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Autonomous right-screw rotation of growth cone filopodia drives neurite turning.

Tamada A, Kawase S, Murakami F, Kamiguchi H - J. Cell Biol. (2010)

Bottom Line: We have developed a technique for monitoring three-dimensional motility of growth cone filopodia and demonstrate that an individual filopodium rotates on its own longitudinal axis in the right-screw direction from the viewpoint of the growth cone body.Furthermore, we provide evidence that the unidirectional rotation of filopodia causes deflected neurite elongation, most likely via asymmetric positioning of the filopodia onto the substrate.Although the growth cone itself has been regarded as functionally symmetric, our study reveals the asymmetric nature of growth cone motility.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Neuronal Growth Mechanisms, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan. tamada@brain.riken.jp

ABSTRACT
The direction of neurite elongation is controlled by various environmental cues. However, it has been reported that even in the absence of any extrinsic directional signals, neurites turn clockwise on two-dimensional substrates. In this study, we have discovered autonomous rotational motility of the growth cone, which provides a cellular basis for inherent neurite turning. We have developed a technique for monitoring three-dimensional motility of growth cone filopodia and demonstrate that an individual filopodium rotates on its own longitudinal axis in the right-screw direction from the viewpoint of the growth cone body. We also show that the filopodial rotation involves myosins Va and Vb and may be driven by their spiral interactions with filamentous actin. Furthermore, we provide evidence that the unidirectional rotation of filopodia causes deflected neurite elongation, most likely via asymmetric positioning of the filopodia onto the substrate. Although the growth cone itself has been regarded as functionally symmetric, our study reveals the asymmetric nature of growth cone motility.

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The involvement of myosin V in inherent neurite turning. (A–D) Hippocampal neurons, which had been transfected with cDNAs for Venus (A), MyoVaHD-Venus (B), MyoVaHD-Venus plus MyoVa/IRES/mRFP (C), or MyoVaHD-Venus plus mRFP (D), were reaggregated and plated on 2D PDL/laminin substrates. Each panel is a composite of 30 fluorescent images acquired under the same optical conditions, which causes both saturation and stray light artifacts in the central images where the neuronal cell bodies are concentrated. In the cases of double transfection (C and D), Venus fluorescence (green) and mRFP fluorescence (magenta) have been superimposed. (E) The y axis represents the curvature of the distal 100 µm of neurites that express the indicated transgene products. Positive values represent rightward turning. ***, P < 0.001 (Bonferroni’s multiple comparison test). (F) The y axis represents the estimated length of neurites that express the indicated transgene products. (E and F) Numbers in parentheses indicate the total number of neurites examined. Data represent mean ± SEM. Bar, 200 µm.
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fig6: The involvement of myosin V in inherent neurite turning. (A–D) Hippocampal neurons, which had been transfected with cDNAs for Venus (A), MyoVaHD-Venus (B), MyoVaHD-Venus plus MyoVa/IRES/mRFP (C), or MyoVaHD-Venus plus mRFP (D), were reaggregated and plated on 2D PDL/laminin substrates. Each panel is a composite of 30 fluorescent images acquired under the same optical conditions, which causes both saturation and stray light artifacts in the central images where the neuronal cell bodies are concentrated. In the cases of double transfection (C and D), Venus fluorescence (green) and mRFP fluorescence (magenta) have been superimposed. (E) The y axis represents the curvature of the distal 100 µm of neurites that express the indicated transgene products. Positive values represent rightward turning. ***, P < 0.001 (Bonferroni’s multiple comparison test). (F) The y axis represents the estimated length of neurites that express the indicated transgene products. (E and F) Numbers in parentheses indicate the total number of neurites examined. Data represent mean ± SEM. Bar, 200 µm.

Mentions: Next we examined the effect of the head domain of myosin V on the growth linearity of neurites on 2D substrates. Dissociated hippocampal neurons were transfected, reaggregated, and incubated for 3 d until the expression level of transgenes became sufficiently high. Then, the reaggregates were replated on 2D PDL/laminin substrates and cultured for an additional 1 d. Examples of a reaggregate transfected with Venus (Fig. 6 A), MyoVaHD-Venus (Fig. 6 B), MyoVaHD-Venus plus MyoVa/IRES/mRFP (Fig. 6 C), or MyoVaHD-Venus plus mRFP (Fig. 6 D) are shown. Turning was evaluated in all of the neurites that had Venus fluorescence or, in case of double transfection, both Venus and mRFP fluorescence. As a measure of neurite turning, we calculated the curvature (radians/millimeter) averaged within the distal 100-µm arc of each neurite (Fig. 6 E) because the proximal neurites were often detached from the substrate and fasciculated. Neurites from Venus-transfected reaggregates turned rightward. In comparison with this control, neurites from reaggregates transfected with either MyoVaHD- or MyoVbHD-Venus grew straighter, indicating that the head domain of myosins Va and Vb inhibited the rightward turning of neurites. MyoVcHD-Venus inhibited the rightward turning of neurites to a lesser degree. Cotransfection with full-length myosin Va (MyoVa/IRES/mRFP) but not with mRFP alone partially rescued the rightward turning of neurites that expressed MyoVaHD-Venus. We also measured the length of neurites that had Venus fluorescence or, in case of double transfection, both Venus and mRFP fluorescence (Fig. 6 F). A correlation between the length of neurites and their turning activity will be evaluated in the following experiment.


Autonomous right-screw rotation of growth cone filopodia drives neurite turning.

Tamada A, Kawase S, Murakami F, Kamiguchi H - J. Cell Biol. (2010)

The involvement of myosin V in inherent neurite turning. (A–D) Hippocampal neurons, which had been transfected with cDNAs for Venus (A), MyoVaHD-Venus (B), MyoVaHD-Venus plus MyoVa/IRES/mRFP (C), or MyoVaHD-Venus plus mRFP (D), were reaggregated and plated on 2D PDL/laminin substrates. Each panel is a composite of 30 fluorescent images acquired under the same optical conditions, which causes both saturation and stray light artifacts in the central images where the neuronal cell bodies are concentrated. In the cases of double transfection (C and D), Venus fluorescence (green) and mRFP fluorescence (magenta) have been superimposed. (E) The y axis represents the curvature of the distal 100 µm of neurites that express the indicated transgene products. Positive values represent rightward turning. ***, P < 0.001 (Bonferroni’s multiple comparison test). (F) The y axis represents the estimated length of neurites that express the indicated transgene products. (E and F) Numbers in parentheses indicate the total number of neurites examined. Data represent mean ± SEM. Bar, 200 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2819689&req=5

fig6: The involvement of myosin V in inherent neurite turning. (A–D) Hippocampal neurons, which had been transfected with cDNAs for Venus (A), MyoVaHD-Venus (B), MyoVaHD-Venus plus MyoVa/IRES/mRFP (C), or MyoVaHD-Venus plus mRFP (D), were reaggregated and plated on 2D PDL/laminin substrates. Each panel is a composite of 30 fluorescent images acquired under the same optical conditions, which causes both saturation and stray light artifacts in the central images where the neuronal cell bodies are concentrated. In the cases of double transfection (C and D), Venus fluorescence (green) and mRFP fluorescence (magenta) have been superimposed. (E) The y axis represents the curvature of the distal 100 µm of neurites that express the indicated transgene products. Positive values represent rightward turning. ***, P < 0.001 (Bonferroni’s multiple comparison test). (F) The y axis represents the estimated length of neurites that express the indicated transgene products. (E and F) Numbers in parentheses indicate the total number of neurites examined. Data represent mean ± SEM. Bar, 200 µm.
Mentions: Next we examined the effect of the head domain of myosin V on the growth linearity of neurites on 2D substrates. Dissociated hippocampal neurons were transfected, reaggregated, and incubated for 3 d until the expression level of transgenes became sufficiently high. Then, the reaggregates were replated on 2D PDL/laminin substrates and cultured for an additional 1 d. Examples of a reaggregate transfected with Venus (Fig. 6 A), MyoVaHD-Venus (Fig. 6 B), MyoVaHD-Venus plus MyoVa/IRES/mRFP (Fig. 6 C), or MyoVaHD-Venus plus mRFP (Fig. 6 D) are shown. Turning was evaluated in all of the neurites that had Venus fluorescence or, in case of double transfection, both Venus and mRFP fluorescence. As a measure of neurite turning, we calculated the curvature (radians/millimeter) averaged within the distal 100-µm arc of each neurite (Fig. 6 E) because the proximal neurites were often detached from the substrate and fasciculated. Neurites from Venus-transfected reaggregates turned rightward. In comparison with this control, neurites from reaggregates transfected with either MyoVaHD- or MyoVbHD-Venus grew straighter, indicating that the head domain of myosins Va and Vb inhibited the rightward turning of neurites. MyoVcHD-Venus inhibited the rightward turning of neurites to a lesser degree. Cotransfection with full-length myosin Va (MyoVa/IRES/mRFP) but not with mRFP alone partially rescued the rightward turning of neurites that expressed MyoVaHD-Venus. We also measured the length of neurites that had Venus fluorescence or, in case of double transfection, both Venus and mRFP fluorescence (Fig. 6 F). A correlation between the length of neurites and their turning activity will be evaluated in the following experiment.

Bottom Line: We have developed a technique for monitoring three-dimensional motility of growth cone filopodia and demonstrate that an individual filopodium rotates on its own longitudinal axis in the right-screw direction from the viewpoint of the growth cone body.Furthermore, we provide evidence that the unidirectional rotation of filopodia causes deflected neurite elongation, most likely via asymmetric positioning of the filopodia onto the substrate.Although the growth cone itself has been regarded as functionally symmetric, our study reveals the asymmetric nature of growth cone motility.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Neuronal Growth Mechanisms, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan. tamada@brain.riken.jp

ABSTRACT
The direction of neurite elongation is controlled by various environmental cues. However, it has been reported that even in the absence of any extrinsic directional signals, neurites turn clockwise on two-dimensional substrates. In this study, we have discovered autonomous rotational motility of the growth cone, which provides a cellular basis for inherent neurite turning. We have developed a technique for monitoring three-dimensional motility of growth cone filopodia and demonstrate that an individual filopodium rotates on its own longitudinal axis in the right-screw direction from the viewpoint of the growth cone body. We also show that the filopodial rotation involves myosins Va and Vb and may be driven by their spiral interactions with filamentous actin. Furthermore, we provide evidence that the unidirectional rotation of filopodia causes deflected neurite elongation, most likely via asymmetric positioning of the filopodia onto the substrate. Although the growth cone itself has been regarded as functionally symmetric, our study reveals the asymmetric nature of growth cone motility.

Show MeSH
Related in: MedlinePlus