growth cones. The regulated localization of specific mRNAs within neuronal processes and growth cones could provide a mechanism to modulate cytoskeletal composition and growth cone dynamics during neuronal development. We have previously shown that beta-actin mRNA is localized in granules that were distributed throughout processes and growth cones of cultured neurons. In this study, we demonstrate that the localization of beta-actin mRNA and protein to growth cones of forebrain neurons is stimulated by neurotrophin-3 (NT-3). A similar response was observed when neurons were exposed to forskolin or db-cAMP, suggesting an involvement of a cAMP signaling pathway. NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA. Localization of beta-actin mRNA was blocked by prior treatment of cells with Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A. Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of beta-actin mRNA. These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones.

Show MeSH
Related in: MedlinePlus