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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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Localization of β-actin protein and actin polymerization in response to NT-3. (A) Immunofluorescence localization of β-actin protein in starved cells. Weak staining was observed within growth cone (arrow) (example of nonlocalized cell). (B) Addition of NT-3 to the medium for 5 min resulted in the presence of focal staining within the peripheral margin of minor neurites and the axonal growth cone (arrow) (example of localized cell). (C) Addition of NT-3 to the medium for 10 min resulted in the localization of β-actin protein throughout the peripheral margin (arrow). (D) Starved cells were extracted with saponin (0.1 mg/ml) in buffer containing rhodamine-actin (0.45 mM) for 1 min, washed, and fixed in paraformaldehyde (4% in 1 PBS). There was no fluorescence staining in these untreated cells. Nucleus was stained with DAPI. (E) By contrast, stimulation of cells with NT-3 for 5 min resulted in visualization of fluorescence signal within the growth cone (arrow). (F) Stimulation with NT-3 for 10 min resulted in the incorporation of rhodamine-actin throughout the peripheral margin (arrow). Bar, 15 mm.
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Figure 7: Localization of β-actin protein and actin polymerization in response to NT-3. (A) Immunofluorescence localization of β-actin protein in starved cells. Weak staining was observed within growth cone (arrow) (example of nonlocalized cell). (B) Addition of NT-3 to the medium for 5 min resulted in the presence of focal staining within the peripheral margin of minor neurites and the axonal growth cone (arrow) (example of localized cell). (C) Addition of NT-3 to the medium for 10 min resulted in the localization of β-actin protein throughout the peripheral margin (arrow). (D) Starved cells were extracted with saponin (0.1 mg/ml) in buffer containing rhodamine-actin (0.45 mM) for 1 min, washed, and fixed in paraformaldehyde (4% in 1 PBS). There was no fluorescence staining in these untreated cells. Nucleus was stained with DAPI. (E) By contrast, stimulation of cells with NT-3 for 5 min resulted in visualization of fluorescence signal within the growth cone (arrow). (F) Stimulation with NT-3 for 10 min resulted in the incorporation of rhodamine-actin throughout the peripheral margin (arrow). Bar, 15 mm.

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)

Localization of β-actin protein and actin polymerization in response to NT-3. (A) Immunofluorescence localization of β-actin protein in starved cells. Weak staining was observed within growth cone (arrow) (example of nonlocalized cell). (B) Addition of NT-3 to the medium for 5 min resulted in the presence of focal staining within the peripheral margin of minor neurites and the axonal growth cone (arrow) (example of localized cell). (C) Addition of NT-3 to the medium for 10 min resulted in the localization of β-actin protein throughout the peripheral margin (arrow). (D) Starved cells were extracted with saponin (0.1 mg/ml) in buffer containing rhodamine-actin (0.45 mM) for 1 min, washed, and fixed in paraformaldehyde (4% in 1 PBS). There was no fluorescence staining in these untreated cells. Nucleus was stained with DAPI. (E) By contrast, stimulation of cells with NT-3 for 5 min resulted in visualization of fluorescence signal within the growth cone (arrow). (F) Stimulation with NT-3 for 10 min resulted in the incorporation of rhodamine-actin throughout the peripheral margin (arrow). Bar, 15 mm.
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Related In: Results  -  Collection

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Figure 7: Localization of β-actin protein and actin polymerization in response to NT-3. (A) Immunofluorescence localization of β-actin protein in starved cells. Weak staining was observed within growth cone (arrow) (example of nonlocalized cell). (B) Addition of NT-3 to the medium for 5 min resulted in the presence of focal staining within the peripheral margin of minor neurites and the axonal growth cone (arrow) (example of localized cell). (C) Addition of NT-3 to the medium for 10 min resulted in the localization of β-actin protein throughout the peripheral margin (arrow). (D) Starved cells were extracted with saponin (0.1 mg/ml) in buffer containing rhodamine-actin (0.45 mM) for 1 min, washed, and fixed in paraformaldehyde (4% in 1 PBS). There was no fluorescence staining in these untreated cells. Nucleus was stained with DAPI. (E) By contrast, stimulation of cells with NT-3 for 5 min resulted in visualization of fluorescence signal within the growth cone (arrow). (F) Stimulation with NT-3 for 10 min resulted in the incorporation of rhodamine-actin throughout the peripheral margin (arrow). Bar, 15 mm.
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