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Capzb2 interacts with beta-tubulin to regulate growth cone morphology and neurite outgrowth.

Davis DA, Wilson MH, Giraud J, Xie Z, Tseng HC, England C, Herscovitz H, Tsai LH, Delalle I - PLoS Biol. (2009)

Bottom Line: We found that silencing Capzb2 in hippocampal neurons resulted in short neurites and misshapen growth cones in which microtubules overgrew into the periphery and completely overlapped with F-actin.In searching for the mechanisms underlying these cytoskeletal abnormalities, we identified beta-tubulin as a novel binding partner of Capzb2 and demonstrated that Capzb2 decreases the rate and the extent of tubulin polymerization in vitro.Together, these data suggest that Capzb2 plays an important role in growth cone formation and neurite outgrowth and that the underlying mechanism may involve direct interaction between Capzb2 and microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

ABSTRACT
Capping protein (CP) is a heterodimer that regulates actin assembly by binding to the barbed end of F-actin. In cultured nonneuronal cells, each CP subunit plays a critical role in the organization and dynamics of lamellipodia and filopodia. Mutations in either alpha or beta CP subunit result in retinal degeneration in Drosophila. However, the function of CP subunits in mammalian neurons remains unclear. Here, we investigate the role of the beta CP subunit expressed in the brain, Capzb2, in growth cone morphology and neurite outgrowth. We found that silencing Capzb2 in hippocampal neurons resulted in short neurites and misshapen growth cones in which microtubules overgrew into the periphery and completely overlapped with F-actin. In searching for the mechanisms underlying these cytoskeletal abnormalities, we identified beta-tubulin as a novel binding partner of Capzb2 and demonstrated that Capzb2 decreases the rate and the extent of tubulin polymerization in vitro. We mapped the region of Capzb2 that was required for the subunit to interact with beta-tubulin and inhibit microtubule polymerization. A mutant Capzb2 lacking this region was able to bind F-actin and form a CP heterodimer with alpha2-subunit. However, this mutant was unable to rescue the growth cone and neurite outgrowth phenotypes caused by Capzb2 knockdown. Together, these data suggest that Capzb2 plays an important role in growth cone formation and neurite outgrowth and that the underlying mechanism may involve direct interaction between Capzb2 and microtubules.

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Comparison of growth cone area not invaded by microtubules in neurons transfected with either control shRNA/pEGFP or Capzb2 shRNA/pEGFP.(A and B) Visualization of microtubules in the growth cones was obtained with tyrosinated α-tubulin antibody signal (Tyr-tubulin, yellow); F-actin (red) upon CytD treatment. The percentage of growth cone area not invaded by microtubules (ROI%, white area) was obtained upon subtraction of Tyr-tubulin signal from the total growth cone area visualized on DIC image. The image left of the panels labeled ROI% shows the ROI border (white line overlay on Tyr-tubulin signal image) composed of the line indicating microtubule most distal position (based on Tyr-tubulin signal) and the outline of the growth cone (based on DIC image). (C) The average ROI% was significantly lower in neurons transfected with Capzb2 shRNA (n = 63, blinded analysis from three experiments) in comparison to controls (n = 85, blinded analysis from three experiments). Mean values ±s.e.m. are depicted; *** = p<0.001.
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pbio-1000208-g003: Comparison of growth cone area not invaded by microtubules in neurons transfected with either control shRNA/pEGFP or Capzb2 shRNA/pEGFP.(A and B) Visualization of microtubules in the growth cones was obtained with tyrosinated α-tubulin antibody signal (Tyr-tubulin, yellow); F-actin (red) upon CytD treatment. The percentage of growth cone area not invaded by microtubules (ROI%, white area) was obtained upon subtraction of Tyr-tubulin signal from the total growth cone area visualized on DIC image. The image left of the panels labeled ROI% shows the ROI border (white line overlay on Tyr-tubulin signal image) composed of the line indicating microtubule most distal position (based on Tyr-tubulin signal) and the outline of the growth cone (based on DIC image). (C) The average ROI% was significantly lower in neurons transfected with Capzb2 shRNA (n = 63, blinded analysis from three experiments) in comparison to controls (n = 85, blinded analysis from three experiments). Mean values ±s.e.m. are depicted; *** = p<0.001.

Mentions: These results suggest that Capzb2 influences microtubule extension into the peripheral domain of a growth cone, raising the question whether Capzb2 may act directly on microtubules or indirectly via actin cytoskeleton [23]. To address this question, we assessed the effect of Capzb2 on microtubules in neurons treated with cytochalasin D (CytD), which removes the actin meshwork and thus prevents inhibitory action of actin retrograde flow on microtubules in growth cones. In a blinded image analysis, we quantified the area of the growth cone not invaded by microtubules in neurons transfected with control shRNA or Capzb2 shRNA and treated with CytD (Figure 3). To visualize microtubules in the growth cones, we used tyrosinated α-tubulin antibody because the tyrosinated form of microtubules is the dominant one in the growth cones [24]. The average percentage of growth cone area not invaded by microtubules (percentage of region of interest, ROI%) was significantly lower in neurons transfected with Capzb2 shRNA than in control (Figure 3C). These data suggest that Capzb2 may directly influence microtubule extension into the peripheral domain of a growth cone.


Capzb2 interacts with beta-tubulin to regulate growth cone morphology and neurite outgrowth.

Davis DA, Wilson MH, Giraud J, Xie Z, Tseng HC, England C, Herscovitz H, Tsai LH, Delalle I - PLoS Biol. (2009)

Comparison of growth cone area not invaded by microtubules in neurons transfected with either control shRNA/pEGFP or Capzb2 shRNA/pEGFP.(A and B) Visualization of microtubules in the growth cones was obtained with tyrosinated α-tubulin antibody signal (Tyr-tubulin, yellow); F-actin (red) upon CytD treatment. The percentage of growth cone area not invaded by microtubules (ROI%, white area) was obtained upon subtraction of Tyr-tubulin signal from the total growth cone area visualized on DIC image. The image left of the panels labeled ROI% shows the ROI border (white line overlay on Tyr-tubulin signal image) composed of the line indicating microtubule most distal position (based on Tyr-tubulin signal) and the outline of the growth cone (based on DIC image). (C) The average ROI% was significantly lower in neurons transfected with Capzb2 shRNA (n = 63, blinded analysis from three experiments) in comparison to controls (n = 85, blinded analysis from three experiments). Mean values ±s.e.m. are depicted; *** = p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2748697&req=5

pbio-1000208-g003: Comparison of growth cone area not invaded by microtubules in neurons transfected with either control shRNA/pEGFP or Capzb2 shRNA/pEGFP.(A and B) Visualization of microtubules in the growth cones was obtained with tyrosinated α-tubulin antibody signal (Tyr-tubulin, yellow); F-actin (red) upon CytD treatment. The percentage of growth cone area not invaded by microtubules (ROI%, white area) was obtained upon subtraction of Tyr-tubulin signal from the total growth cone area visualized on DIC image. The image left of the panels labeled ROI% shows the ROI border (white line overlay on Tyr-tubulin signal image) composed of the line indicating microtubule most distal position (based on Tyr-tubulin signal) and the outline of the growth cone (based on DIC image). (C) The average ROI% was significantly lower in neurons transfected with Capzb2 shRNA (n = 63, blinded analysis from three experiments) in comparison to controls (n = 85, blinded analysis from three experiments). Mean values ±s.e.m. are depicted; *** = p<0.001.
Mentions: These results suggest that Capzb2 influences microtubule extension into the peripheral domain of a growth cone, raising the question whether Capzb2 may act directly on microtubules or indirectly via actin cytoskeleton [23]. To address this question, we assessed the effect of Capzb2 on microtubules in neurons treated with cytochalasin D (CytD), which removes the actin meshwork and thus prevents inhibitory action of actin retrograde flow on microtubules in growth cones. In a blinded image analysis, we quantified the area of the growth cone not invaded by microtubules in neurons transfected with control shRNA or Capzb2 shRNA and treated with CytD (Figure 3). To visualize microtubules in the growth cones, we used tyrosinated α-tubulin antibody because the tyrosinated form of microtubules is the dominant one in the growth cones [24]. The average percentage of growth cone area not invaded by microtubules (percentage of region of interest, ROI%) was significantly lower in neurons transfected with Capzb2 shRNA than in control (Figure 3C). These data suggest that Capzb2 may directly influence microtubule extension into the peripheral domain of a growth cone.

Bottom Line: We found that silencing Capzb2 in hippocampal neurons resulted in short neurites and misshapen growth cones in which microtubules overgrew into the periphery and completely overlapped with F-actin.In searching for the mechanisms underlying these cytoskeletal abnormalities, we identified beta-tubulin as a novel binding partner of Capzb2 and demonstrated that Capzb2 decreases the rate and the extent of tubulin polymerization in vitro.Together, these data suggest that Capzb2 plays an important role in growth cone formation and neurite outgrowth and that the underlying mechanism may involve direct interaction between Capzb2 and microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.

ABSTRACT
Capping protein (CP) is a heterodimer that regulates actin assembly by binding to the barbed end of F-actin. In cultured nonneuronal cells, each CP subunit plays a critical role in the organization and dynamics of lamellipodia and filopodia. Mutations in either alpha or beta CP subunit result in retinal degeneration in Drosophila. However, the function of CP subunits in mammalian neurons remains unclear. Here, we investigate the role of the beta CP subunit expressed in the brain, Capzb2, in growth cone morphology and neurite outgrowth. We found that silencing Capzb2 in hippocampal neurons resulted in short neurites and misshapen growth cones in which microtubules overgrew into the periphery and completely overlapped with F-actin. In searching for the mechanisms underlying these cytoskeletal abnormalities, we identified beta-tubulin as a novel binding partner of Capzb2 and demonstrated that Capzb2 decreases the rate and the extent of tubulin polymerization in vitro. We mapped the region of Capzb2 that was required for the subunit to interact with beta-tubulin and inhibit microtubule polymerization. A mutant Capzb2 lacking this region was able to bind F-actin and form a CP heterodimer with alpha2-subunit. However, this mutant was unable to rescue the growth cone and neurite outgrowth phenotypes caused by Capzb2 knockdown. Together, these data suggest that Capzb2 plays an important role in growth cone formation and neurite outgrowth and that the underlying mechanism may involve direct interaction between Capzb2 and microtubules.

Show MeSH
Related in: MedlinePlus