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. 2018 Jun 4;217(6):2033-2046.
doi: 10.1083/jcb.201703205. Epub 2018 Mar 27.

Myosin 1b Promotes Axon Formation by Regulating Actin Wave Propagation and Growth Cone Dynamics

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Free PMC article

Myosin 1b Promotes Axon Formation by Regulating Actin Wave Propagation and Growth Cone Dynamics

Olga Iuliano et al. J Cell Biol. .
Free PMC article

Abstract

Single-headed myosin 1 has been identified in neurons, but its function in these cells is still unclear. We demonstrate that depletion of myosin 1b (Myo1b), inhibition of its motor activity, or its binding to phosphoinositides impairs the formation of the axon, whereas overexpression of Myo1b increases the number of axon-like structures. Myo1b is associated with growth cones and actin waves, two major contributors to neuronal symmetry breaking. We show that Myo1b controls the dynamics of the growth cones and the anterograde propagation of the actin waves. By coupling the membrane to the actin cytoskeleton, Myo1b regulates the size of the actin network as well as the stability and size of filopodia in the growth cones. Our data provide the first evidence that a myosin 1 plays a major role in neuronal symmetry breaking and argue for a mechanical control of the actin cytoskeleton both in actin waves and in the growth cones by this myosin.

Figures

Figure 1.
Figure 1.
Distribution of Myo1b in cultured cortical neurons. (A) After 1, 3, 5, and 8 d of culture, lysates of cortical neurons were analyzed by SDS-PAGE and immunoblotting with anti-Myo1b antibodies. GAPDH was used as a loading control. (B) The amount of Myo1b detected in the lysates has been quantified as described in Materials and methods and expressed as AU. Data are shown as the mean of three experiments. Error bars represent ± SEM. (C) Cortical neurons DIV2 were immunolabeled with anti-Myo1b and anti–β3-tubulin antibodies, fluorescently labeled for F-actin with phalloidin, and analyzed by confocal microscopy. A representative overlay of z stack for F-actin (red), Myo1b (green), and β3-tubulin (magenta), and the independent z stack for Myo1b are shown for cortical neurons at stage 2 and stage 3. Bars, 20 µm. (D) Cortical neurons DIV2 immunolabeled with anti-Myo1b and anti–β3-tubulin antibodies and fluorescently labeled with phalloidin were analyzed by SIM. Representative images of the growth cones of one short and one long neurite labeled for Myo1b and its overlay (green) with the corresponding image for β3-tubulin (magenta) and phalloidin (red) are shown. Bars, 5 µm.
Figure 2.
Figure 2.
Depletion of Myo1b delays neuronal differentiation and inhibits the formation of axon. (A) 48 h after transfection with control or Myo1b-siRNAs, lysates of cortical neurons were analyzed by SDS-PAGE and immunoblotting with anti-Myo1b antibodies. GAPDH was used as loading control. (B) The amount of Myo1b was quantified and normalized to the amount of GAPDH. Data are shown as a mean of three experiments. (C) Cortical neurons were transfected with control or Myo1b-siRNAs, fluorescently labeled for F-actin, and immunolabeled for Myo1b and β3-tubulin at DIV2 or Tau-1 at DIV4. Representative confocal z stacks for Myo1b and merged confocal z stacks for F-actin (green) and β3-tubulin or Tau-1 (magenta) are shown. Bars, 20 µm. (D) The percentages of neurons showing each of the different stages of differentiation were quantified for neurons transfected with control siRNAs, Myo1b-siRNA, Myo1b-siRNA+Flag-HA-Myo1b5M, and Myo1b-siRNA+Flag-HA-Myo1b5MR based on their morphologies observed at DIV2 after immunolabeling for β3-tubulin and fluorescently labeled for F-actin. Data are shown as the mean of three independent experiments for each condition. Error bars represent ± SEM. n = 30 cells/experiment. (E) The number of neurites per cell was quantified in cortical neurons transfected with control siRNA or Myo1b-siRNA at DIV2 and represented as box plots. n = 3 independent experiments; n = 30 cells/experiment. (F) The length of the longest neurite in cortical neurons transfected with control siRNA or Myo1b-siRNA was measured at DIV2 and represented as box plots. n = 3 independent experiments; n = 30 cells/experiment. (G) The percentages of neurons with none, single, and multiple axons were quantified after transfection of control and Myo1b-siRNAs, Myo1b-siRNA+FlagHA-Myo1b-5M, or Myo1b-siRNA+FlagHA-Myo1b-5MR and immunolabeling at DIV4 for Tau-1 to identify nascent axons. Data are shown as the mean of three independent experiments for each condition. Error bars represent ± SEM. n = 30 cells/experiment. Data distribution was assumed to be normal. χ2 test (D and G); unpaired t test (F and G). *, P < 0.05; **, P < 0.01; ***, P < 0.0001.
Figure 3.
Figure 3.
Myo1b overexpression induces multiple axon-like processes and requires the integrity of its PH motif. (A) Cortical neurons transfected with plasmids encoding EGFP, EGFP-Myo1b, and EGFP-Myo1b-K966A were immunolabeled for Tau-1 to identify nascent axons at DIV4 and analyzed by confocal microscopy. Representative z stacks are shown. Bars, 20 µm. (B) The percentages of neurons with none, single, or multiple axons were quantified after transfection of plasmids encoding EGFP, EGFP-Myo1b, and EGFP-Myo1b-K966A and immunolabeling at DIV4 for Tau-1. Data are shown as the mean of three independent experiments for each condition. Error bars represent ± SEM. n = 30 cells/experiment. Data distribution was assumed to be normal. χ2 test. *, P < 0.05; ***, P < 0.001.
Figure 4.
Figure 4.
Growth cones and actin waves share important structural similarities. (A–D) Distribution of F-actin cytoskeleton of growth cones (A and B) and actin waves (C and D) of DIV2 cortical neurons visualized by SIM (A and C) and platinum replica EM (B and D). Boxed regions in B and D are enlarged in the right panel. Bars: (A–D, left) 2 µm; (B and D, right) 1 µm.
Figure 5.
Figure 5.
Myo1b is associated with and propagates with actin waves. (A) Merged fluorescence images acquired with SIM of an actin wave costained with anti-Myo1b antibodies, Alexa Fluor 488 phalloidin, and anti–β3-tubulin antibody and images of the 2.5× enlargement of the region marked by a white box are shown. (B) The propagation of F-actin structures and mCherry-Myo1b has been analyzed in cortical neurons expressing LifeAct-EGFP and mCherry-Myo1b at DIV1 by time-lapse spinning confocal microscopy. The first frame of Video 1 (one frame per minute) for LifeAct-EGFP and mCherry-Myo1b and the merged kymographs of 36 frames for the region marked on the first frame by a white lane for the two recombinant proteins are shown. The increase of fluorescence intensity for LifeAct-EGFP correlated with the increase of fluorescence intensity for mCherry-Myo1b. (C) The propagation of PIP3 and mCherry-Myo1b has been analyzed in cortical neurons expressing AKT-PH-EGFP and mCherry-Myo1b at DIV1 by time-lapse spinning confocal microscopy. The first frame of Video 2 and kymographs of 36 frames for the region marked on the first frame by a white lane for the two recombinant proteins are shown. The increase of fluorescence intensity for AKT-PH-EGFP correlated with the increase of fluorescence intensity for mCherry-Myo1b. Bars: (A) 10 µm; (B and C) 20 µm. T, time
Figure 6.
Figure 6.
Myo1b, its motor activity, and its PH motif control the anterograde propagation of actin waves. (A and B) The propagation of actin waves has been analyzed in cortical neurons transfected with control (A) or Myo1b-siRNAs (B) at DIV1 by time-lapse spinning confocal microscopy (see also Video 3, a and b). Images of the first frame and the kymographs of the Video 6 (a and b) for the region marked by a purple lane on the first frames for 13 min are shown (time lapse, 1 min). T, time. Two anterograde waves are observed in control cells (A1 and A2), whereas in Myo1b-depleted neurons, actin waves are formed, but they either migrate to the cell body (retrograde; B1) or oscillate and collapse along the shaft of neurite (B2). Bars, 10 µm. (C and D) Ratio of actin waves per neurite (C) and anterograde, retrograde, and abortive actin waves normalized to the total number of waves (D) observed by time-lapse spinning confocal microscopy in cells cotransfected with LifeAct-EGFP and control or Myo1b-siRNA. Data are shown as the mean of three independent experiments for each condition. n = 13, 9, and 6 cells for control siRNA treatment; n = 9, 8, and 3 cells for Myo1b-siRNA treatment per experiments. (E and F) Ratio of actin waves per neurite (E) and anterograde, retrograde, and abortive waves normalized to the total number of waves (F) observed by time-lapse spinning confocal microscopy in cells transfected with LifeAct-EGFP and treated with DMSO or PClP. Data are shown as the mean of three independent experiments for each condition. n = 8, 15, and 16 cells for DMSO treatment; n = 11, 17, and 7 for PClP treatment per experiment. (G and H) Ratio of actin waves/neurite (G) and anterograde, retrograde, and abortive actin waves normalized to the total number of waves (H) observed in neurons transfected with LifeAct-EGFP together with mCherry, mCherry-Myo1b, or mCherry-Myo1b-K966A. Data are shown as the mean of three independent experiments for each condition. Error bars represent ± SEM. n = 6, 10, and 7 cells expressing mCherry; n = 7, 11, and 10 cells expressing mCherry-Myo1b; n = 11, 8, and 8 cells expressing mCherry-Myo1b-K966A per experiment. Data distribution was assumed to be normal. χ2 test. *, P < 0.05; ***, P < 0.001.
Figure 7.
Figure 7.
Myo1b expression regulates the trafficking of Kif5C560 in neurites. The dynamics of Kif5C560 have been analyzed by time-lapse spinning confocal microscopy in cells expressing Kif5C560-EGFP and control or Myo1b-mCherry-shRNA at DIV1 (A and B) or mCherry or mCherry-Myo1b (C and D). The merged first frames and the merged kymograph (one frame/2 min) for Kif5C560-EGFP (green) and mCherry (red) of the Video 4 (a and b) for shRNAs and Video 5 (a and b) for mCherry and mCherry-Myo1b are shown. T, time. Bars, 20 µm. The purple lanes on the first frame mark the regions for which the kymographs illustrate the dynamics of Kif5C560 within 1 h.
Figure 8.
Figure 8.
Myo1b controls the size of the F-actin–enriched area in the growth cones. (A) Cortical neurons were transfected with control or Myo1b-siRNAs, immunolabeled for Myo1b and β3-tubulin, and fluorescently labeled for F-actin at DIV2. Representative confocal z stacks for Myo1b, merged confocal z stacks for F-actin (green) and β3-tubulin (magenta), and images of the 3× enlargements of the regions marked by the white boxes are shown. (B) The measurements of the area occupied by F-actin in the growth cone of the longest neurite as judged by β3-tubulin labeling in cells transfected with Myo1b- or control siRNAs are represented as box plots. n = 51 in three independent experiments. (C) Cortical neurons expressing EGFP, EGFP-Myo1b, or EGFP-Myo1b-K966A, immunolabeled for β3-tubulin, and fluorescently labeled for F-actin at DIV2. Representative merged confocal z stacks for EGFP (green), β3-tubulin (magenta), and F-actin (red) and images of the 2.5× enlargements of the regions marked by the white boxes are shown. Bars, 20 µm. (D) The measurements of the area occupied by F-actin in the growth cone of the longest neurite as judged by β3-tubulin labeling in cells expressing EGFP, EGFP-Myo1b, or EGFP-Myo1b-K966A are represented as box plots. n = 25 in three independent experiments. Data distribution was assumed to be normal. Unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 9.
Figure 9.
Depletion of Myo1b affects the length and lifespan of filopodia. (A–E) Cortical neurons transfected with plasmid encoding mGFP-F-tractin-P and control siRNA (A) or Myo1b-siRNA (B) were analyzed at DIV1 during 3 min (one frame/s) using spinning confocal microscopy. Images of the first frames of Video 7 (a and b) and the kymographs of 22 frames for the region marked by a purple lane are shown. Note that the two filopodia in a Myo1b-depleted cell collapse within 22 s, whereas those in the control cell remain stable. The ratio of filopodia per growth cone observed within 3 min (n = 18 or 15; C), the length of the filopodia (n = 199 or 153; D), and the ratio of filopodia per growth cone collapsing within 3 min (n = 18 or 15; E) have been quantified and represented as box plots for three independent experiments. T, time. (F–K) Cortical neurons expressing mGFP-F-tractin-P and control mCherry-shRNA (F) or Myo1b-mCherry-shRNA (G) were analyzed at DIV1 for 3 min (one frame/s) using spinning confocal microscopy. Merged images of the first frames of Video 8 (a and b) showing F-actin (gray) and mCherry (red) in the growth cone are shown. The magenta lanes mark the regions for which kymographs of the dynamics of filopodia for 22 s are shown. Bars, 13 µm. The ratio of filopodia per growth cone observed within 3 min (n = 10 or 12; H), the length of the filopodia (n = 58 or 91; I), the ratio of filopodia per growth cone collapsing within 3 min (n = 10 or 15; J), and the velocity of the elongation (n = 21 or 29; K) have been quantified and represented as box plots in three independent experiments. Data distribution was assumed to be normal. Unpaired t test. **, P < 0.01; ***, P < 0.001.
Figure 10.
Figure 10.
Myo1b depletion decreases F-actin in filopodia and increases F-actin in the interfilopodial veils. (A) Representative confocal z stacks of cortical neurons expressing control mCherry-shRNA or Myo1b-Cherry-shRNA and mGFP-F-tractin-P. Bars, 11 µm. (B) The fluorescent intensity of F-actin within the area occupied by filopodia has been quantified on all sections of the stacks and normalized to the fluorescent intensity measured on the area occupied by the total growth cone on all sections and represented as box plots. n = 23 or 25 in three independent experiments. Data distribution was assumed to be normal. Unpaired t test. **, P < 0.01. (C) Cortical neurons transfected with plasmid encoding control mCherry-shRNA or Myo1b-mCherry-shRNA (B) were analyzed with SIM. Representative merged images of growth cones showing F-actin (gray) and mCherry (red) are shown. Bars, 5 µm.

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