Limits...
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.

Show MeSH

Related in: MedlinePlus

The head domain of myosin Va or Vb inhibits the filopodial rotation. (A–D) The line sketches in each panel show trajectories of filopodial tips of a single growth cone that expresses Venus (A), MyoVaHD-Venus (B), MyoVbHD-Venus (C), or MyoVcHD-Venus (D). Each color corresponds to a single filopodium. All of the filopodial tips that appeared in the focal plane for a period of 5-min imaging were included in this study. The numbered end of each line represents the point where a filopodial tip first appeared in the focal plane, and the other end of the line is the point at which it moved out of the focal plane. The mean angular velocity (ω) and the mean velocity (v) of filopodial tips for each growth cone are shown. Positive and negative values of the angular velocity indicate right- and left-screw rotation, respectively.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: The head domain of myosin Va or Vb inhibits the filopodial rotation. (A–D) The line sketches in each panel show trajectories of filopodial tips of a single growth cone that expresses Venus (A), MyoVaHD-Venus (B), MyoVbHD-Venus (C), or MyoVcHD-Venus (D). Each color corresponds to a single filopodium. All of the filopodial tips that appeared in the focal plane for a period of 5-min imaging were included in this study. The numbered end of each line represents the point where a filopodial tip first appeared in the focal plane, and the other end of the line is the point at which it moved out of the focal plane. The mean angular velocity (ω) and the mean velocity (v) of filopodial tips for each growth cone are shown. Positive and negative values of the angular velocity indicate right- and left-screw rotation, respectively.

Mentions: To test our hypothesis that the right-screw rotation of filopodia involves myosin V, we monitored the movement of the filopodial tips of hippocampal neurons that overexpressed a truncated mutant of myosin V consisting of its head domain only. The mutant should interfere with the binding between actin filaments and endogenous myosin V in a dominant-negative manner. To visualize transfected cells, the YFP Venus was fused to the C terminus of the head domain of the three members in the class V myosins, myosin Va, Vb, and Vc. These three constructs are designated in this paper as MyoVaHD-, MyoVbHD-, and MyoVcHD-Venus, respectively. Growth cone filopodia of Venus-transfected neurons (Fig. 3 A and Video 5) exhibited the right-screw rotation. In contrast, the filopodial rotation was blocked in neurons transfected with either MyoVaHD-Venus (Fig. 3 B and Video 6) or MyoVbHD-Venus (Fig. 3 C). However, MyoVcHD-Venus had no substantial effect on the filopodial rotation (Fig. 3 D). Given the structural similarity of the head domain among different forms of myosin, the observed inhibition of filopodial rotation by MyoVaHD- and MyoVbHD-Venus may have been mediated by dominant-negative effects on other myosin motors. Therefore, we tested whether overexpression of full-length myosin V rescues the filopodial rotation in neurons transfected with MyoVaHD-Venus. As expected, the filopodial rotation was rescued partially by cotransfection with either myosin Va (Fig. 4 A and Video 7) or myosin Vb (Fig. 4 B) using a vector for bicistronic expression of full-length myosin V and monomeric RFP (mRFP) designated as MyoVa/internal ribosomal entry site (IRES)/mRFP or MyoVb/IRES/mRFP, respectively. In contrast, full-length myosin Vc plus mRFP (MyoVc/IRES/mRFP) failed to rescue the filopodial rotation in neurons transfected with MyoVaHD-Venus (Fig. 4 C). As a control, cotransfection with mRFP alone had no detectable effect (Fig. 4 D and Video 8). Furthermore, the filopodial rotation was analyzed in neurons expressing the Venus-fused head domain of myosin IIa, IIb, or IIc. None of these three proteins showed a detectable effect on filopodial rotation (Figs. S1–S3).


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

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

The head domain of myosin Va or Vb inhibits the filopodial rotation. (A–D) The line sketches in each panel show trajectories of filopodial tips of a single growth cone that expresses Venus (A), MyoVaHD-Venus (B), MyoVbHD-Venus (C), or MyoVcHD-Venus (D). Each color corresponds to a single filopodium. All of the filopodial tips that appeared in the focal plane for a period of 5-min imaging were included in this study. The numbered end of each line represents the point where a filopodial tip first appeared in the focal plane, and the other end of the line is the point at which it moved out of the focal plane. The mean angular velocity (ω) and the mean velocity (v) of filopodial tips for each growth cone are shown. Positive and negative values of the angular velocity indicate right- and left-screw rotation, respectively.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: The head domain of myosin Va or Vb inhibits the filopodial rotation. (A–D) The line sketches in each panel show trajectories of filopodial tips of a single growth cone that expresses Venus (A), MyoVaHD-Venus (B), MyoVbHD-Venus (C), or MyoVcHD-Venus (D). Each color corresponds to a single filopodium. All of the filopodial tips that appeared in the focal plane for a period of 5-min imaging were included in this study. The numbered end of each line represents the point where a filopodial tip first appeared in the focal plane, and the other end of the line is the point at which it moved out of the focal plane. The mean angular velocity (ω) and the mean velocity (v) of filopodial tips for each growth cone are shown. Positive and negative values of the angular velocity indicate right- and left-screw rotation, respectively.
Mentions: To test our hypothesis that the right-screw rotation of filopodia involves myosin V, we monitored the movement of the filopodial tips of hippocampal neurons that overexpressed a truncated mutant of myosin V consisting of its head domain only. The mutant should interfere with the binding between actin filaments and endogenous myosin V in a dominant-negative manner. To visualize transfected cells, the YFP Venus was fused to the C terminus of the head domain of the three members in the class V myosins, myosin Va, Vb, and Vc. These three constructs are designated in this paper as MyoVaHD-, MyoVbHD-, and MyoVcHD-Venus, respectively. Growth cone filopodia of Venus-transfected neurons (Fig. 3 A and Video 5) exhibited the right-screw rotation. In contrast, the filopodial rotation was blocked in neurons transfected with either MyoVaHD-Venus (Fig. 3 B and Video 6) or MyoVbHD-Venus (Fig. 3 C). However, MyoVcHD-Venus had no substantial effect on the filopodial rotation (Fig. 3 D). Given the structural similarity of the head domain among different forms of myosin, the observed inhibition of filopodial rotation by MyoVaHD- and MyoVbHD-Venus may have been mediated by dominant-negative effects on other myosin motors. Therefore, we tested whether overexpression of full-length myosin V rescues the filopodial rotation in neurons transfected with MyoVaHD-Venus. As expected, the filopodial rotation was rescued partially by cotransfection with either myosin Va (Fig. 4 A and Video 7) or myosin Vb (Fig. 4 B) using a vector for bicistronic expression of full-length myosin V and monomeric RFP (mRFP) designated as MyoVa/internal ribosomal entry site (IRES)/mRFP or MyoVb/IRES/mRFP, respectively. In contrast, full-length myosin Vc plus mRFP (MyoVc/IRES/mRFP) failed to rescue the filopodial rotation in neurons transfected with MyoVaHD-Venus (Fig. 4 C). As a control, cotransfection with mRFP alone had no detectable effect (Fig. 4 D and Video 8). Furthermore, the filopodial rotation was analyzed in neurons expressing the Venus-fused head domain of myosin IIa, IIb, or IIc. None of these three proteins showed a detectable effect on filopodial rotation (Figs. S1–S3).

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