Kinesin-1-powered microtubule sliding initiates axonal regeneration in Drosophila cultured neurons - PubMed (original) (raw)
Kinesin-1-powered microtubule sliding initiates axonal regeneration in Drosophila cultured neurons
Wen Lu et al. Mol Biol Cell. 2015.
Abstract
Understanding the mechanism underlying axon regeneration is of great practical importance for developing therapeutic treatment for traumatic brain and spinal cord injuries. Dramatic cytoskeleton reorganization occurs at the injury site, and microtubules have been implicated in the regeneration process. Previously we demonstrated that microtubule sliding by conventional kinesin (kinesin-1) is required for initiation of neurite outgrowth in Drosophila embryonic neurons and that sliding is developmentally down-regulated when neurite outgrowth is completed. Here we report that mechanical axotomy of Drosophila neurons in culture triggers axonal regeneration and regrowth. Regenerating neurons contain actively sliding microtubules; this sliding, like sliding during initial neurite outgrowth, is driven by kinesin-1 and is required for axonal regeneration. The injury induces a fast spike of calcium, depolymerization of microtubules near the injury site, and subsequent formation of local new microtubule arrays with mixed polarity. These events are required for reactivation of microtubule sliding at the initial stages of regeneration. Furthermore, the c-Jun N-terminal kinase pathway promotes regeneration by enhancing microtubule sliding in injured mature neurons.
© 2015 Lu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Figures
FIGURE 1:
Larval motor neuron regeneration after dissociation. (A) Motor neuron pattern in a Drosophila third-instar larva. (B) Motor neurons in a live third-instar larva labeled with a GFP-tagged Nrv2. The larva was oriented anterior up, and the ventral side view is imaged. Scale bar, 500 μm. (C, D) Motor neurons in culture 2 h after dissociation, with only one (C) or several (D) very short neurites (gold arrowheads). Scale bar, 5 μm. (E) A motor neuron in culture for 72 h after dissociation, with a long neurite, >50 μm. Scale bar, 5 μm.
FIGURE 2:
Microtubule sliding promotes neurite outgrowth during larval motor neuron regeneration. (A, B) Motor neurons expressing photoconvertible tdEOS-tagged α-tubulin 84B under the motor neuron–specific D42 driver (_D42_>tdEOS-αtub84B) at 2–4 h in culture after plating, showing a high level of microtubule sliding. (C, D) Motor neurons (_D42_>tdEOS-αtub84B) at >48 h in culture after plating, showing no detectable microtubule sliding in the cell body (C) or in the neurite (D). Column 1 shows the green signal of tdEOS-αtub84B before photoconversion, with the photoconversion zone marked by a purple circle; columns 2–4 show the red signal of tdEOS-αtub84B after photoconversion in the indicated areas (purple circles). Note that in A and B, photoconverted microtubules move outside the photoconversion area. Scale bar, 5 μm.
FIGURE 3:
KHC drives microtubule sliding during larval neuron regeneration. (A–A′′) A freshly dissociated Khc mutant (Khc27/Khc23) neuron (∼2–4 h in culture after plating) has little motility of microtubules labeled by _ubi_-Jupiter-mCherry. (B) A control neuron (∼72 h after plating) has a long axon. (C, C′) A Khc mutant (Khc27/Khc23) neuron (∼72 h after plating) has very short neurites. (D) A DHC64C-RNAi mutant neuron (∼72 h after plating) has a long axon. (E) Quantification of the axon length in control, Khc mutant (Khc27/Khc23), and DHC64C-RNAi (∼72 h after plating). The average (±95% confidence interval [CI]) for the control is 86.8 ± 5.5 μm (n = 56); the average (±95% CI) for the Khc mutant is 14.7 ± 2.2 μm (n = 80); the average (±95% CI) for DHC64C-RNAi is 88.5 ± 5.6 μm (n = 76). Unpaired t test between control and Khc mutant neurons: ***p < 0.0001; unpaired t test between Khc mutant and DHC64C-RNAi neurons: ***p < 0.0001; unpaired t test between control and DHC64C-RNAi neurons: p = 0.6693 (nonsignificant). Scale bar, 5 μm.
FIGURE 4:
Microtubule sliding is reactivated upon axonal injury and powers axonal regrowth. (A) An embryonic Jupiter-GFP neuron after axotomy. White arrowhead shows the axotomy site. (B, B′) Different time points of the regeneration event at the axotomized tip, indicated by the white dashed box in A. (B) Phase-contrast images; (B′) inverted fluorescence images of Jupiter-GFP. (C–D′) Two examples of microtubule sliding in injured neurons expressing photoconvertible tdEOS-tagged α-tubulin 84B (_elav_>tdEOS-αtub84B). Purple circle, photoconverted area; white arrowhead, axotomy position; green dashes, outline of the cell indicated by transmitted light images (not shown); green asterisk, nucleus position; orange arrowheads, anterograde movement of photoconverted microtubules; blue arrowheads, retrograde movement of photoconverted microtubules. Scale bar, 5 μm.
FIGURE 5:
Axotomy leads to fast Ca2+ influx and microtubule depolymerization. (A–A′′′) Signal heat maps of a neuron-specific calcium reporter (_elav_>GCaMP3) before and after axotomy, showing that a fast spike of calcium occurs upon mechanical axotomy. Axotomy is indicated by the white arrowheads (A′–A′′′). (B, B′) Fast microtubule depolymerization occurs upon axonal injury in an _elav_>tdEOS-αtub84B neuron (green channel). (B′′) Postcutting phase image of B′. (C) Quantification of relative fluorescence intensity of green tdEOS-αtub84B signal at two sites of the axon, near the cutting site and near the cell body, in control neurons (red and blue boxes in B and B′, respectively) and in 10 mM EGTA–treated neurons (purple and green boxes in Supplemental Figure S4 C–C′′′, respectively). All intensities were normalized to the intensity of the first frame (−5 s). Axotomy occurred at 0 s, indicated as the black arrowhead. Scale bar, 5 μm.
FIGURE 6:
Reorganization of microtubules after axotomy. (A) Quantification of axonal regeneration percentage in control, EGTA-treated, vinblastine-treated, elav>KHC-RNAi, and ciliobrevin D–treated neurons. The _y_-axis represents the regeneration ratio (≥5-μm neurite regrowth overnight is counted as a regeneration event) of all the samples examined. (B, C) Microtubules of mixed polarity formed after axotomy. (B) A phase-contrast image of the neuron, with white arrowhead showing the axotomy site. (C) Kymographs of EB1-GFP comets in the segment of the axon next to the axotomy site marked with white dashed line in B: before (top), 1 min after (middle), and 25 min after (bottom) axotomy. (D–D′′′) Inhibition of microtubule dynamics by 10 nM vinblastine 4 h after axotomy does not prevent Jupiter-GFP–labeled microtubule movement and looping. Looping microtubules are indicated by orange arrowheads, and sprouting microtubules are indicated by blue arrowheads. The time of adding 10 nM vinblastine is 0 min (120 min after imaging). Scale bar, 5 μm.
FIGURE 7:
JNK regulates neuronal regeneration by enhancing microtubule sliding. (A, B) JNK pathway activity is stimulated by axonal injury. JNK transcriptional activity is visualized by an enhancer trap of JNK target gene, _pucE69_-lacZ, in a control intact neuron (A) and an axotomized neuron (B). (A′, B′) Boxed area in A and B, respectively. Fluorescence images in A′ and B′ were acquired and adjusted identically. White arrowhead, axotomy position. (C) Quantification of axonal regeneration percentage in constitutively active JNK mutant (CA JNKK), JNK inhibitor (SP600125)-treated, and actinomycin D–treated neurons. The _y_-axis represents the regeneration ratio (≥5-μm neurite regrowth overnight is counted as a regeneration event) of all the samples examined. (D) Quantification of fluorescence signal outside of the photoconversion zone after 21 min in control and CA JNKK neurons, both 4 d in culture. Unpaired t test: p < 0.005. (E–E′′′′) A 4-d control neuron has no detectable microtubule sliding. (F–F′′′′) A 4-d CA JNKK neuron slides microtubules from the cell body to neurites (indicated by orange arrowheads). Purple circle, photoconverted area; scale bar, 5 μm.
FIGURE 8:
A model of initial axonal regeneration powered by KHC-driven microtubule sliding. 1) Long-extending axons are susceptible to cutting or severing by mechanical forces (axotomy). 2) Axotomy causes fast calcium–induced microtubule depolymerization near the injury site. 3) Mixed-polarity microtubule arrays are formed after the microtubule depolymerization near the injury site. 4) Axotomy activates the JNK pathway and subsequently drives transcription of key positive regulator(s) of microtubule sliding. 5) KHC slides antiparallel microtubules apart, which provides mechanical force for membrane extension during initial axonal regrowth. 6) Long-range regeneration after initial axonal regrowth reestablishes axonal functions.
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