Limits...
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.

Show MeSH

Related in: MedlinePlus

β-actin mRNA and protein localization is influenced by neurotrophin-3 (NT-3). (A) β-actin mRNA was prominent in the cell body and localized within the axonal process and growth cone in the form of spatially distinct granules (arrow; example of localized cell). (B) Cells that were starved in minimal essential medium (MEM) for 6 h showed hybridization within the cell body but growth cones did not reveal mRNA granules (arrow; example of nonlocalized cell). (C) Cells that were starved for 6 h and then stimulated with NT-3 for 10 min were observed to relocalize β-actin mRNA granules within growth cones (arrow). (D) Relocalization of β-actin mRNA within growth cones after 2 h in NT-3–supplemented MEM (arrow). (E) Cells grown in normal defined medium showed intense staining for β-actin protein within the minor neurites and the peripheral margin of the axonal growth cone (arrow) (example of localized cell). (F) Starvation resulted in reduced staining for β-actin protein within growth cones (arrow) (example of nonlocalized cell). (G and H) NT-3 stimulation for 10 min and 2 h restored the localization of β-actin protein within the peripheral margin (arrows). Bar, 15 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2164987&req=5

Figure 1: β-actin mRNA and protein localization is influenced by neurotrophin-3 (NT-3). (A) β-actin mRNA was prominent in the cell body and localized within the axonal process and growth cone in the form of spatially distinct granules (arrow; example of localized cell). (B) Cells that were starved in minimal essential medium (MEM) for 6 h showed hybridization within the cell body but growth cones did not reveal mRNA granules (arrow; example of nonlocalized cell). (C) Cells that were starved for 6 h and then stimulated with NT-3 for 10 min were observed to relocalize β-actin mRNA granules within growth cones (arrow). (D) Relocalization of β-actin mRNA within growth cones after 2 h in NT-3–supplemented MEM (arrow). (E) Cells grown in normal defined medium showed intense staining for β-actin protein within the minor neurites and the peripheral margin of the axonal growth cone (arrow) (example of localized cell). (F) Starvation resulted in reduced staining for β-actin protein within growth cones (arrow) (example of nonlocalized cell). (G and H) NT-3 stimulation for 10 min and 2 h restored the localization of β-actin protein within the peripheral margin (arrows). Bar, 15 μm.

Mentions: For quantitative analysis using a visual scoring method, 100 cells per coverslip were analyzed for each cell culture condition. Experiments were done with duplicate coverslips for each variable and each experiment was repeated at least three times. The scoring method involved visualization of the presence or absence of β-actin mRNA granules in the axon-like growth cone from each cell. Cells were scored as localized if several granules were observed, and scored as nonlocalized if the signal was not distinguishable from background levels (hybridization with control probe). Localized cells would be expected to have a higher amount of fluorescent signal in growth cones compared with nonlocalized cells. Examples of localized and nonlocalized cells are shown in Fig. 1. This scoring method was used to show that NT-3 promotes an increase in localization (see Fig. 2) and that the results were comparable to the quantitation of fluorescence intensity using the CCD camera (see Fig. 3). The value for each bar within the histogram reports the mean and standard deviation between independent samples (see Fig. 2). The Student's t test was used to compare the percentage of localized cells under a number of experimental conditions. This scoring method produced similar results to that of quantitation of fluorescence intensity within each growth cone using digital imaging analysis (see above). The advantage of the scoring method is that one can rapidly score hundreds of cells within a population and evaluate multiple variables.


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

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

β-actin mRNA and protein localization is influenced by neurotrophin-3 (NT-3). (A) β-actin mRNA was prominent in the cell body and localized within the axonal process and growth cone in the form of spatially distinct granules (arrow; example of localized cell). (B) Cells that were starved in minimal essential medium (MEM) for 6 h showed hybridization within the cell body but growth cones did not reveal mRNA granules (arrow; example of nonlocalized cell). (C) Cells that were starved for 6 h and then stimulated with NT-3 for 10 min were observed to relocalize β-actin mRNA granules within growth cones (arrow). (D) Relocalization of β-actin mRNA within growth cones after 2 h in NT-3–supplemented MEM (arrow). (E) Cells grown in normal defined medium showed intense staining for β-actin protein within the minor neurites and the peripheral margin of the axonal growth cone (arrow) (example of localized cell). (F) Starvation resulted in reduced staining for β-actin protein within growth cones (arrow) (example of nonlocalized cell). (G and H) NT-3 stimulation for 10 min and 2 h restored the localization of β-actin protein within the peripheral margin (arrows). Bar, 15 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: β-actin mRNA and protein localization is influenced by neurotrophin-3 (NT-3). (A) β-actin mRNA was prominent in the cell body and localized within the axonal process and growth cone in the form of spatially distinct granules (arrow; example of localized cell). (B) Cells that were starved in minimal essential medium (MEM) for 6 h showed hybridization within the cell body but growth cones did not reveal mRNA granules (arrow; example of nonlocalized cell). (C) Cells that were starved for 6 h and then stimulated with NT-3 for 10 min were observed to relocalize β-actin mRNA granules within growth cones (arrow). (D) Relocalization of β-actin mRNA within growth cones after 2 h in NT-3–supplemented MEM (arrow). (E) Cells grown in normal defined medium showed intense staining for β-actin protein within the minor neurites and the peripheral margin of the axonal growth cone (arrow) (example of localized cell). (F) Starvation resulted in reduced staining for β-actin protein within growth cones (arrow) (example of nonlocalized cell). (G and H) NT-3 stimulation for 10 min and 2 h restored the localization of β-actin protein within the peripheral margin (arrows). Bar, 15 μm.
Mentions: For quantitative analysis using a visual scoring method, 100 cells per coverslip were analyzed for each cell culture condition. Experiments were done with duplicate coverslips for each variable and each experiment was repeated at least three times. The scoring method involved visualization of the presence or absence of β-actin mRNA granules in the axon-like growth cone from each cell. Cells were scored as localized if several granules were observed, and scored as nonlocalized if the signal was not distinguishable from background levels (hybridization with control probe). Localized cells would be expected to have a higher amount of fluorescent signal in growth cones compared with nonlocalized cells. Examples of localized and nonlocalized cells are shown in Fig. 1. This scoring method was used to show that NT-3 promotes an increase in localization (see Fig. 2) and that the results were comparable to the quantitation of fluorescence intensity using the CCD camera (see Fig. 3). The value for each bar within the histogram reports the mean and standard deviation between independent samples (see Fig. 2). The Student's t test was used to compare the percentage of localized cells under a number of experimental conditions. This scoring method produced similar results to that of quantitation of fluorescence intensity within each growth cone using digital imaging analysis (see above). The advantage of the scoring method is that one can rapidly score hundreds of cells within a population and evaluate multiple variables.

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