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. 2009 Oct;69(12):761-79.
doi: 10.1002/dneu.20734.

Growth Cone-Like Waves Transport Actin and Promote Axonogenesis and Neurite Branching

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Growth Cone-Like Waves Transport Actin and Promote Axonogenesis and Neurite Branching

Kevin C Flynn et al. Dev Neurobiol. .
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Axonogenesis involves a shift from uniform delivery of materials to all neurites to preferential delivery to the putative axon, supporting its more rapid extension. Waves, growth cone-like structures that propagate down the length of neurites, were shown previously to correlate with neurite growth in dissociated cultured hippocampal neurons. Waves are similar to growth cones in their structure, composition and dynamics. Here, we report that waves form in all undifferentiated neurites, but occur more frequently in the future axon during initial neuronal polarization. Moreover, wave frequency and their impact on neurite growth are altered in neurons treated with stimuli that enhance axonogenesis. Coincident with wave arrival, growth cones enlarge and undergo a marked increase in dynamics. Through their engorgement of filopodia along the neurite shaft, waves can induce de novo neurite branching. Actin in waves maintains much of its cohesiveness during transport whereas actin in nonwave regions of the neurite rapidly diffuses as measured by live cell imaging of photoactivated GFP-actin and photoconversion of Dendra-actin. Thus, waves represent an alternative axonal transport mechanism for actin. Waves also occur in neurons in organotypic hippocampal slices where they propagate along neurites in the dentate gyrus and the CA regions and induce branching. Taken together, our results indicate that waves are physiologically relevant and contribute to axon growth and branching via the transport of actin and by increasing growth cone dynamics.


Figure 1
Figure 1. Waves have growth cone-like characteristics and propagate along the lengths of neurites
A. DIC images of a wave (black arrowhead) advancing down a neurite shaft. Note that as the wave approaches the distal neurite, the growth cone (white arrowhead) retracts and then engorges and advances following wave arrival. Relative times in min are indicated in lower right corner. B. Fluorescent images of F-actin (phallodin), tubulin, and cofilin in a hippocampal neuron with two waves (arrowheads). Note the high levels of F-actin and cofilin and the splaying of microtubules in the waves. Magnified views of a growth cone and a wave are shown in the right panel. Note the similar features including filopodia, lamellipodia and splaying microtubules. C. DIC time-lapse images (10 s intervals) of growth cone (top panel) and wave (bottom panel), which both contain dynamic filopodial and lamellipodial protrusions. Relative times are shown in seconds in the lower right of the images. D. Quantification of change in growth cone and wave area over ten second intervals (ΔA/10s) indicates that growth cones and waves have similar dynamics. Growth cones show 23.5±7.5% ΔA/10s and waves show 27.5±7.3% ΔA/10s (non-significant difference (ns)). E. Wave (arrowhead) stained for acetylated and tyrosinated tubulin and phalloidin stainable F-actin. Insets show higher magnification of wave region. Color image is overlay of acetylated and tyrosinated tubulin.
Figure 2
Figure 2. Waves are associated with axonogenesis
A. Phase images of waves in stage 2–3 neurons. Multiple waves occur in the developing axon (arrowheads). Time shown is in h:min. B. Quantification of the frequency and impact of anterograde waves in neurites of stage 2, stage 2–3 and stage 3 neurons (see definition in methods) followed for 24–72 h. Wave frequency increases in the developing axon during the stage 2–3 transition. During stage 2, the neurite destined to become the axon averages 1.14±0.45 waves/h and minor neurites average 0.63±0.31 waves/h. During the stage 2–3 transition, the developing axon averages 1.55±0.67 waves/h while the minor neurites average 0.55±0.22 waves/h. During stage 3, the newly formed axon averages 1.2±0.53 waves/h while the minor neurites average 0.59±0.22 waves/h. Significance is indicated for axon compared to minor processes: ** p<0.01; *** p<0.005. (n= 10 neurons, Error bars = sd). The impact of waves to bursts of neurite outgrowth is different for developing axons and minor processes and varies at different stages. During the stage 2–3 transition the impact of waves on neurite growth is greater in the developing axon compared to the minor processes (1.5±0.2 fold greater). During stage 3 the relative contribution of waves to neurite growth remains high in the axon compared to the minor processes (1.7±0.3 fold greater). Significance is indicated for axon compared to minor processes: ** p<0.01 (n=6 neurons, Error bars = sd). C. Neurite branch formation. The bottom row of images shows magnified views of the branch point in the images above. Time shown is min. A small filopodia protruding from the shaft before wave arrival (arrowhead, bottom row) undergoes engorgement upon wave arrival (0:05 min) inducing the subsequent elongation of a new branch.
Figure 3
Figure 3. Cofilin activity may be involved in wave propagation
A. The ratio of total cofilin/phospho-cofilin (pseudocolor intensity profile) shows the relative distribution of dephosphorylated (putatively active) cofilin. Waves (arrowhead) have a high level of dephosphorylated cofilin, similar to that observed in the growth cone periphery. B. Hippocampal neurons expressing RFP (top row- control infected), Cofwt (second row) and CofA3 (third row). F-actin was stained with Alexa-phalloidin (green) and neurons expressing Cofwt and CofA3 were also stained for Tau1 (red). Note that the neurons expressing Cofwt and CofA3 have longer axons and more wave-like structures (arrowheads). C. Expression of either wild type (wt) or active (A3) cofilin, but not the inactive pseudophosphorylated (E3) cofilin enhances wave formation in neurons by 48.1±7.4% (*p<0.05) and 48.8±10.8% (ns), respectively (n=3 separate experiments, >75 neurons for each condition).
Figure 4
Figure 4. Inhibition of myosin II enhances axonogenesis, neurite branching and increases the prevalence of wave-like structures
A. Control neurons (left) and those treated with the myosin II inhibitor blebbistatin (right) were stained for the axonal marker Tau1 (red) and for F-actin with Alexa-phalloidin. Blebbistatin-treated neurons have longer axons and more extensive neuritic arbors. Many blebbistatin-treated neurons have multiple long axons (right). Wave-like structures are highlighted by arrowheads. B. Quantification of neuronal polarity phenotypes under different culture conditions (n = 3 separate experiments, >125 neurons). Neurons treated with 2.5 μM blebbistatin have 8.4±3.3% with no axon compared to 24.8±2.2% in controls (***p<0.005), 52.6±6.8% with one axon compared to 69.1±1.6% for controls, and 39.0±9.8% with multiple axons compared to 6.1±1.3% for controls (**p<0.01). C. Quantification of neurite branches. Control neurons average 0.48±0.1 branches/neuron whereas blebbistatin-treated neurons have an average of 1.56±0.45 branches/neuron (*p<0.05). D. Neurons exhibiting wave-like structures increase 1.46 fold when treated with blebbistatin (**p<0.01).
Figure 5
Figure 5. Inhibition of myosin II influences wave dynamics
A. Images of a neuron before and after treatment with blebbistatin. Blebbistatin reduces the retraction of the neurite preceding wave arrival. Before blebbistatin addition, as waves (black hollow arrowhead) approach the neurite tip (white arrowhead) there is a retraction of the neurite, followed by a small outburst of growth. After blebbistatin addition, a wave propagating down the same neurite does not cause retraction preceding outgrowth and the subsequent burst in neurite outgrowth (solid black arrowhead) is greater and longer lasting. B. Myosin inhibition increases wave frequency 22.2±0.1% (n=27 neurons, error bars= SEM). C. Myosin II inhibition decreases the average retraction distance induced by waves. Before wave arrival, control neurites retract 5.6±0.7 μm and blebbistatin-treated neurites retract 2.3±0.5 μm (**p<0.01) (error bars = SEM). D. Myosin II inhibition increases wave impact on neurite growth. Upon wave arrival, control neurites extend an average of 9.9±0.2 μm, whereas blebbistatin-treated neurites extend an average of 15.9±1.6 μm (**p<0.01) (n=13 neurons, error bars = SEM).
Figure 6
Figure 6. Waves increase growth cone size and dynamics
A. DIC images of a neuron with propagating wave (top row, arrowhead). Magnified view of time-lapse images of the same growth cone for 1 min before (top row) and 1 min after (bottom row) wave arrival. Time shown is in seconds. The growth cone enlarges and becomes more dynamic following wave arrival. B. Relative growth cone size increases 2.6±0.7 fold after wave arrival (n=18, error bars=sd; ****p<0.001). C. Growth cone activity (relative change in area every ten seconds (ΔA/10s) increases from 16.1±6.6% ΔA/10s to 22.5±6.2% ΔA/10s after wave arrival (n = 15, error bars=sd; ****p<0.001).
Figure 7
Figure 7. Waves transport actin
A. Fluorescence time-lapse images of a hippocampal neuron expressing RFP-actin. Time shown in min:s. The net fluorescence of RFP-actin in the growth cone increases following wave arrival as shown in pseudocolor images (hot scale) in right panels. B. Time-lapse images of hippocampal neurons expressing pcDendra-actin with no wave structures (top row) and within a wave (bottom row). The region photo-converted is within the highlighted box. Following photo-conversion, images were acquired at 561 nm excitation every 7.5 s. The fluorescence dissipates rapidly in a non-wave region but remains associated with the wave when photo-converted within a wave (insets). C. Line-scans of peak fluorescence intensity of pcDendra-actin following photo-conversion of a neurite with no wave (left) and of a wave (right). Photo-converted pcDendra-actin decays to <30% of the original peak intensity-value within 15 s in the non-wave region but remains high in the region of a wave (>90% of original peak intensity-value at 22.5 s). D. Fluorescence images (561 nm excitation) showing movement of pcDendra-actin in a wave as the wave moves down the neurite. E. Line-scans of pcDendra-actin over longer periods than in C. Photo-converted Dendra-actin dissipates almost completely within 2 min (green symbols) in non-wave region of a neurite but >30% of the total original wave fluorescence (red) remains after 8 min (dark blue) by which time the wave has traveled ~15 μm. The fluorescence distribution changes over time due to the changes in wave shape.
Figure 8
Figure 8. Waves occur in Thy1-YFP labeled neurons in hippocampal slices
Inverted fluorescence time-lapse images of a YFP-expressing neuron near the CA3 region of a P1 hippocampal slice from the Thy1-YFP transgenic mouse line H (Feng et al., 2000). Time shown is in min:s. Black arrowhead follows an anterograde wave which moves ~30μm at an average speed of 1.5μm/min toward the tip (gray arrowhead in first panel). The images are maximum projections of 6, 1μm steps of a Z series. The soma of this neuron is not within the field of view. This wave travels ~30μm in 20 min (average speed of 1.5μm/min). Upon reaching the distal neurite, the growth cone enlarges and the neurite extends (bottom row). Bar=30 μm.

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