Summary
Inhibition of kinesin-5, a mitotic motor also expressed in neurons [1], causes axons to grow faster due to alterations in the forces on the microtubules (MTs) in the axonal shaft [2-4]. Here, we investigated whether kinesin-5 plays a role in growth cone guidance. Growth cone turning requires that MTs in the central (C-) domain enter the peripheral (P-) domain in the direction of the turn. We found that inhibition of kinesin-5 in cultured neurons prevents MTs from polarizing within growth cones and causes them to grow past cues that would normally cause them to turn. We found that kinesin-5 is enriched in the transition (T-) zone of the growth cone and that kinesin-5 is preferentially phosphorylated on the side opposite to the invasion of MTs. Moreover, when a growth cone encounters a turning cue, phospho-kinesin-5 polarizes even before the growth cone turns. Additional studies indicate that kinesin-5 works in part by antagonizing cytoplasmic dynein, and that these motor-driven forces function together with the dynamic properties of the MTs to determine whether MTs can enter the P-domain. We propose that kinesin-5 permits MTs to selectively invade one side of the growth cone by opposing their entry into the other side.
Results and Discussion
Effects of kinesin-5 inhibition on growth cone turning
To test whether kinesin-5 (also called Eg5) is involved in growth cone turning, we first used the “border assay,” which utilizes a glass coverslip with a sharp border between a laminin-coated side and a polylysine-coated side. When explants of rat sympathetic ganglia are plated on the laminin side, growth cones normally turn when they reach the border. Indeed, almost all axons turned at the border in the case of DMSO-treated control neurons. However, very few axons turned in the presence of monastrol, an allosteric inhibitor of kinesin-5 [5] (Fig 1A-C). Instead, the growth cones ignored the border, crossed it, and continued growing on the polylysine side. This effect was quantified by measuring the fluorescence for the immunostained MTs on each side of the coverslip. The ratio of MT fluorescence per unit pixel area of axons that crossed the border and axons that turned at the border was 0.0232 ± 0.008 (n=7) and 0.790 ± 0.046 (n=11) respectively for control and monastrol-treated cultures, a 40-fold difference (p ≤ 0.0001, two tailed t-test). The same result was obtained using explants of chicken dorsal root ganglia (DRG) (data not shown).
Figure 1. Inhibition of kinesin-5 inhibits growth cone turning at a laminin/polylysine border, inhibits turning toward NGF-coated beads, and prevents reorganization of MTs in response to NGF-coated beads.
(A, B) Axons (green, tubulin) grow on laminin (red) and their growth cones reach the border. (A) Growth cones of control explants treated with 0.1% DMSO turn at the border. (B) In the presence of 100 μM monastrol, most growth cones cross the border. Scale bar, 15 μm. (C) Quantification of the ratio of MT fluorescence on polylysine (axons that crossed the border) and laminin (axons that turned at the border) shows a significant increase in cultures treated with monastrol (red bar) compared to DMSO (blue bar), *, p ≤ 0.0001. (Data represented as mean ± SEM). (D) Quantification of the ratio of MT fluorescence as above shows that when kinesin-5 was depleted using siRNA, a similar result as with monastrol treatment was observed. *, p ≤ 0.001. (E) and (F) Time-lapse phase-contrast images of growth cones of chicken DRG explants treated with DMSO (as the control) or monastrol to inhibit kinesin-5 respectively. (E) Example of a growth cone treated with 0.1% DMSO turning toward the NGF-coated bead. (F) Example of a growth cone treated with 100 μM monastrol that had contacted the NGF-coated bead but did not turn toward the bead. Scale bar, 10 μm. (G) Percentage of growth cones that turned toward the NGF-coated bead. *, p ≤ 0.001. (H) and (I) Examples of MT (green) and actin (red) organization in a DMSO-treated growth cone (H and H’) and monastrol-treated (I and I’) growth cone contacting an NGF-coated bead. The axonal axis (see Experimental Procedures) is depicted as a blue line. Bead locations are shown as dotted circles denoting the circumference of the beads. The control growth cone shows an increased MT distribution toward the contact point with the bead, but the monastrol-treated growth cone does not show any marked asymmetry in MT distribution. (J) Quantification of the MDR (as defined in Experimental Procedures) in control and monastrol-treated growth cones. The blue bar indicates the MDR in control growth cones that contact the bead. The red bar indicates the monastrol-treated growth cones that contact the bead. The yellow and green bars indicate growth cones that do not contact beads in the case of control and monastrol-treated growth cones, respectively. *, p < 0.009. Scale bar, 10 μm.
Qualitatively similar results were obtained when siRNA was used to deplete kinesin-5 from the neurons. Previous quantitative studies demonstrated that this approach depletes over 95% of kinesin-5 [4]. The ratio of MT fluorescence for controls (in this case, control siRNA) and kinesin-5-depleted cultures was 0.091 ± 0.005 (n=7) and 0.768 ± 0.039 (n=7) respectively (p ≤ 0.001, two-tailed t-test), which is an 8.44 fold increase (Fig 1D). Thus, either depletion or inhibition of kinesin-5 results in a dramatic inhibition of growth cone turning in response to substrate cues.
The growth cones of chicken DRGs turn toward NGF-coated polystyrene beads [7,8]. In DMSO-treated control cultures (n=19), the growth cones that made contact with an NGF-coated bead typically turned toward the bead (in 79% of the cases). In monastrol-treated cultures (n=14), growth cones that contacted beads turned only in 14% of the cases (p ≤ 0.001, chi-square test). The growth cones in these monastrol-treated cultures showed no reaction to encountering the bead, but instead advanced forward, past the bead, without turning (Fig 1E-G). Thus, inhibition of kinesin-5 curtails growth cone turning in response to either growth factors or substrate cues.
Effect of kinesin-5 inhibition on MT distribution during growth cone turning
We next studied MT distribution in the bead assay. Growth cones of control neurons displayed a polarized MT array with abundant MTs invading the P-domain on the side of the growth cone in the direction of the bead. There were fewer MTs invading the other side of the growth cone, away from the bead (Fig 1H, 1H’). By contrast, the monastrol-treated neurons displayed no such bias on one side of the growth cone or the other, with respect to MT distribution (Fig 1I, 1I’). The MDR (“MT Distribution Ratio”; see Experimental Procedures) was 2.43 in control growth cones (n=13) and 1.10 in monastrol-treated growth cones (n=14) (p < 0.009) (Fig 1J). Analysis of the percentage of NGF-coated beads contacting axons that elicited filopodial sprouting [6], and the number of filopodia associated with each NGF-coated bead failed to reveal a difference suggesting that monastrol did not alter the response of the filamentous actin to the NGF-coated beads (data not shown). These results suggest that kinesin-5 is crucial for the ability of growth cones to alter their MT distribution in response to a turning cue.
MT behaviors in growth cones after experimental manipulations of kinesin-5 and cytoplasmic dynein
To visualize and quantify MT behaviors in growth cones, we quantified the number and velocity of the EGFP-EB3 “comets” within filopodia of EGFP-EB3-transfected neurons, as described previously [7-9]. Quantification over the course of 3 minutes showed significantly more comets (and with a higher velocity) in filopodia of kinesin-5-depleted growth cones as compared to control growth cones. These results, shown in supplemental figure 1, suggest that kinesin-5 normally opposes the entry of MTs into filopodia.
Recent studies have shown that forces generated by cytoplasmic dynein enable MTs to overcome myosin II-based retrograde flow and enter the P-domain, including the filopodia [7-9]. Forces generated by kinesin-5 have been shown to oppose forces generated by cytoplasmic dynein during mitosis [10] and in MT asters [11]. To investigate whether the same is true in growth cones, we treated rat sympathetic neurons with control siRNA or siRNA to dynein heavy chain (DHC) for 2 days as described previously [12], and then re-plated the neurons and allowed them to grow axons anew for 7 hours either in DMSO or monastrol. Under these conditions, DHC levels were reduced by roughly 90% by Western blot [12]. After re-plating, the cultures were exposed to either monastrol or DMSO.
In results similar to those obtained in the siRNA-based depletion studies, quantification of the number of comets that invaded per filopodium (Fig 2E) during the course of 3 minutes showed a significant increase in case of monastrol-treated growth cones (22.78 ± 2.72, n=9) as compared to DMSO-treated growth cones (6.4 ± 0.86, n=15) (p ≤ 0.001, two-tailed t-test) (Fig 2A and 2C). In the case of DHC-depleted neurons, the number of comets was 1.3 ± 0.51 (n=12) in the presence of the DMSO and increased nearly 16-fold to 16.55 ± 1.01 (n=11) in the presence of monastrol (Fig 2B, 2D, and 2E (p ≤ 0.001, two-tailed t-test)). Also, the velocity of EB3 comets in the filopodia (Fig 2F) was increased in monastrol-treated growth cones (0.0357 ± 0.005 μm/sec) compared to the control (0.014 ± 0.003 μm/sec) (p ≤ 0.005, two-tailed t-test). When dynein-depleted neurons were treated with monastrol, EB3 comet velocity was higher (0.023 ± 0.005 μm/sec) than comet velocities measured in growth cones of neurons treated with either control or DHC siRNA alone (0.0065 ± 0.002 μm/sec) (p ≤ 0.01, two-tailed t-test). These results are consistent with a mechanism wherein kinesin-5 and cytoplasmic dynein work in an antagonistic relationship to regulate MT entry into filopodia. A similar antagonistic relationship is observed in measurements of axonal length (see supplemental figure 2).
Figure 2. MT behaviors in growth cones after experimental manipulations of kinesin-5 and cytoplasmic dynein.
Panels A-D show examples of fluorescence micrographs. (A) Neuron transfected with control siRNA and treated with DMSO. (B) Neuron transfected with DHC siRNA and treated with DMSO. (C) Neuron transfected with control siRNA and treated with monastrol. (D) Neuron transfected with DHC siRNA and treated with monastrol. Scale bar, 5 μm. (E) Graph showing the number of EB3 comets that enter the filopodia in growth cones. *, p ≤ 0.01; **, p ≤ 0.001. (F) Graph showing the velocity of EB3 comets that enter the filopodia. *, p ≤ 0.01; **, p ≤ 0.005. In (E) and (F), red bars indicate neurons transfected with control siRNA and treated with DMSO. Blue bars indicate neurons simultaneously depleted of DHC and treated with DMSO. Yellow bars indicate neurons treated with the combination of control siRNA and monastrol, and the combination of DHC siRNA and monastrol is represented by the green bars.
Several earlier studies demonstrated that the dynamic properties of MTs are important for determining whether they can enter filopodia during growth cone turning [13-16]. We analyzed the relative roles of motor-driven forces and MT dynamics in determining MT distribution in growth cones by determining the MT levels in the P-domain of growth cones that had been treated with 5 nM vinblastine after depletion of dynein by siRNA or inhibition of kinesin-5 with monastrol. At this low concentration, vinblastine dampens MT dynamics without causing detectable MT loss [7]. The results show that no matter what the experimental manipulation of DHC or kinesin-5 is, treatment with vinblastine results in a statistically indistinguishable low level of MTs in the P-domain (see supplemental figure 3). These results suggest that forces generated by molecular motors are one element in a multi-tiered mechanism that also includes the dynamic properties of the MTs.
The fact that MTs are able to overcome the retrograde flow of actin when both cytoplasmic dynein and kinesin-5 are suppressed may indicate that the assembly properties of the MTs are sufficient to overcome the retrograde flow, as long as the forces generated by the two opposing motors are eliminated. Alternatively, there may be other motors that contribute to the capacity of the MTs to overcome the retrograde flow. During mitosis, MTs are regulated by a variety of motor-driven forces together with the dynamic properties of the MTs [17], and the same might be true of growth cones.
Distribution and phosphorylation of kinesin-5 in the growth cone
Kinesin-5 is a homotetrameric motor that functions by generating forces between neighboring MTs [18]. In mitosis, the interaction of the motor with MTs is promoted by phosphorylation at amino acid 927 [26-28]. To investigate whether phosphorylation at this site regulates kinesin-5 in growth cones, we conducted studies with an antibody that recognizes total kinesin-5 and an antibody that recognizes only phospho-kinesin-5. Virtually all growth cones showed prominent staining for total kinesin-5, while the intensity of staining for phospho-kinesin-5 varied. Our general sense was that axons that had been growing straight displayed the least phospho-kinesin-5 in their growth cones, while axons that were in the process of turning showed the most. Fig 3A-B show micrographs of growth cones of cultured neurons double-labeled for MTs and with each of the two kinesin-5 antibodies. Immunostaining with the total kinesin-5 antibody shows that kinesin-5 is present in the axonal shaft and diffusely throughout the growth cone but is strongly enriched in the T-zone (Fig 3A). Staining with the total kinesin-5 antibody shows a significantly higher ratio of kinesin-5 to MTs in the T-zone (0.744 ± 0.11) as compared to the C-domain (0.145 ± 0.03) (p ≤ 0.005, two-tailed t-test) as well as the axonal shaft (0.222 ± 0.03) (n=5) (p ≤ 0.05, two-tailed t-test). The ratio of phospho-kinesin-5 to MTs was also higher in the T-zone (1.48 ± 0.37) as compared to the C-domain (0.138 ± 0.012) (p ≤ 0.05, two-tailed t-test) and the axonal shaft (0.099 ± 0.02) (n=6) (p ≤ 0.05, two-tailed t-test). Also, in the T-zone, the ratio of the phospho-kinesin-5 to MTs is much higher than the ratio of total kinesin-5 to MTs. This suggests that a far greater proportion of the kinesin-5 in the T-zone is phosphorylated compared to the kinesin-5 in the axonal shaft and C-domain (Fig 3D).
Figure 3. Distribution and phosphorylation of kinesin-5 in growth cones.
(A) and (B) Fluorescence micrographs of growth cones stained with the total kinesin-5 antibody (A) and phospho-kinesin-5 antibody (B). (A) shows a growth cone stained with the total kinesin-5 antibody; (A’) shows the same growth cone stained for MTs; and (A”) shows the overlay of A and A’. (B) shows a growth cone stained with the phospho-kinesin-5 antibody. (B’) shows the same growth cone stained for MTs, and (B”) shows the overlay of kinesin-5 and MT images. The box in (B’) is shown in higher magnification in ‘P’. Green arrowheads in ‘P’ shows the MTs in the P-domain and the red region shows phospho-kinesin-5. The blue squiggly line indicates the perimeter of the P-domain. Scale bar, 5 μm. (C) Quantification of the polarized distribution of kinesin-5 in growth cones. The ratio of kinesin-5 levels of one side to the other is shown for growth cones immunostained with antibody for total kinesin-5 (brown bar) and phospho-kinesin-5 antibody (orange bar). *, p ≤ 0.01. (D) Quantification of the levels of kinesin-5 in the T-zone, C-domain and axonal shaft. The values are ratioed against the levels of MTs in the same region. Brown bars indicate total kinesin-5 and the orange bar indicates phospho-kinesin-5. *, p ≤ 0.05. (E) Quantification of the levels of phospho-kinesin-5 and MTs on each half of the growth cones. Red bars indicate the levels of phospho-kinesin-5 and green bars indicate the levels of MTs on that side of the growth cone in the P-domain. *, p ≤ 0.05; **, p ≤ 0.005.
Whereas the total kinesin-5 antibody stained throughout the T-zone, the phospho-kinesin-5 antibody tended to stain more strongly in the T-zone on one side or the other of the growth cone (Fig 3B). To quantify this, we divided the growth cone into equal halves, and calculated the ratio of fluorescence intensity between the two halves. The ratio for total kinesin-5 is 1.15 (n=10), showing a slight tendency to polarize, whereas the ratio for phospho-kinesin-5 is 1.74 (n=9), showing a stronger tendency to polarize (Fig 3C) (p ≤ 0.01, two-tailed t-test). Of particular interest, when we quantified MT levels on each half of the growth cones, we found that the half that shows an enrichment of phospho-kinesin-5 (832.49 ± 110.61) shows lower levels of MTs entering the adjacent P-domain (249.04 ± 48.86) as compared to the MT levels in the P-domain on the other half of the growth cone (485.11 ± 80.44), (n=9) (p ≤ 0.05, two-tailed t-test) that shows lower levels of phospho-kinesin-5 (259.55 ± 52.66) (p ≤ 0.005, two-tailed t-test) (Fig 3E, see also Fig 3B‘P’).
Based on these observations, we hypothesize that kinesin-5 is mainly non-phosphorylated before the growth cone is challenged to turn and after a turn has been completed, but that it becomes phosphorylated when the growth cone is presented with a turning cue. If this is correct, polarization of phospho-kinesin-5 should occur even before the MT array becomes polarized and before the growth cone displays morphological indications of a turning response. To test these predictions, we analyzed the localization of phospho-kinesin-5 in a modified border assay (see Experimental Procedures). We chose axons that (i) had been growing straight toward the border but had either not yet reached it; (ii) had just reached the border but showed no indication of turning yet; or (iii) had completed turning in response to the border. As predicted, the first (n=13) and third (n=19) categories displayed very little phospho-kinesin-5 in their growth cones (Figs 4A, 4C). In sharp contrast, and also as predicted, prominent and highly polarized staining for phospho-kinesin-5 was detected in the second category of growth cones (n=16) (p ≤ 0.001, chi-square test) (Fig 4B; see also Fig 4D). These observations are consistent with a mechanism by which kinesin-5 is phosphorylated in a polarized fashion when the growth cone is challenged to turn, and that this event is critical for the subsequent polarization of the MT array underlying growth cone turning (Fig 4E).
Figure 4. Distribution of phospho-kinesin-5 in growth cones in the border assay.
(A-C) Growth cones stained for MTs (green) and phospho-kinesin-5 (red) at different stages on the laminin (blue)/polylysine border (white dotted line) preparation. The leading edges of the growth cones are shown by pink dotted line. White arrow denotes the localization of phospho-kinesin-5. (A) Growth cone that has not encountered the border. (B) Growth cone that has contacted the border and (C) Growth cone that has made a turn at the border. (D) Quantification of the percentage of growth cones that show localization of phospho-kinesin-5. Red bar indicates growth cones that have not reached the border, green bar indicates growth cones that have contacted the border and the blue bar indicates the growth cones that have already turned. *, p ≤ 0.001. Scale bar, 5 μm (E) Illustration of how forces exerted by different motor proteins may act during growth cone turning. Arrows show the direction of the forces exerted by each motor. Dynein-based forces (red arrows) enable MTs to enter into the P-domain by opposing myosin-II based forces (blue arrowheads). Kinesin-5 (purple and pink) is enriched in the T-zone but its phosphorylation state may exhibit asymmetry across the growth cone (purple = high phosphorylation, pink = low phosphorylation). It becomes more phosphorylated on the side opposite to the direction of turning (purple). When phosphorylated, kinesin-5 is able to generate forces (purple with blue arrows) in the T-zone that oppose the dynein-based forces. As a result, the MTs preferentially invade the P-domain on the side of the growth cone where kinesin-5 is less phosphorylated (pink). This scenario is consistent with our previously proposed model in which the forces that influence the distribution of long MTs in growth cones are generated by the same motors that regulate the transport of short MTs within the axonal shaft [19].
In conclusion, the studies reported here demonstrate that kinesin-5 plays an essential role in polarizing the MT array in response to cues that cause the growth cone to turn.
Supplementary Material
The growth cone makes contact with the bead and then turns to grow toward the bead. For more details, see figure 1 and its legend.
The growth cone makes contact with the bead but shows no apparent reaction to it, and then grows past the bead rather than toward it. For more details, see figure 1 and its legend.
The EB3 comets enter the filopodia. For more details, see figure 2 and its legend.
The EB3 comets do not enter the filopodia, but are restricted to the C-domain of the growth cone. For more details, see figure 2 and its legend.
The EB3 enter the filopodia in greater numbers as compared to the growth cone in movie 3. For more details, see figure 2 and its legend.
The EB3 comets robustly enter the filopodia, demonstrating that inhibition of kinesin-5 restores the entry of the comets suppressed by DHC depletion. For more details, see figure 2 and its legend.
Acknowledgments
This work was funded by grants from the National Institutes of Health (NIH), the Spastic Paraplegia Foundation, the Alzheimer’s Association, the Department of Defense, the Christopher and Dana Reeve Foundation, and the Craig H. Nielsen Foundation to P.W. Baas; and a grant from the NIH to G. Gallo. K.A. Myers was supported by a pre-doctoral NRSA from the NIH. We thank Niels Galjart for providing the EGFP-EB3 construct. P.W. Baas and G. Gallo are co-senior authors. Authors declare no competing financial interest.
Footnotes
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Associated Data
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Supplementary Materials
The growth cone makes contact with the bead and then turns to grow toward the bead. For more details, see figure 1 and its legend.
The growth cone makes contact with the bead but shows no apparent reaction to it, and then grows past the bead rather than toward it. For more details, see figure 1 and its legend.
The EB3 comets enter the filopodia. For more details, see figure 2 and its legend.
The EB3 comets do not enter the filopodia, but are restricted to the C-domain of the growth cone. For more details, see figure 2 and its legend.
The EB3 enter the filopodia in greater numbers as compared to the growth cone in movie 3. For more details, see figure 2 and its legend.
The EB3 comets robustly enter the filopodia, demonstrating that inhibition of kinesin-5 restores the entry of the comets suppressed by DHC depletion. For more details, see figure 2 and its legend.