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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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NT-3 stimulated localization of β-actin mRNA and protein analyzed using quantitative digital imaging microscopy. Neurons were fixed for in situ hybridization to β-actin mRNA (A) and immunofluorescence detection of β-actin protein (B). DIC and fluorescence images were captured using a cooled CCD camera. 20 growth cones were imaged for each condition with identical exposure times. Data expressed as fluorescence density (total intensity/growth cone area). NT-3 was observed to increase the density of fluorescence signal for both β-actin mRNA and protein within growth cones. #, P < 0.01 when MEM was compared with N2, or MEM was compared with NT-3, 10 min or NT-3, 2 h. *, P < 0.05 when MEM was compared to NT-3 at 10 min. N2, normal culture medium. MEM, starvation in minimum essential medium.
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Figure 3: NT-3 stimulated localization of β-actin mRNA and protein analyzed using quantitative digital imaging microscopy. Neurons were fixed for in situ hybridization to β-actin mRNA (A) and immunofluorescence detection of β-actin protein (B). DIC and fluorescence images were captured using a cooled CCD camera. 20 growth cones were imaged for each condition with identical exposure times. Data expressed as fluorescence density (total intensity/growth cone area). NT-3 was observed to increase the density of fluorescence signal for both β-actin mRNA and protein within growth cones. #, P < 0.01 when MEM was compared with N2, or MEM was compared with NT-3, 10 min or NT-3, 2 h. *, P < 0.05 when MEM was compared to NT-3 at 10 min. N2, normal culture medium. MEM, starvation in minimum essential medium.

Mentions: Immunofluorescence signal was viewed using an Olympus-IX70 microscope equipped with a 60× Plan-Neofluar objective and Nomarski (DIC) optics. Cells were viewed using a 100 watt mercury arc lamp and light was filtered using HiQ bandpass filters (ChromaTech). The images were captured with a cooled CCD camera (Photometrics) using a 35-mm shutter and processed using IP Lab Spectrum (Scanalytics) running on a Macintosh G3. After identification of growth cones using DIC optics, a fluorescence image was immediately acquired. All exposure times with the CCD camera were kept constant (1 s for β-actin mRNA, 0.5 s for β-actin protein) and below grey scale saturation to permit a linear response to light intensity and quantitative analysis of differences in fluorescence intensities. The perimeter of each growth cone was traced using the DIC image and IP Lab software to identify a region of interest (ROI) and measure total fluorescence intensity. For quantitative image analysis of β-actin mRNA and protein localization using this method (see Fig. 3 and Fig. 4), 20 cells were imaged for each cell culture condition.


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

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

NT-3 stimulated localization of β-actin mRNA and protein analyzed using quantitative digital imaging microscopy. Neurons were fixed for in situ hybridization to β-actin mRNA (A) and immunofluorescence detection of β-actin protein (B). DIC and fluorescence images were captured using a cooled CCD camera. 20 growth cones were imaged for each condition with identical exposure times. Data expressed as fluorescence density (total intensity/growth cone area). NT-3 was observed to increase the density of fluorescence signal for both β-actin mRNA and protein within growth cones. #, P < 0.01 when MEM was compared with N2, or MEM was compared with NT-3, 10 min or NT-3, 2 h. *, P < 0.05 when MEM was compared to NT-3 at 10 min. N2, normal culture medium. MEM, starvation in minimum essential medium.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: NT-3 stimulated localization of β-actin mRNA and protein analyzed using quantitative digital imaging microscopy. Neurons were fixed for in situ hybridization to β-actin mRNA (A) and immunofluorescence detection of β-actin protein (B). DIC and fluorescence images were captured using a cooled CCD camera. 20 growth cones were imaged for each condition with identical exposure times. Data expressed as fluorescence density (total intensity/growth cone area). NT-3 was observed to increase the density of fluorescence signal for both β-actin mRNA and protein within growth cones. #, P < 0.01 when MEM was compared with N2, or MEM was compared with NT-3, 10 min or NT-3, 2 h. *, P < 0.05 when MEM was compared to NT-3 at 10 min. N2, normal culture medium. MEM, starvation in minimum essential medium.
Mentions: Immunofluorescence signal was viewed using an Olympus-IX70 microscope equipped with a 60× Plan-Neofluar objective and Nomarski (DIC) optics. Cells were viewed using a 100 watt mercury arc lamp and light was filtered using HiQ bandpass filters (ChromaTech). The images were captured with a cooled CCD camera (Photometrics) using a 35-mm shutter and processed using IP Lab Spectrum (Scanalytics) running on a Macintosh G3. After identification of growth cones using DIC optics, a fluorescence image was immediately acquired. All exposure times with the CCD camera were kept constant (1 s for β-actin mRNA, 0.5 s for β-actin protein) and below grey scale saturation to permit a linear response to light intensity and quantitative analysis of differences in fluorescence intensities. The perimeter of each growth cone was traced using the DIC image and IP Lab software to identify a region of interest (ROI) and measure total fluorescence intensity. For quantitative image analysis of β-actin mRNA and protein localization using this method (see Fig. 3 and Fig. 4), 20 cells were imaged for each cell culture condition.

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