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Article |
Correspondence to M. Laura Feltri: feltri.laura{at}hsr.it
Myelin is a multispiraled extension of glial membrane that surrounds axons. How glia extend a surface many-fold larger than their body is poorly understood. Schwann cells are peripheral glia and insert radial cytoplasmic extensions into bundles of axons to sort, ensheath, and myelinate them. Laminins and ß1 integrins are required for axonal sorting, but the downstream signals are largely unknown. We show that Schwann cells devoid of ß1 integrin migrate to and elongate on axons but cannot extend radial lamellae of cytoplasm, similar to cells with low Rac1 activation. Accordingly, active Rac1 is decreased in ß1 integrin–null nerves, inhibiting Rac1 activity decreases radial lamellae in Schwann cells, and ablating Rac1 in Schwann cells of transgenic mice delays axonal sorting and impairs myelination. Finally, expressing active Rac1 in ß1 integrin–null nerves improves sorting. Thus, increased activation of Rac1 by ß1 integrins allows Schwann cells to switch from migration/elongation to the extension of radial membranes required for axonal sorting and myelination.
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Introduction |
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 TopAbstract Introduction ResultsDiscussionMaterials and methodsReferences |
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6ß1, 6ß4 integrin, and dystroglycan (for review see Feltri and Wrabetz, 2005). Among these, ß1 integrins play a pivotal role in radial sorting, as its absence in SCs causes a defect similar to that of laminin mutants (Feltri et al., 2002). The signaling cascades activated by ß1 integrins to promote sorting are poorly known. Small Rho GTPases, such as Rac, Cdc42, and RhoA, are signaling molecules that cycle between an active (GTP bound) and an inactive (GDP bound) state. They influence cell shape by regulating actin upon activation from various stimuli, including integrin engagement (Hall et al., 1993; Nobes and Hall, 1995; Del Pozo et al., 2002, 2004). Rac1 promotes actin polymerization to produce lamellipodia and ruffles. Low levels of active Rac1 produce axial (at the two extremities of the main cell axis) lamellae, favoring directional cell migration, whereas higher levels of Rac1 produce radial (around the whole cell perimeter) lamellae (Pankov et al., 2005). Cdc42 regulates the formation of filopodia, whereas RhoA leads to the assembly of stress fibers and of focal adhesions (Nobes and Hall, 1995). Small GTPases are active in the peripheral nervous system (Terashima et al., 2001). Studies in Drosophila and in vitro proposed a role for Rac1 in glial migration and oligodendrocyte differentiation and for RhoA in internodal and nodal organization (Sepp and Auld, 2003; Liang et al., 2004; Melendez-Vasquez et al., 2004; Yamauchi et al., 2005). Little is known on the role of small GTPases in mammalian peripheral nerves and during sorting and myelination.
Here, we first determine that ß1 integrin–null SCs display normal cytoskeletal dynamics during migration and elongation on axons, but cannot produce radial lamellipodia, similar to cells with reduced levels of active Rac1. Second, we show that the levels of active Rac1 are reduced in nerves lacking ß1 integrin in SCs and that Rac1 is not targeted to the membrane of ß1 integrin–null SCs. Third, we generate a mouse with specific Rac1 deletion in SCs and show that Rac1 regulates radial lamellipodia, segregation of axons, and myelination. Finally, we show that exogenous activation of Rac1 in ß1 integrin–null nerves ameliorates the sorting defects. We conclude that SCs longitudinally oriented and elongated on axons produce radial processes that segregate and then myelinate axons upon ß1 integrin–mediated activation of Rac1.
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Results |
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We first characterized mutant DRG cultures explanted at E14.5. Mutant DRG reached a maximum of 60% ß1-negative SCs after 4 wk in culture (Fig. 1 A'), in contrast to postnatal nerves, where the extent of P0-Cre–mediated recombination was nearly complete (Feltri et al., 2002). ß1-null SCs migrated at distances from DRG (Fig. 1 A''). The number of SC nuclei was not reduced in mutant cultures (Fig. 1 E), although mutant SCs have a slight increase in the fraction of apoptotic nuclei (not depicted). Because of the presence of both ß1-negative and -positive SCs, the absence of ß1 integrin was always confirmed by direct or retrospective staining (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200610014/DC1).
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ß1 integrin–null SCs maintain proper association with axons
The inability of ß1 integrin–null SCs to sort axons could be due to impaired longitudinal migration on axons; lack of recognition, orientation, or association with axons; or inability to send processes toward or radially between axons. To distinguish between these possibilities, we visualized static and dynamic SC–axon interactions by videomicroscopy.
To evaluate whether mutant SCs maintained proper orientation and association with axons, we analyzed these parameters at 3–5 wk in culture. Cells were classified based on their relationship with axons (Fig. 1 G), and their proportions were quantified. ß1 integrin–null SCs presented a slight decrease in the percentage of cells associated with axons (Fig. 1 G). Despite the statistically significant difference, this decrease is probably not sufficient to explain the dramatic impairment of mutant SCs to form myelin.
ß1 integrin–null SCs recognize and explore axons normally using growth cone–like processes and migrate on axons
To evaluate the dynamic interactions between mutant SCs and axons, we used time-lapse microscopy. Previous experiments showed that SC tips remodel resembling growth cones, with filopodia and lamellipodia (Gatto et al., 2003). We asked whether mutant SCs manifest this behavior near axons in the presence or absence of ascorbic acid. In both conditions, mutant and wt SCs behaved similarly, as they organized dynamic processes at one extremity that interacted with and moved around the axon (Fig. S2 and Videos 3 and 4, available at http://www.jcb.org/cgi/content/full/jcb.200610014/DC1). The time frame during which these growth cone–like processes were dynamically reorganized was similar in both genotypes (Fig. S2 and Videos 1–4).
Next, we analyzed the ability of mutant SCs to migrate along axons in the presence or absence of ascorbic acid. No qualitative differences were observed between the migration of wt and mutant SCs. Both the cells sent filopodia-like processes to "sample" the environment, extended an axial lamellipodium, translocated the nucleus, and retracted the uropod. The time, extent of migration, and uropod retraction was similar (Fig. S2 and Videos 1 and 2). This suggests that ß1 integrin does not determine the ability of SCs to move longitudinally along axons.
In conclusion, mutant SCs perform correct "early" axonal interactions, namely, orientation, longitudinal migration, and association. After these steps, SCs must sort and myelinate axons by inserting processes radially within bundles and then around single axons. We modeled this phenomenon using isolated SCs spreading on a substrate.
ß1 integrin–null SCs have a defect in radial lamellipodia
To visualize the ability of mutant SCs to extend radial processes, we asked if they were able to spread on laminin, vitronectin, or poly-L-lysine (PLL). ß1 integrin–null SCs spread less when plated on laminin (Fig. 2, A and B).
In contrast, the spreading of mutant and wt cells plated on vitronectin and PLL was similar. This is consistent with the fact that ß1 integrin is contained in all laminin receptors expressed by cultured SCs except dystroglycan (Einheber et al., 1993; Feltri et al., 1994; Tsiper and Yurchenco, 2002; Saito et al., 2003), whereas ß1 integrin–null and wt SCs express the vitronectin receptor vß3 (unpublished data) and PLL promotes non–receptor-specific cell adhesion. Thus, deficient spreading by ß1-null SCs results from loss of ß1 integrin binding to laminin.
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Rac1 activity controls radial lamellipodia in SCs
It was recently shown that the formation of radial/peripheral versus axial lamellae in other cell types depends on levels of activation of the small GTPase Rac1 (Pankov et al., 2005). To ask directly if the levels of Rac1 affect the formation of radial and peripheral lamellae in SCs, we inhibited Rac1 using the specific NSC23766 inhibitor (Gao et al., 2004). NSC23766 inhibited the levels of active Rac in rat SCs in a dose-dependent way (Fig. 3, A–C).
In the absence of inhibitor, SCs spreading on laminins had both radial and axial lamellipodia (Fig. 3 D). At 100 µM of inhibitor (intermediate levels of active Rac1), the number of radial lamellipodia was reduced (P < 0.0001; n = 100), whereas the number of axial lamellipodia was unchanged (P = 0.6; n = 100; Fig. 3, E and G). Decreasing further the activity of Rac1 reduced the number of both radial and axial lamellipodia (Fig. 3, F and G). Few axial, and nearly no radial, lamellipodia were produced on vitronectin or PLL. Thus, laminins and high levels of Rac1 are required for the formation of radial lamellipodia in SCs. Having shown the effect of NSC23766 on SC lamellipodia, we asked if Rac1 inhibition also affected myelination by SCs in DRG explants. Low doses of inhibitor were sufficient to almost completely inhibit myelination, as shown by the absence of MBP-positive internodes (Fig. 3 Q), without affecting axons and SC number (Fig. 3 S).
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60% of wt nerves (Fig. 4 D). One way by which integrins regulate Rac1 is by promoting the translocation of GTP-bound Rac1 to the cell membrane, allowing interaction with the effector p21-activated kinase (PAK) 1 (Del Pozo et al., 2000). To test if GTP-bound Rac was able to target to the SC membrane in the absence of ß1 integrin, we added purified GST–PAK binding domain (PBD) to wt or mutant SCs spreading on laminin and visualized its localization using anti-GST antibodies. We favored internalization of PBD-GST using saponin. Addition of GST alone resulted in no immunostaining (unpublished data). As expected, PBD was enriched at the surface of lamellipodia in wt cells, suggesting that active Rac1 is recruited at the leading edge of SC spreading on laminin (Fig. 4, E, E', and G). In contrast, PBD-GST was not enriched in processes of mutant SCs (Fig. 4, I and I'). The fraction of processes with membrane enrichment was significantly lower in ß1 integrin cells (121 out of 208 processes in 47 wt cells and 33 out of 114 processes in 31 null cells; P < 0.005 by 
2 analysis). This suggests that Rac1 translocation is inhibited in the absence of ß1 integrin. PBD can bind also Cdc42. To evaluate whether PBD could be interacting with Cdc42 in lamellipodia, we stained wt and mutant SCs for Cdc42. Cdc42 is excluded from lamellipodia of SCs spreading on laminin (Fig. 4 K), and its localization is similar between ß1 integrin–negative and –positive cells (Fig. 4 M). Thus, Cdc42 does not colocalize with PAK during lamellipodia formation on laminin. We conclude that SCs recruit active Rac1 to the membrane of lamellipodia when spreading on laminin and that this translocation is impaired in the absence of ß1 integrin.
Cdc42 and RhoA activity in ß1 integrin–null nerves
We next assayed the activity Cdc42 and RhoA in mutant nerves. The levels of active Cdc42 were not substantially different (Fig. 4 D), consistent with the normal localization of Cdc42 in ß1-null SCs. This suggests that Cdc42 is activated independently of ß1 integrins in SCs.
RhoA activity was markedly reduced in ß1 integrin–null and control sciatic nerves (Fig. 4 D). Because RhoA in other cell types controls the formation of focal adhesion and stress fibers (Hall et al., 1993), we visualized them in wt and mutant SCs using phalloidin and antibodies against talin and paxillin. We could not detect any qualitative difference in focal adhesion and stress fiber formation between wt and mutant cells (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200610014/DC1).
Rac1 conditional null mice manifest delayed axonal sorting and hypomyelination
Because the absence of ß1 integrin in SCs causes a reduction in lamellipodia formation and Rac1 activity and Rac1 promotes radial lamellipodia formation in SCs, we hypothesized a role for Rac1 in the formation of radial processes by SCs during axonal sorting (Webster et al., 1973). To test this, we generated mice with an SC-specific deletion of the Rac1 gene, by crossing mice bearing a conditional loxP-flanked allele of Rac1 (Rac1F) with P0-Cre transgenic mice (Feltri et al., 1999; Walmsley et al., 2003). Rac1 conditional null mice were viable but developed severe clenching and occasional paralysis of posterior limbs (Fig. 5 A).
We analyzed recombination of the Rac1 locus by PCR on genomic DNA from different tissues of Rac1F/F//P0-Cre mice. As shown for other floxed loci (Feltri et al., 2002; Saito et al., 2003; Bolis et al., 2005), P0-Cre mediated specific and robust inactivation of Rac1 (Fig. 5 B) in sciatic nerves, but not liver and kidney. Recombination was also observed in brain, as previously reported for some floxed loci using this P0-Cre (Bolis et al., 2005). By Western blot analysis, the levels of Rac1 protein were reduced in mutant nerves (Fig. 5 C). We could not demonstrate the complete absence of Rac1 protein or activity in SCs by Western blot, pull-down assay, or immunohistochemistry of nerves (Fig. 5 D and not depicted), likely because Rac1 is expressed in axons and because all available anti-Rac1 antibodies also recognized the highly related Rac3 (Haataja et al., 1997). To ask whether Rac1 affected SC number, we counted the number of nuclei in P45 Rac1 mutant nerves. The number of nuclei per sciatic nerve segment was not reduced (Fig. 5, F and G). Instead, the number of nuclei was increased, as well as their density, as the mutant nerve was smaller. Cdc42 and RhoA activities were not substantially different between mutant and wt mice (Fig. 5 D).
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Delay of radial sorting and myelination in mice with SC-specific ablation of Rac1
We next analyzed sciatic nerve morphology by semithin and ultrathin sections during the postnatal development of Rac1F/F//P0-Cre mice and controls. In wt animals, P5 nerves contain few bundles of unsorted axons, axons in a one-to-one relationship with SCs and many thinly myelinated fibers (Fig. 7 A).
In contrast, in P5 Rac1 mice, many axons were unsorted in bundles, some axons were in a one-to-one relationship, and no myelinated fibers were present (Fig. 7 D). Ultrastructural analysis at P5 confirmed that many axons were unsorted in more numerous and larger bundles in mutant than in wt littermates (Fig. 7, G and H, asterisks). In P10 wt nerves, occasional unsorted bundles were present, and myelin becomes thicker (Fig. 7 B). In contrast, several unsorted bundles of axons remained in Rac1-null nerves (Fig. 7 E, arrows), which contain axons >3 µm (Fig. 7 I, double asterisks). Many SCs began to segregate large caliber axons in a one-to-one relationship (promyelinating SCs), and several thinly myelinated fibers appeared (Fig. 7 E). Thus, developing Rac1-null nerves present a delay in axonal sorting and myelination. Many promyelinating SCs in Rac-null nerves showed bizarre and disoriented cytoplasmic processes directed away from axons (Fig. 7 I), strikingly similar to those observed in ß1 integrin–null nerves (Feltri et al., 2002). By P28, unsorted axons were not detectable in mutant nerves, and there was a progressive increase in the number of myelinated fibers (Fig. 7 F). However, many large caliber axons were devoid of myelin or had thin myelin sheaths. Thus, Rac1 is required for timely radial sorting of axons by SCs, but the sorting defect can be overcome, possibly by compensation or redundancy with other GTPases such as Rac3, Cdc42, or RhoG. After reaching the promyelinating stage, albeit with delay, many Rac1-null SCs are still unable to myelinate.
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2, 4, and 
1 laminin chain and to collagen IV did not reveal abnormalities in mutant nerves. A subset of fibers in Rac-null sciatic nerves showed higher 4 laminin immunoreactivity, probably because of their immaturity (Fig. S5 F, available at http://www.jcb.org/cgi/content/full/jcb.200610014/DC1). Basal laminae appeared normal ultrastructurally (Fig. S5, I and J). Similarly, we asked if Rac activation in rescued ß1 integrin–null nerves had an effect on the basal lamina. ß1 integrin–null nerves contain both fibers with normal and redundant basal lamina (Fig. S5, K and M; Feltri et al., 2002). Similarly, CA-Rac rescued nerves contained both normal (Fig. S5, L') and redundant (Fig. S5, N') basal laminae around nerve fibers. Thus, basal laminae abnormalities do not seem to account for the delay in myelination seen in Rac-null nerves or for the rescue by Rac1 seen in ß1 integrin–null nerve. |
Discussion |
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Cell shape abnormalities in ß1-null SCs
Inactivation of laminins or ß1 integrin in SCs of transgenic mice causes an arrest in radial sorting (Feltri et al., 2002). Radial sorting requires first that the number of SCs match the number of axons and then that SCs change their shape to allow extension of processes around axons. Although laminin deficiency in SCs impairs proliferation (Yang et al., 2005) and survival (Yu et al., 2005b), these parameters are preserved in the absence of ß1 integrins, suggesting that a ß1-containing receptor promotes sorting by regulating changes in shape (Feltri et al., 2002). Here, we show that one abnormality in ß1 integrin–deficient SCs is indeed in their ability to spread. Specifically, the surface area and the number of lamellipodia are decreased in ß1 integrin–null cells and peripheral, but not axial, lamellipodia are affected. Because SCs must insert cytoplasmic lamellae radially among axons during radial sorting, we suggest that this is one major impairment in ß1-null nerves. It is possible that laminins use a different receptor to regulate SC proliferation and survival.
Spatial localization of Rac1 activation in SCs during axonal sorting
Rac1 activation and membrane translocation are impaired in ß1 integrin–deficient SCs. This suggests that ß1 integrins in SCs regulate Rac1 activation and membrane targeting and is consistent with data in several cell systems showing that ß1 integrins regulate Rac1 (Cox et al., 2001; Suzuki-Inoue et al., 2001; Hirsch et al., 2002; Miao et al., 2002; Laforest et al., 2005; Pankov et al., 2005). These data could imply that ß1 integrin in SCs is located at the leading edge of the advancing lamellipodia, which is in contact with axons during radial sorting. However, this is hard to reconcile with the fact that laminin mutants have a similar sorting phenotype (for review see Colognato et al., 2005), and laminins are in the basal lamina and thus located on the outer surface of the SC, away from the axon. At least three possibilities can explain this discrepancy. First, laminins at the time of radial sorting (E17.5 to P5 in the mouse) could also be present at the SC surface contacting the axon. Although the resolution of staining in SC processes contacting an axon bundle precludes a certain answer, this hypothesis is supported by the fact that SCs secrete laminins (Bunge et al., 1986) and that laminins may not need to be organized in a basal lamina for radial sorting to occur (for review see Feltri and Wrabetz, 2005). Second, laminins could initiate a transcellular signal that affects apical (in this case near the axon) surfaces. This has been shown in epithelial cysts, where laminin polymerization at the basal surface directs polarization of the apical surface via Rac1 (Ojakian and Schwimmer, 1994; Yu et al., 2005a). Another example showing that laminins in SCs control events at the adaxonal surface, possibly through transcellular signaling, is the clustering of voltage-gated sodium channels on the axon (Occhi et al., 2005). The final possibility is that laminins mediate sorting through a different mechanism (e.g., regulation of proliferation) and that ß1 participates in sorting via a nonlaminin receptor, such as 4ß1 or 5ß1 integrin.
Role of Rac1 and other Rho GTPases in nerve development
The generation of Rac1 conditional null mice shows that Rac1 is involved both in axonal sorting and myelination. The block in axonal sorting is transient and then some cells are blocked again at the promyelinating stage. As a result, many axons remain devoid of myelin, and the remaining axons are hypomy elinated. Probably the absence of Rac1 first impairs the formation of radial/peripheral processes that are necessary for the first steps of axonal sorting. A successive up-regulation or activation of other small GTPases (such as Rac3 or RhoG) may compensate at activity levels that are sufficient for the formation of radial lamellae of the appropriate size for sorting, but not large enough for wrapping during myelination. A redundancy with Rac3 or RhoG may thus potentially explain the differences in the severity of sorting observed between Rac1 and ß1 integrin mutant nerves. In addition, other molecules are likely to promote radial sorting downstream of ß1 integrins. Mice lacking focal adhesion kinase in SCs have recently been reported and have a radial sorting phenotype very similar to that of ß1 integrin mice (except for minor differences probably due to the timing of Cre recombination; Grove et al., 2007). Because Cdc42 and Rac1 cooperate in many cell types, it was surprising to find that Cdc42 activity was not reduced in ß1 integrin conditional null mice. Cdc42 controls filopodia formation (Nobes and Hall, 1995), and SCs use filopodia-like structures to explore axons during initial interactions (Gatto et al., 2003). We showed that both these filopodia-like structures and Cdc42 localization are normal in ß1-null SCs. Because Cdc42 is important for a cell to initiate its polarization, by acting on microtubule (for review see Hall, 2005), it is possible that these events are required for axonal sorting, but they precede the activation of ß1 integrin and Rac1 in SCs. Thus, we may have not observed an effect on Cdc42 because P0-Cre–mediated ß1 recombination occurred after E17.5. Alternatively, these results indicate that ß1 integrins activate Rac1 during axonal sorting, whereas Cdc42 is activated through another signaling pathway (see Benninger et al. on p. 1051 of this issue). Finally, the levels of active RhoA were markedly reduced in ß1 integrin–null nerves, suggesting that RhoA is also involved in SC development downstream of ß1 integrins. The role of RhoA in nerve development remains to be examined.
ß1 integrins signal via Rac1 to regulate SC shape during radial sorting of axons
Small interfering RNA–mediated reduction of Rac1 showed that modulation of Rac1 activity controls the decision between directionally persistent and random migration in cultured epithelial cells and fibroblasts. Low levels of active Rac1 promote the formation of axial lamellae at the extremities of the cell-long axis and favor directed migration. In contrast, increasing levels of active Rac1 favor the formation of peripheral lamellae with random migration and spreading (Pankov et al., 2005). Here, we show that this is also true for cultured SCs. More important, we provide the first application of this model in vivo in developing SCs. We propose that a progressive rise in Rac1 activation regulates the transition from migration/elongation to the arrest of migration and the wrapping of axons (Fig. 9).
Interestingly, the cellular phenotype of ß1 integrin–null cells is opposite to that of SCs deficient in merlin/Schwannomin, the tumor suppressor mutated in neurofibromatosis 2. Merlin is a negative regulator Rac1 (Shaw et al., 2001; Kissil et al., 2003), and loss of merlin in patients leads to Rac1 hyperactivation in SCs (Kaempchen et al., 2003), impeding elongation on axons (Nakai et al., 2006). Merlin is recruited to the cell membrane, where it interacts with ß1 integrin (Fernandez-Valle et al., 2002). We suggest that the association with ß1 integrin is inhibitory for merlin, permitting controlled activation of Rac1.
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Materials and methods |
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Organotypic neuron/SC cocultures
Mouse E14.5 DRG were dissected as described previously (Kleitman et al., 1991) and maintained in N/D Sato medium (modified from Cosgaya et al., 2002). Myelination was induced with 50 µg/ml ascorbic acid (Sigma-Aldrich). Time-lapse analysis was performed on glass-bottomed 35-mm plates (Mat-Tek), using an inverted microscope (Axiovert; Carl Zeiss Microimaging, Inc.). Images were captured every 60 s, for a maximum of 2 h, by a charge-coupled device camera (Hamamatsu) and analyzed with ImageJ. Recorded cells were identified by drawing on the plate with a diamond tip and retrospectively stained for ß1 integrin.
Cold jet
SCs were purified from DRG cultures by the cold jet technique (Jirsova et al., 1997), plated in N/D Sato, and stained after 24 h.
SC purification
Mouse SCs were isolated from P5 sciatic nerves stripped of perineurium using 4% collagenase and 2.5% trypsin, plated on coverslip in defined medium (Parkinson et al., 2001) with 10 ng/ml ß-neuregulin-1 (R&D Systems) and 2 µm forskolin (Calbiochem), and analyzed after 24 h.
Rac1 pharmacological inhibition
Rat SCs were plated on laminin in DME and 10% fetal calf serum for 2 h, treated with NSC23766 (Gao et al., 2004; provided by Drug Synthesis and Chemistry Branch, Developmental Therapeutic Program, Division of Cancer Treatment and Diagnosis, Bethesda, MD) in defined medium for 30 or 120 min, and stained with phalloidin or assayed for Rac1 activity. For myelination, NSC23766 was added to mouse DRG during ascorbic acid treatment.
Immunofluorescence
Immunohistochemistry was performed as described previously (Feltri et al., 2002). Immunocytochemistry was performed on glass coverslips, coated with PLL alone (Sigma-Aldrich), 0.1 mg/ml, or followed by coating with 10 µg/ml vitronectin or laminin 1 (Sigma-Aldrich). Purified mouse SCs or DRG were fixed in 4% PFA or in dodecyl-trimethylammonium chloride + 1% PFA (Nakamura, 2001) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) or 100% methanol. To reveal active Rac1 localization, recombinant PAK1 PBD-GST protein was detected with an anti-GST antibody. PAK1 PBD-GST was added at a final concentration of 0.01 µg/µl. After 5–6 h, cells were fixed with 4% PFA for 15 min at RT, permeabilized with 0.2% saponin (Sigma-Aldrich)/0.2% gelatin gepulvert (Merck), and reexposed to PAK PBD-GST at 0.01 µg/µl diluted in 0.1% saponin/0.2% gelatin overnight at 4°C. Primary and secondary antibodies were diluted in 0.1% saponin/0.2% gelatin. GST alone was used as a negative control. Negative controls for ß1 integrin staining were ß1 integrin–deficient "TKO" embryoid cells (Stephens et al., 1993).
Antibodies
The following antibodies were used: rabbit anti–Neurofilament H (Chemicon); rat anti-Neurofilament (TA-51; a gift from V. Lee, University of Pennsylvania, Philadelphia, PA); mouse anti–Neurofilament M (Roche and Chemicon); rabbit anti–ß1 integrin (a gift from K. Rubin, University of Uppsala, Uppsala, Sweden); goat anti-paxillin antibody, goat anti-talin antibody, rabbit anti-Rac1, mouse anti-RhoA, and rabbit anti-Cdc42 (Santa Cruz Biotechnology, Inc.); goat anti-GST (GE Healthcare); mouse anti-Rac1 (Upstate Biotechnology and BD Biosciences); mouse anti–ß-tubulin (Sigma-Aldrich); rabbit anti–S-100 (DakoCytomation); rat anti–laminin 2 (a gift from L. Sorokin, Lund University, Lund, Sweden); rabbit anti–laminin 4 (a gift from J. Miner); and peroxidase-conjugated anti-HA antibodies (Roche). Secondary antibodies were conjugated with FITC, TRITC, or Cy5 fluorochromes (Jackson ImmunoResearch Laboratories and Sothern Biotechnology Associates, Inc.).
Western blotting and Rho GTPase assays
GST pull-down assays for Rho GTPases were performed for Rac and Cdc42 as described previously (Benard et al., 1999) using a pGEX-cRac1A plasmid (a gift from G. Bokoch, The Scripps Research Institute, La Jolla, CA), and for RhoA using Rhotekin RBD-GST plasmid (a gift from C. Laudanna, University of Verona, Verona, Italy) as described by Giagulli et al. (2004), with few modifications. Lysates from sciatic nerves at P1, P5, and adult mice or from SCs were triturated in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, and 5 mM MgCl2 with protease and phosphatase inhibitors. After centrifugation, 500 µg of lysates were incubated either with 17 µg PAK1 PBD-GST or 35 µg Rhotekin RBD-GST bound to glutathione agarose for 60 min at 4°C. Lysates activated or inactivated with GTP-s or GDP-ßs were used as positive and negative controls, respectively. Beads were washed once in lysis buffer and, together with total lysates (10 µg for Rac1 and Cdc42 and 20 µg for RhoA), heated for 5 min at 100°C in reducing sample buffer and processed for Western blotting by standard methods. For quantification, films were digitalized and analyzed using ImageQuant v1.2 for Mac software (Molecular Dynamics). Images for comparison were always on the same gel.
Adenovirus injection
1.5 µl adenovirus-Rac1CA (1012 virions/ml; a gift from M. Resh [Memorial Sloan-Kettering Cancer Center, New York, NY]; Liang et al., 2004), saline, or adenoviruses expressing GFP of LacZ were injected in the endoneurium of P10 sciatic nerve of anesthetized mice (seven animals total). Nerves were collected after 12 d. To quantify the rescue, the mean area of all bundles of unsorted axons present in transverse semithin section of the complete –1 levels from seven different experiments were measured using ImageJ. Promyelinating and myelinating axons were counted as a proportion of total nerve fibers in 10 random electron microscopic fields from the –1 levels in seven CA-Rac1–injected and seven control nerves.
Morphological analysis
Morphological analyses of nerves were conducted as described previously (Wrabetz et al., 2000).
Image acquisition and analysis
Images were acquired using confocals (UltraView ERS spinning disk confocal microscope [PerkinElmer] equipped with a Plan Apochromat 63x/1.4 oil-immersion objective and using the UltraView acquisition software; TCS-SP5 [Leica] equipped with a Plan Apochromat 63x/1.4 oil-immersion objective and using the LCS confocal acquisition software [Leica]; or Confocal-MRC 1024 laser-scanning confocal microscope [Bio-Rad Laboratories, Inc.] equipped with a Plan Neofluar 40x/1.3 oil-immersion objective and using the Laser Sharp 2000 acquisition software) or a camera (DFC480 R2; Leica) mounted on a fluorescence microscope (DM 5000 B; Leica) equipped with N Plan 10x/0.25, HC PL Fluotar 20x/0.50, and HCX PL Fluotar 40x/0.75 objectives, using Firecam software (Leica). Videos were acquired using a camera (Orca II; Hamamatsu) mounted on a microscope (Axiovert S100 TV2; Carl Zeiss MicroImaging, Inc.) equipped with a Plan Neofluar 40x/1.3 oil-immersion objective at 37°C in Eagle's minimum essential medium with 10% FCS and 25 mM Hepes, without phenol red, with or without ascorbic acid, and using the Image Pro-Plus 4.5 acquisition software. Electron microscopy sections were visualized and photographed using a transmission electron microscope (EM900; Carl Zeiss MicroImaging, Inc.). Films were digitalized using a scanner (Arcus II; Agfa-Gevaert). Image processing and quantification was performed using Photoshop 7.0 (Adobe) or ImageJ (v1.33u). Adjustment of brightness or contrast was used in some cases but without obscuring, eliminating, or misrepresenting information. Statistical analysis was performed using Excel (Office X; Microsoft), Stat View (v5.0), and SPSS v11.
Online supplemental material
Videos 1 and 2 show a wt and a ß1 integrin–null SC, respectively, migrating on a DRG axon in a qualitatively similar manner. Videos 3 and 4 show a wt and a ß1 integrin–null SC, respectively, interacting with axons in a similar fashion. Fig. S1 shows that the cells in Videos 2 and 4 were negative for ß1 integrin. Fig. S2 shows still frames from Videos 1–4. Fig. S3 shows that stress fibers and focal adhesions appear normal in ß1 integrin–null SCs. Fig. S4 shows the method used to quantify PAK PBD-GST enrichment in the cell membrane. Fig. S5 shows that basal laminae are normal in Rac1-null nerves and in CA-Rac rescued ß1 integrin–null nerves. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200610014/DC1.
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Acknowledgments |
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This work was supported by the National Institutes of Health (NS045630 and NS055256), Telethon, Italy (GGP04019 and GGP030074), and the Medical Research Council, UK.
Submitted: 3 October 2006
Accepted: 16 May 2007
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Jessen, K.R., and R. M
