Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Aug 26;29(34):10488-98.
doi: 10.1523/JNEUROSCI.2355-09.2009.

Negative Guidance Factor-Induced Macropinocytosis in the Growth Cone Plays a Critical Role in Repulsive Axon Turning

Affiliations
Free PMC article

Negative Guidance Factor-Induced Macropinocytosis in the Growth Cone Plays a Critical Role in Repulsive Axon Turning

Adrianne L Kolpak et al. J Neurosci. .
Free PMC article

Abstract

Macropinocytosis is a type of poorly characterized fluid-phase endocytosis that results in formation of relatively large vesicles. We report that Sonic hedgehog (Shh) protein induces macropinocytosis in the axons through activation of a noncanonical signaling pathway, including Rho GTPase and nonmuscle myosin II. Macropinocytosis induced by Shh is independent of clathrin-mediated endocytosis but dependent on dynamin, myosin II, and Rho GTPase activities. Inhibitors of macropinocytosis also abolished the negative effects of Shh on axonal growth, including growth cone collapse and chemorepulsive axon turning but not turning per se. Conversely, activation of myosin II or treatment of phorbol ester induces macropinocytosis in the axons and elicits growth cone collapse and repulsive axon turning. Furthermore, macropinocytosis is also induced by ephrin-A2, and inhibition of dynamin abolished repulsive axon turning induced by ephrin-A2. Macropinocytosis can be induced ex vivo by high Shh, correlating with axon retraction. These results demonstrate that macropinocytosis-mediated membrane trafficking is an important cellular mechanism involved in axon chemorepulsion induced by negative guidance factors.

Figures

Figure 1.
Figure 1.
A high concentration of Shh increases dextran+ vesicles in RGC axons. A, RGC axons were labeled with FITC–dextran for 15 min, with vehicle control, high Shh, or Slit2. The dex+ vesicles correspond to visible structures in DIC images, including reverse shadow-cast vesicles (arrow, inset), or protrusive vesicles (arrowhead). The insets are a twofold enlargement of the images. Scale bar, 5 μm. B, The percentages of dextran-positive axons were quantified and normalized to control. An inhibitor of Shh signaling, cyclopamine (cyc), abolished the effects of high concentrations of Shh on dextran labeling. **p < 0.01, ***p < 0.001, Student's t test. Numbers in parentheses indicate the total number of axons scored.
Figure 2.
Figure 2.
The dex+ vesicles are independent of clathrin. A, RGC axons were labeled with dextran, followed by staining with an anti-clathrin heavy chain antibody. Clathrin-positive puncta appeared excluded from the dex+ vesicles. B, An inhibitor to clathrin-mediated endocytosis, MDC, inhibited transferrin uptake in the cultured retinal cells via clathrin-dependent vesicles. C, MDC did not inhibit dextran uptake induced by high Shh or in the basal condition. Another inhibitor of clathrin-mediated endocytosis, chlorpromazine, also did not inhibit dextran uptake.
Figure 3.
Figure 3.
Characterization of the dex+ vesicles in RGC axons. A, RGC axons labeled with high Shh and dextran for 5 min were stained with phalloidin. By confocal microscopy analysis, some dex+ vesicles appeared surrounded by actin filaments (arrowheads). B, RGC axons labeled by dextran in the presence of high Shh were subsequently stained by antibodies specific to myosin IIA and IIB heavy chains. C, RGC axons were pretreated with vehicle control, cytochalasin D (CD), jasplakinolide (Jasp), blebbstatin (Blebb), or LY294002, wortmannin, or C3 transferase before incubation with dextran for 2 min in the presence or absence of high Shh. The percentages of dextran-positive axons were quantified and normalized to the controls. Data are represented as mean ± SEM. **p < 0.01, Student's t test. Numbers in parentheses indicate the total number of axons scored.
Figure 4.
Figure 4.
A, Time-lapse microscopy was performed to analyze growth cone collapse in the presence of vehicle control, high Shh or high Shh with blebbistatin, or C3 transferase. Note that blebbistatin and C3 transferase inhibited growth cone collapse in response to high Shh. B, Percentages of growth cone collapse in the presence of various inhibitors were scored. Growth cone collapse was defined as loss of lamellipodia and reduction of filopodia number to less than three per axon. Data are represented as mean ± SEM. **p < 0.01, Student's t test. Numbers in parentheses indicate the total number of axons scored. C, To analyze the Rho or Rac GTPases activities, cell extracts from the retinal tissues treated with vehicle control or high concentration of Shh for 2 min were immunoprecipitated by RBD domain of Rhotekin or PBD domain of Pak and blotted by anti-Rho or anti-Rac antibodies, respectively. Cell extracts treated with control (CTL) or high Shh for 2 min were also analyzed by Western blot using anti-phospho-myosin light chain antibody. D, Immunofluorescent staining using anti-phospho-myosin light chain antibody was performed in RGC axonal cultures. Insets represent 2× magnification of the images.
Figure 5.
Figure 5.
Dynamin is required for dextran uptake and growth cone collapse. A, RGC axons were transfected with constructs expressing GFP or DN dyn2. Note that DN dyn2 appears to surround some large vesicles in the growth cones (arrows). B, Transfected RGCs were tested for their response to high Shh on stripe assay. Note that the GFP-transfected axons mostly turned away from high Shh, whereas DN dyn2-transfected axons ignored the boundary between high Shh/BSA. C, RGC axons were pretreated with vehicle control (CTL), dynasore, dynamin inhibitory peptide (Dyn Pep) before incubation with dextran for 2 min in the presence or absence of high Shh. The percentages of dextran-positive axons were quantified and normalized to the controls. D, Growth cone collapse was scored, and data are represented as mean ± SEM. **p < 0.01, ***p < 0.001, Student's t test. Numbers in parentheses indicate the total number of axons scored.
Figure 6.
Figure 6.
Macropinocytosis is important for chemorepulsive axon turning. A, Turning assay was performed by providing vehicle control (CTL) or high Shh through a micropipette at 45° angle to the direction of axon extension (arrows). B, Composite drawings of the paths of axonal extension during the 20 min filming with various inhibitors in the culture media and high Shh or vehicle control provided via the micropipette (arrows). The origin represents the position of the center of the growth cone at the beginning of recording. The original direction of neurite extension, as defined by the last 20 μm segment of the axonal shaft at the beginning of the experiment, was aligned with the vertical scale line. Tick marks along the x- and y-axis represent 5 μm. C, Average turning angles and length of net axon extension for each condition are shown. D, Cumulative distributions of turning angles summarize the effects of various inhibitors on the repulsive turning of the RGC axons in response to high Shh. Each point depicts the percentage of growth cones bearing a turning angle equal to or less than the value indicated on the x-axis. Positive angles indicate turning toward the pipette, whereas negative angles indicate turning away from the pipette. E, FM1-43+ vesicles (arrowheads) were imaged by fluorescence time-lapse microscopy while Shh protein was provided through a micropipette positioned at 45° angle to the direction of axon extension (arrow). Blebb, Blebbistatin; LY, LY294002.
Figure 7.
Figure 7.
A, Percentages of axons undergoing dextran uptake were scored in the presence of vehicle control (CTL), calyculin A (CalA), and PMA. B, Axon turning assays were performed by providing calyculin A or PMA through a micropipette positioned at 45° angle to the direction of axon extension (arrow). C, RGC axons were treated with vehicle control or low concentration of Shh (0.5 μg/ml) for 2 or 15 min in the absence or presence of cyclopamine (cyc). Percentages of axons positive for dextran were scored and normalize to control. Soluble laminin was also tested for its effect on dextran uptake. Note that both low Shh and laminin (LM) significantly decrease dextran uptake in the RGC axons. D, Macropinocytosis is also required for ephrin-A2-induced repulsive axon turning. Increase of dextran uptake by ephrin-A2 in the RGC axons is significantly inhibited by coaddition of dynasore (p < 0.001). E, Turning assay was performed by applying ephrin-A2 through the micropipette. Note that repulsive axon turning induced by ephrin-A2 was abolished by addition of dynasore in bath. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, Student's t test. Numbers in parentheses indicate the total number of axons scored.
Figure 8.
Figure 8.
High Shh induces dextran uptake and axon retraction ex vivo. A, Dextran uptake can occur ex vivo in retinal explants. Flat-mount retinas were incubated with FITC–dextran and vehicle control (CTL) or high Shh for 2 min. The tissues were then washed, fixed, and photographed at the RGC side by fluorescence microscopy. Note that treatment with high Shh induced a marked increase in dextran uptake. B, RGC axons were labeled by DiI in the explant culture. The movement of RGC axons in the explant culture was filmed by time-lapse microscopy in the presence of vehicle control or high Shh in the culture media. Note that high Shh substantially increased the number of axons undergoing retraction compared with the control samples. Scale bars, 10 μm.

Similar articles

See all similar articles

Cited by 29 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback