cells by triggering ca2+-dependent cofilin activation and ... · the migration of oecs is guided...

186 Research Article

IntroductionOlfactory ensheathing cells (OECs) are a unique type of glial cellsin the olfactory system, and have been discovered to promote thegrowth of olfactory sensory axons during development and theregeneration of injured axons after being transplanted into nerveinjury sites (Cao et al., 2004; Li et al., 1998; Raisman and Li, 2007;Ramon-Cueto et al., 1998; Vincent et al., 2005). Derived from theolfactory placode, OECs migrate out of the olfactory epithelium(OE) together with growing olfactory sensory axons from the laminapropria (LP) and accumulate as a superficial mass upon reaching thetelencephalic vesicle at embryonic day (E) E13–E18 in rat,contributing to the formation of the presumptive olfactory nervelayer (Chuah and West, 2002; Valverde et al., 1992). However, howthe migration of OECs is guided during development remains unclear.

Slit and Robo are a pair of conserved repulsive ligands andreceptors for axon pathfinding (Dickson and Gilestro, 2006). Slitwas first identified in Drosophila as a molecule secreted by midlinecells, and was later shown to repel the extension of axons expressingRobo receptors (Brose et al., 1999; Kidd et al., 1999; Seeger et al.,1993) and to regulate the migration of cells such as neuronalprecursors (Wu et al., 1999). In the olfactory system, members ofthe Slit and Robo families are expressed in a specific spatio-temporal pattern and play important roles in the guidance ofolfactory sensory axons (Cho et al., 2007; Marillat et al., 2002;Nguyen-Ba-Charvet et al., 2008; Yuan et al., 1999). Whether OECs,which have the same developmental origin as olfactory sensoryneurons, are also responsive to Slits is unknown.

The signal transduction underlying the guidance of axonpathfinding and neuronal migration has been studied in cell culture.In dissociated culture of cerebellar granule cells, a frontal gradientof Slit-2 triggers the elevation of the intracellular concentration ofCa2+ ([Ca2+]i) in the leading growth cone, and the subsequentpropagation of a Ca2+ wave from the growth cone to the somamediates the reversal of soma translocation by inhibiting the activityof the small GTPase RhoA (Guan et al., 2007; Xu et al., 2004).However, the molecular mechanism underlying the Slit-2-inducedcollapse of neuronal growth cones is unclear.

In the present study, we tested the guidance effect of Slit on themigration of cultured OECs. We found that a Slit-2 gradient infront of migrating OECs triggered a Ca2+-dependent collapse andreversal of OEC migration. Furthermore, Slit-2 triggered theactivation of the F-actin severing protein cofilin and the inhibitionof RhoA, leading to the collapse of the leading front and thesubsequent reversal of the polarity of OEC migration.

ResultsRobo1 is expressed in OECs both in vitro and in vivoTo explore the potential effects of Slits on OEC migration, weexamined the expression of Robos in cultured OECs that exhibitactive cell migration (Huang et al., 2008). In purified OEC culture,RT-PCR experiments revealed that mRNAs of both Robo1 andRobo2 were highly expressed. However, mRNA of Robo3 wasundetectable (Fig. 1A). Robo1 protein could also be detected incultured OECs by western blotting, and its expression could be

Accepted 16 September 2010Journal of Cell Science 124, 186-197 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.071357

SummaryOlfactory ensheathing cells (OECs) migrate from the olfactory epithelium towards the olfactory bulb during development. However,the guidance mechanism for OEC migration remains a mystery. Here we show that migrating OECs expressed the receptor of therepulsive guidance factor Slit-2. A gradient of Slit-2 in front of cultured OECs first caused the collapse of the leading front, then thereversal of cell migration. These Slit-2 effects depended on the Ca2+ release from internal stores through inositol (1,4,5)-triphosphatereceptor channels. Interestingly, in response to Slit-2 stimulation, collapse of the leading front required the activation of the F-actinsevering protein cofilin in a Ca2+-dependent manner, whereas the subsequent reversal of the soma migration depended on the reversalof RhoA activity across the cell. Finally, the Slit-2-induced repulsion of cell migration was fully mimicked by co-application ofinhibitors of F-actin polymerization and RhoA kinase. Our findings revealed Slit-2 as a repulsive guidance factor for OEC migrationand an unexpected link between Ca2+ and cofilin signaling during Slit-2-triggered repulsion.

Key words: Ca2+, Cofilin, Migration, Olfactory ensheathing cells, RhoA, Slit-2

Slit-2 repels the migration of olfactory ensheathingcells by triggering Ca2+-dependent cofilin activationand RhoA inhibitionZhi-hui Huang1,2,3, Ying Wang1,2,3, Zhi-da Su1, Jian-guo Geng4, Yi-zhang Chen1,3, Xiao-bing Yuan5,* and Cheng He1,3,*1Institute of Neuroscience and Key Laboratory of Molecular Neurobiology of Ministry of Education, Neuroscience Research Center of ChangzhengHospital, Second Military Medical University, Shanghai 200433, China2Institute of Hypoxia Medicine, Wenzhou Medical College, Wenzhou, Zhejiang 325035, China3Institute of Neuroscience, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China4Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China5Institute of Neuroscience and State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China*Author for correspondence ([email protected]; [email protected])

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knocked down by transfection with specific short interfering RNA(siRNA) against Robo1 (Fig. 1B; supplementary material Fig.S1C,D). To examine the subcellular distribution of Robo receptorsin OECs, we transfected OECs with a construct encoding Robo2fused with GFP. As shown in Fig. 1C, GFP fluorescence(normalized by membrane marker Dil) was present mostly in thecell surface and enriched at the leading front. The distribution ofRobo2–GFP at the leading front suggests that migrating OECsmight be responsive to Slit. To examine the expression of Slit indeveloping OE, we stained the brain sections from E16 mice usingthe anti-Slit-2 antibody. Western blotting and immunocytochemistry

187Slit-2 repels OEC migration

confirmed the specificity of this antibody by detecting the specificSlit-2 signal in a HEK293 cell line stably expressing Slit-2, but notin the control cell line (supplementary material Fig. S1A,B).Consistent with previous reports (Nguyen-Ba-Charvet et al., 2008),immunostaining in E16 brain sections from mice showed that Slit-2 was highly expressed in apical cells of the OE (Fig. 1D). However,at the LP, Robo1 was highly expressed in cells that could belabeled by the antibody against the specific OEC marker p75;presumably these cells were migrating OECs (Fig. 1E). Theseresults suggest that the Slit receptor Robo1 is expressed in migratingOECs both in vitro and in vivo.

Slit-2 gradient repels the migration of cultured OECsTo test whether OECs are responsive to Slit-2, single-cell migrationassays were first performed in a low-density culture of OECsobtained from olfactory bulb (OB) tissue. A gradient of Slit-2 wasproduced in front of isolated migrating OECs by using amicropipette loaded with Slit-2 with repetitive injection by airpressure (Huang et al., 2008). As shown in Fig. 2C,F andsupplementary material Movie 1, after the application of a Slit-2gradient, the leading front of migrating OECs with elaboratedlamellipodia was inhibited in their motility and showed collapseand retraction within 20 minutes. For 23 out of 26 of tested cells,the soma later reversed their direction of translocation, with theoriginal trailing tail becoming a new leading front. Interestingly,after OEC migration had been reversed by the Slit-2 gradient,when the Slit-2 gradient was applied to the same OEC from thereverse direction, OEC migration was reversed again (Fig. 2C). Bycontrast, OECs appeared to have enhanced motility toward thegradient of lysophophatidic acid (LPA), a known attractant forOECs (Huang et al., 2008; Yan et al., 2003) (Fig. 2B,F); whereasOEC migration was not affected by the gradient of another axonguidance molecule Netrin-1, or by phosphate-buffered saline (PBS)(Fig. 2A,F).

To quantify the effect of tested factors on OEC migration, wemeasured migration rates of each cell during the period before andafter the application of factors and calculated their ratio(after/before). A ratio >1 implies accelerated cell migration inresponse to the treatment. Conversely, a ratio <1 implies inhibitionof migration, and a ratio <0 implies a reversal of migration (Huanget al., 2008). As shown by the cumulative distribution graph (Fig.2D), ratios of migration rate for most OECs under Slit-2 gradientwere <0, as indicated by a significant left shift in the cumulativedistribution curve of migration ratios compared with that of thePBS-treated group (P<0.001, Kolmogorov–Smirnov test), with theaverage migration ratio under Slit-2 gradient markedly decreased(Fig. 2E). By contrast, ratios of migration rate for most OECsunder LPA gradient were >1, and there was a significant right shiftin the distribution curve of migration ratios under LPA treatmentcompared with that of the PBS-treated group (P<0.001,Kolmogorov–Smirnov test) (Fig. 2D). The average migration ratiowas markedly increased under LPA gradient, but not changedunder Netrin-1 gradient (Fig. 2E). Furthermore, in the presence ofanti-Slit-2 antibody or the soluble protein of the Robo1 ectodomain(RoboN), a competitive inhibitor for Slit–Robo interaction (Wu etal., 1999), spontaneous migration of OECs was not affected.However, the Slit-2-induced collapse and reversal of migration ofOECs were both blocked, compared with treatment with a normalIgG or control media, respectively (Fig. 2E,F). These resultsstrongly suggest that Slit-2 repels the migration of these culturedOECs.

Fig. 1. Robo1 is expressed in OECs in vitro and in vivo. (A)RT-PCRanalysis of the expression of mRNAs encoding Robo1, Robo2 and Robo3 incultured OECs. (B)Western blotting detected the expression of Robo1 proteinin cultured OECs, OE and cerebellum tissue (a).The expression of Robo1 atboth mRNA level (left) and protein level (right) was downregulated by RNAinterference (RNAi) in cultured OECs (b). (C)Images of example cellshowing the distribution of Robo2–GFP in OECs. The ratio of fluorescenceintensity of GFP to Dil represents the Robo2–GFP density, coded bypseudocolors in a linear scale. (D,E)Sagittal brain sections of OE from E16mice were immunostained for Slit-2 (green) and Toto-3 to visualize nuclei(blue) (D), and for Robo1 (green) and p75 (red) (E). Images of selectedregions are shown at high magnification. Scale bars: 20m.

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Similar collapse and reversal of migration of OECs in responseto a Slit-2 gradient were observed in cultured OECs obtained frompostnatal day 0 (P0) OE tissue (supplementary material Fig. S2A–D). Moreover, in the high-density culture, when Slit-2 gradientwas applied toward a group of OECs, cells exhibited the obviouscollapse and retraction and escaped from the tip of the micropipettewithin 35 minutes in a distance-dependent manner (supplementarymaterial Fig. S2E). Taken together, these results show that Slit-2 isa repulsive guidance factor for the migration of cultured OECs.

Ca2+ release from Ins(1,4,5)P3 receptor channels isessential for the effect of Slit-2We next examined whether Ca2+ signaling was involved in Slit-2-induced repulsion of OECs. In OECs loaded with the Ca2+-sensitivefluorescent dyes Fluo-4 and Fura-Red, we observed a transientelevation in the ratio of the fluorescent intensity (Fluo-4/Fura-Red)both in the leading process and in the soma in response to the Slit-2 gradient, indicating that Slit-2 triggers the elevation of [Ca2+]i inthese cultured OECs (Fig. 3A,B). By contrast, when a PBS gradientwas applied at a similar distance from the OEC, no [Ca2+]i elevationwas observed in either the leading process or the soma (Fig. 3C,D).The elevation of [Ca2+]i was abolished by pretreatment withthapsigargin, a drug that depletes the intracellular Ca2+ stores, orwith 2-APB, an inhibitor of inositol (1,4,5)-trisphosphate

188 Journal of Cell Science 124 (2)

[Ins(1,4,5)P3] receptors at the internal stores, but not by removingthe extracellular Ca2+ (Fig. 3C,D). Furthermore, the repulsive effectof Slit-2 on OEC migration was totally blocked by bath applicationof thapsigargin or 2-APB, but not by removing the extracellularCa2+ (Fig. 3E,F). Thus, Ca2+ release from internal stores throughIns(1,4,5)P3 channels is essential for both Slit-2-induced collapseand reversal of migration of OECs.

Cytoskeletal reorganization induced by Slit-2Because cytoskeletal reorganization is essential for cell motility(Suetsugu and Takenawa, 2003), we next examined the cytoskeletalreorganization in OECs after Slit-2 exposure by usingimmunofluorescent staining. As shown in Fig. 4, in untreatedOECs, a thin layer of F-actin was evenly distributed in theperipheral region of the leading front, and F-actin bundles spannedthe central region of the fan-like lamellipodia of the leading frontand at the soma. By contrast, microtubules localized to the centraldomain of lamellipodia, parallel to the F-actin bundles, with a fewmicrotubules penetrating the peripheral region of the leading front.After Slit-2 stimulation, F-actin in the peripheral region of theleading front disappeared rapidly (within 10 minutes), and F-actindensity in the central domain of the lamellipodia reduced gradually.However, microtubules remained extending in the central andperipheral region of the lamellipodia before the full collapse of the

Fig. 2. Slit-2 gradient induces the collapse and reversal of migration of OECs in single-cell migration assay. (A–C) Images of OECs before and after frontalapplication of a gradient of PBS (A), 500M LPA (B) or 4g/ml Slit-2 (C). White arrowheads indicate the direction of the micropipette. Scale bars: 20m.(D)Cumulative distribution of migration ratios of OECs exposed to a gradient of PBS, Slit-2 or LPA. Migration ratio is the migration rate after loading of the factorgradient divided by that of control period (after/before). Each point represents the result from one OEC (n12). A ratio >1 and <0 means that the treatment attractsand repels OEC migration, respectively. **P<0.01, Kolmogorov–Smirnov test. (E)Average migration ratios under various conditions, as shown in F. Data aremean + s.e.m.; **P<0.01, Student’s t-test. (F)Histograms show the percentages of collapsed or reversed cells in total observed cells under various conditions.Netrin-1, 50g/ml; IgG, normal IgG; Anti-Slit, antibody against Slit-2 (1:200); CM, control medium; RoboN, medium containing ectodomain of Robo1.Jo

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leading front, even though F-actin had disappeared in the peripheralregion (Fig. 4B,C). Thus, the F-actin in the peripheral region ismore sensitive to the Slit-2 stimulation than microtubules, and thedisruption of F-actin in the peripheral region of the leading frontis implicated in initiating the collapse of leading front in OECs.

Cofilin activation through Ca2+–calcineurin signaling isrequired for Slit-2-induced collapse of the leading frontand reversal of migrationWhat causes the loss of F-actin in the peripheral region of the cellin response to Slit-2? Because cofilin is a key F-actin severingprotein (Theriot, 1997), we therefore examined whether cofilin isinvolved in the Slit-2-induced loss of F-actin in the leading frontof OECs. As shown in Fig. 5A, western blot analysis showed thatstimulation of OECs with Slit-2 markedly decreased the level ofphosphorylated cofilin (p-cofilin, the inactive form of cofilin) in atime-dependent manner, without affecting the total cofilin level.Immunofluorescent staining showed that in untreated OECs, theinactive p-cofilin was mainly localized at the soma and colocalizedwith F-actin at the peripheral region of the leading front. After Slit-2 incubation, the p-cofilin level in the whole cell dramaticallydecreased, and its signal in the peripheral region of the leadingfront disappeared, a process correlated with the loss of F-actin in


the peripheral region (Fig. 5B). Furthermore, anti-p-cofilin andanti-cofilin double-staining showed that the p-cofilin signal rapidlydecreased at the peripheral ruffles in Slit-2-stimulated OECs (10minutes) before the full collapse of the leading front; however,cofilin signal still localized at the peripheral ruffles (Fig. 5C).These results suggest that Slit-2 activates cofilin bydephosphorylation at the peripheral region, which could beresponsible for the disruption of F-actin at the peripheral region inresponse to Slit-2.

To further examine whether the activation of cofilin depends onthe Ca2+ signal, we added the intracellular Ca2+ chelator BAPTA-AM, or cyclosporin A (CsA), a specific inhibitor of the Ca2+- andcalmodulin-dependent phosphatase (calcineurin), to the bath beforeSlit-2 treatment. Interestingly, the Slit-2-induced dephosphorylationof cofilin in OECs was blocked by either BAPTA-AM or CsA(Fig. 5D). These results suggest that Slit-2 activates cofilin in aCa2+- and calcineurin-dependent manner.

To further examine the direct role of cofilin in Slit-2-inducedcollapse of the leading front, we transfected GFP-tagged wild-typecofilin (GFP–cofilin-WT) and a non-phosphorylatable andconstitutively active mutant of cofilin (GFP–cofilin-S3A) (Arberet al., 1998) into OECs and analyzed the morphology and motilityof OECs transfected with these constructs. After transfection of

Fig. 3. Intracellular Ca2+ release through the Ins(1,4,5)P3 channel is essential for Slit-2-induced collapse and reversal of migration of OECs.(A)Representative images showing [Ca2+]i at various time points (in seconds) before and after frontal application of a Slit-2 gradient (arrowheads). The [Ca2+]i wasdetermined by the ratio of Fluo-4 to Flura-Red fluorescence and coded by pseudocolors in a linear scale. White arrowheads and arrows indicate the gradientdirection and leading process and soma, respectively. Note the [Ca2+]i rise in response to Slit-2 at both the soma and the leading process. The selected sampleregions indicate the leading process (1) and the soma (2) for quantification of [Ca2+]i signal. Scale bar: 20m. (B)Changes in [Ca2+]i at the leading process and thesoma of OECs before and after exposure to a frontal Slit-2 gradient. The traces are based on averaging of [Ca2+]i signal of 8 cells at each time point.(C,D)Summary of average peak levels of Ca2+ signal based on the maximum [Ca2+]i signal from individual cells in the leading process (C) and the soma (D) undervarious conditions. (E,F)Average migration ratios of OECs (E) and the percentages of collapsed or reversed cells in total observed cells (F) in response to Slit-2gradient under various conditions. Thaps, thapsigargin. Data are mean + s.e.m.; **P<0.01, Student’s t-test.

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OECs with either GFP–cofilin-WT or GFP–cofilin-S3A, we didnot observe obvious changes in cell morphology and F-actinorganization, but observed marked elevation in migration speedcompared with cells transfected with GFP alone (supplementarymaterial Fig. S3A–D). When Slit-2 gradient was applied in frontof migrating OECs transfected with GFP–cofilin-S3A, it failed toinduce the collapse and reversal of migration of these cells.However, Slit-2 still induced the collapse and reversal of migrationin OECs transfected with GFP–cofilin-WT (Fig. 5E,F;supplementary material Fig. S3E,F). Taken together, these resultsindicate that cofilin dephosphorylation in response to Slit-2 throughthe Ca2+–calcineurin signaling pathway is essential for Slit-2-induced collapse of the leading front.

RhoA is required for the reversal of migration triggered bySlit-2What causes the reversal of migration of OECs in response to Slit-2? Because RhoGTPases (RhoA, Cdc42, Rac1) play a key role indirectional cell migration (Fukata et al., 2003) and the signaltransduction of Slit in axon guidance (Wong et al., 2001), we next


tested whether RhoGTPases are involved in the Slit-2-inducedreversal of migration of OECs. In the presence of a generalRhoGTPase inhibitor, toxin-B (10 ng/ml), the spontaneousmigration of OECs was not affected (Fig. 6B). Frontal applicationof the Slit-2 gradient on these toxin-B-treated OECs still triggeredthe collapse of the leading front, but failed to reverse the migrationof the soma, which stopped their forward migration after the fullcollapse of the leading front (Fig. 6B–D). By contrast, pretreatmentwith the specific phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 (20 M) did not affect Slit-2-induced repulsion of OECs(Fig. 6C–D). These results suggest that RhoGTPase activity isessential for the reversal of soma migration of OECs, but not forthe collapse of leading process triggered by Slit-2.

Further experiments were carried out in OECs transfected withconstructs expressing fusion proteins of GFP and various dominant-negative (DN) forms of RhoGTPases. We selected single OECs ofsimilar level of GFP fluorescence intensity for cell migration assays(supplementary material Fig. S4A,B). We found that in culturedOECs expressing DN-RhoA–GFP, frontal application of Slit-2gradient failed to induce the reversal of migration, although theleading process still exhibited collapse and retraction (Fig. 6C,D;supplementary material Fig. S4E). By contrast, Slit-2-inducedcollapse and reversal were not affected in cells expressing DN-Rac1–GFP or DN-Cdc42–GFP (Fig. 6C,D; supplementary materialFig. S4C,D). Thus, RhoA is specifically required for the somareversal. In support of this notion, we found that Slit-2-inducedreversal of migration was abolished by bath incubation with Y-27632 (20 M), a specific inhibitor of RhoA-dependent kinase(Rho kinase) (Fig. 6C,D), with the spontaneous migration of theseOECs being unaffected.

To further elucidate the role of RhoA in the reversal of migration,we used pull-down assays to examine the activity of RhoA incultured OECs. We found that treatment with either serum (10%)or the RhoA activating agent LPA (10 M) (Yan et al., 2003)resulted in marked elevation of RhoA activity in these culturedOECs (Fig. 7A,B). Interestingly, bath application of Slit-2 causeda reduction of RhoA activity in a time-dependent manner (Fig.7C,D), suggesting that the reversal in the direction of somatranslocation might be related to the inhibition, rather than theactivation, of RhoA. This notion was further supported byphotometric analysis of the active RhoA in migrating OECs byusing a FRET (fluorescence resonance energy transfer)-basedbiosensor pRichu-RhoA, which was constructed by linking CFP-conjugated RhoA and the YFP-conjugated RhoA-binding domain(RBD) of Rhotekin, a configuration sensitive to RhoA activationby guanine nucleotide exchange factor (Yoshizaki et al., 2003). Asshown in Fig. 7E, the FRET signal for the active RhoA displayeda polarized distribution in a migrating OEC, with the leading frontexhibiting higher activity than the soma and the trailing end.Application of a Slit-2 gradient in front of migrating OECstransfected with pRichu-RhoA led to a reduction in the FRETsignal of RhoA activity in the leading front and a reversal of thepolarity of active RhoA in this OEC (Fig. 7E,F). Taken together,these results suggest that downregulation of RhoA signaling isspecifically required for the soma reversal of OECs triggered bySlit-2.

Collapse of the leading front with simultaneous inhibitionof RhoA triggers the reversal of soma translocationTo further examine how the coordination between the collapse ofthe leading front and the reversal of soma translocation in response

Fig. 4. Slit-2 induces cytoskeletal reorganization in the leading front ofOECs. OECs stimulated by Slit-2 for indicated times were labeled for F-actin(red), acetylated microtubules (ace-MTs) (green) and p75 (blue). Images ofselected regions are shown below at higher magnification. Scale bar: 20m.

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to Slit-2 gradient is achieved, we applied a gradient of latrunculinA (LA) or cytochalasin D (CD), to mimic the Slit-2-triggeredcollapse of the leading front. After the application of a gradient ofLA or CD (50 M and 5 mM, respectively, in the pipette), theleading front of most OECs (15 out of 16 in CD; 14 out of 16 inLA) was inhibited in their motility and showed obvious collapsewithin 20 minutes. Only a few of these cells (4 out of 16 in 5 mMCD; 4 out of 16 in 50 M LA) later reversed their somatranslocation (Fig. 8B,E,F). Thus the collapse of the leading frontdoes not seem to be sufficient to trigger the reversal of somatranslocation, and other signals triggered by Slit-2 should berequired.

We have shown that the Slit-2 gradient inhibits RhoA activity inOECs. To further examine whether the downregulation of RhoAactivity in the leading front is sufficient to trigger the reversal ofsoma translocation, we applied a gradient of the Rho kinaseinhibitor Y-27632 (10 mM in the pipette) in front of migratingOECs to mimic a gradient of RhoA inhibition across the cell. Asshown in Fig. 8C,E,F, the Y-27632 gradient did not cause thecollapse of the leading front nor the reversal of soma migration,


but dramatically reduced the soma motility. Surprisingly, when weapplied a gradient of the mixture of Y-27632 and LA in front ofmigrating OECs, most cells (14 out of 16) quickly reversed theirmigration after the collapse of the leading front (Fig. 8D–F), aprocess reminiscent of the collapse and reversal of migrationtriggered by Slit-2 (Fig. 2C). These results suggest that Slit-2triggers a coordinated collapse of the leading front along with agradient of RhoA inhibition across the cell to reverse OECmigration.

DiscussionIt has been shown that the OB secretes some soluble factors thatmight attract OEC migration from the OE towards the OB (Liu etal., 1995). Another potential guidance mechanism for OECmigration is that the OE might secrete some repellants to promoteOEC migration toward the OB during early development. Recentstudies have shown that glial cell migration can also be directed bysome axon guidance molecules (Tsai and Miller, 2002). BecauseOECs share a common origin (olfactory placode) with olfactorysensory neurons and migrate out from the OE to the OB together

Fig. 5. Activation of cofilin through Ca2+–calcineurin signaling is required for Slit-2-induced collapse and reversal of migration of OECs. (A)Western blotanalysis for Slit-2-induced cofilin dephosphorylation and quantitative analysis of the ratio of p-cofilin to cofilin, normalized to the baseline level (0 minutes).(B)Immunocytochemical analysis of the distribution of p-cofilin (green) and F-actin (red) after Slit-2 treatment. Images of selected regions (1) and (2) are shown as(1�) and (2�) at higher magnification. (C)Immunocytochemical analysis of the distribution of cofilin (green) and p-cofilin (red) after Slit-2 treatment. (D)BAPTA-AM or CsA blocked the cofilin dephosphorylation triggered by Slit-2. (E,F)Average migration ratios (E) and the percentages of collapsed or reversed cells (F) inresponse to Slit-2 in total observed OECs transfected with GFP–cofilin-WT or GFP–cofilin-S3A. Data are mean + s.e.m.; *P<0.05, **P<0.01, Student’s t-test.Scale bars: 20m.

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with olfactory sensory axons during the same developing period(Valverde et al., 1992), it is likely that guidance cues for olfactorysensory axons, like Slit (Cho et al., 2007; Nguyen-Ba-Charvet etal., 2008), might also guide the migration and distribution of OECs.

In the present study, we have shown that Robo proteins areexpressed in cultured OECs and exhibit enriched distribution at theleading edge. A Slit-2 gradient indeed strongly repelled themigration of these cultured OECs. To our knowledge, this is thefirst guidance factor discovered to repel OEC migration. BecauseSlit-2 is highly expressed in the apical cells of OE, it is likely thatit might help Robo-expressing OECs and olfactory axons migrateout of the OE through chemorepulsion during early development.Slits expressing in the OB might also regulate the stop andscattering of OECs that have arrived at the surface of the OB.OECs have been reported to pioneer the olfactory sensory nervesand provide a conductive substrate for the growth of olfactorysensory axons during development (Tennent and Chuah, 1996;Tisay and Key, 1999). An intriguing possibility is that the guidanceof OECs by Slits might contribute to the guidance of axons becauseof the close interaction between neurons and glia. Extensive futurestudies using Slit and Robo mutant mice are needed to addressthese possibilities. Because multiple Slit and Robo family membersare expressed in the developing olfactory system, double or eventriple mutants of Slits and Robos might be required to clarifywhether and how Slits guide the migration and distribution ofOECs in developing olfactory system. Conditional knockoutmutants with OEC-specific deletion of Robos are required toaddress whether Slit proteins could directly guide the migration ofOECs in vivo and whether this guidance of OEC migration by Slitcontributes to the pathfinding of olfactory sensory axons.

The intracellular signal transduction for Slit-2-induced collapseof the leading front of migrating cells has been largely unclear.Cultured OECs provide a good experimental system for addressing


this issue because of their large cell size, active migration and highresponsiveness to Slit-2. In the present work, we found that cofilinis a major downstream target of Slit-2 in triggering the collapse ofthe leading front of migrating OECs. ADF/cofilin family membersare key regulators of F-actin dynamics and mediate the rapidturnover of F-actin by severing F-actin near the pointed ends(Bamburg, 1999; Moon and Drubin, 1995; Theriot, 1997). Recentstudies have shown that cofilin has complicated regulation of F-actin dynamics and diverse effects on F-actin polymerization (VanTroys et al., 2008). Whether cofilin promotes F-actin assembly ordisassembly depends upon the concentration of cofilin relative toactin and the relative concentrations of other actin-binding proteins.A low cofilin to actin ratio promotes actin disassembly, whereas ahigh ratio promotes actin assembly in vitro (Andrianantoandro andPollard, 2006; Chan et al., 2009; Van Troys et al., 2008). Duringchemotaxis, spatial and temporal regulation of cofilin activity isrequired for cell directional migration (Mouneimne et al., 2006;Nishita et al., 2005). A previous study has shown that Slit-2 couldinduce the local synthesis of cofilin in Xenopus retinal growthcones (Piper et al., 2006). In our studies, we observed thatstimulation of OECs with Slit-2 significantly decreased the p-cofilin level, without affecting the total cofilin level. These resultssuggest that Slit-2-induced collapse of OECs is mediated bychanges of the ratio of p-coflin to non-p-cofilin, but not by anincrease of total cofilin proteins in these migratory glial cells. Wealso found that cofilin proteins were mainly the phosphorylatedinactive form at the peripheral ruffle of normal migratory OECs.Upon Slit-2 stimulation, cofilin was quickly activated at cellperipheral ruffles, a process that might be responsible for thedisruption of F-actin in this region and the subsequent collapse ofleading front. In support of this notion, F-actin at cell peripheralruffles was lost upon Slit-2 stimulation (Fig. 4) and a gradient ofCD or LA, which promotes F-actin de-polymerization, fully

Fig. 6. RhoA activity is required for thereversal of migration triggered by Slit-2.(A,B)Images of migrating OECs beforeand after exposure to a Slit-2 gradientwithout (A) or with (B) toxin-B (10 ng/ml)incubation. White arrowheads and arrowsindicate the direction of micropipette andthe leading front of OECs, respectively.Scale bars: 20m. (C,D)Summary ofaverage migration ratios (C) and thepercentages of collapsed or reversed cells intotal observed cells (D) in response to Slit-2gradient under various conditions (see textfor details). Data are mean + s.e.m.;**P<0.01, Student’s t-test.

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mimicked the Slit-2-induced collapse of the leading front (Fig. 8).Furthermore, overexpression of the constitutive active cofilin-S3Amutant could block the Slit-2-triggered collapse of OECs. Theglobal presence of cofilin-S3A might set a new balance of F-actinturnover at a very high speed, override any local regulation ofcofilin activity triggered by Slit-2 gradient, and thus abolish theSlit-2-induced collapse of the leading front of OECs. It is likelythat similar signaling mechanisms might happen during Slit-triggered collapse and repulsion of neuronal growth cones.Interestingly, other repulsive guidance molecules, such asSemaphorin-3A, Semaphorin-3F, bone morphogenic protein (BMP)and myelin-associated inhibitors including Nogo-66, have beenshown to trigger the collapse and repulsion through cofilinactivation (Aizawa et al., 2001; Hsieh et al., 2006; Shimizu et al.,2008; Wen et al., 2007). Thus, activation of cofilin and the


consequent fast severing of F-actin at the leading front might be ageneral molecular mechanism for the collapse of the leading frontin response to different repulsive factors.

The F-actin-severing activity of cofilin can be regulated byreversible phosphorylation at the Ser3 residue, with the loss ofactivity upon phosphorylation. It has been shown that LIM kinases(LIMK) specifically phosphorylate cofilin at Ser3, whereasphosphatases Slingshot and calcineurin are capable ofdephosphorylating it (Arber et al., 1998; Huang et al., 2006). Previousstudies have shown that regulation of cofilin activity by LIMK andSlingshot is involved in regulating growth cone motility andmorphology in response to extracellular cues, including Semaphorin-3A, BMP, brain-derived neurotrophic factor, and myelin-associatedinhibitors (Aizawa et al., 2001; Gehler et al., 2004; Hsieh et al.,2006; Meberg and Bamburg, 2000; Wen et al., 2007). It is alsoshown that the Ca2+–calcineurin signaling pathway candephosphorylate cofilin and mediate BMP-triggered repulsion of

Fig. 7. RhoA inhibition by Slit-2 is required for reversal of migration.(A–D) Representative western blots (A,C) and quantitative results (B,D)showing RhoA activity in response to treatment with serum (10%, 30minutes), LPA (10M, 30 minutes) and Slit-2 (4g/ml, 0–20 minutes) in apull-down assay. For quantitative analysis, the ratio of active RhoA to totalRhoA was normalized to the baseline level (untreated control or 0 minutes).Data are mean + s.e.m.; **P<0.01, Student’s t-test. (E)Representative FRETimages (in pseudocolors) showing the inhibition and redistribution of activeRhoA in response to a Slit-2 gradient. Scale bar: 20m. (F)Changes in theFRET signal at leading process of cultured OECs before and after theapplication of PBS or Slit-2 gradients.

Fig. 8. Collapse of leading front with simultaneous inhibition of RhoAtriggers reversal of migration. (A–D) Images of migrating OECs before andafter the frontal application of a gradient of DMSO (A), LA (B), Y-27632 (C),or Y-27632 and LA mixture (D). Scale bars: 20m. (E,F)Percentages ofcollapsed or reversed cells in total observed cells (E) and migration rates of thesoma (F) after the frontal application of various drug gradients. Data are mean+ s.e.m.; **P<0.01, Student’s t-test.

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growth cones. Interestingly, calcineurin also promotes the activity ofanother cofilin-dephosphorylation enzyme Slingshot, which alsodirectly de-phosphorylates and suppresses LIMK activity(Soosairajah et al., 2005; Wang et al., 2005; Wen et al., 2007). Thus,inhibition of LIMK activity is one potential mechanism for thereduction in cofilin phosphorylation upon activation of the Ca2+–calcineurin signaling pathway. Consistent with these previous reports,the present study showed that the Ca2+–calcineurin signaling pathwayactivated cofilin and mediated the collapsing effect of Slit-2 to theleading front of OECs. How Ca2+–calcineurin signaling pathway isactivated and whether Slingshot and LIMK are involved in thecofilin activation by Slit remains to be further clarified.

RhoGTPases including Cdc42, Rac1 and RhoA can also regulatecofilin through regulation of LIMK activity by phosphorylation ofits kinase activation loop, with Rac/Cdc42 acting through p21-activated kinases 1 and 4, Cdc42 through mytotonic-dystrophy-related cdc42-binding kinase, and RhoA though ROCK (Dan et al.,2001; Edwards et al., 1999; Ohashi et al., 2000; Sumi et al., 2001).However, our study showed that none of the dominant negativemutants of Cdc42, Rac1 and RhoA could block Slit-2-triggeredcollapse of the leading front in OECs. Thus, RhoGTPases do notplay a significant role in cofilin regulation and the leading frontcollapse in response to Slit-2 in these OECs. Instead, the Ca2+–calcineurin signaling pathway should be the major downstreamevent that leads to the cofilin activation and the consequent collapseof the leading front in response to Slit-2 stimulation.

In the present study, we found that collapse of the leading frontis necessary, but not sufficient, to trigger the reversal of somatranslocation, suggesting that other signals independent of thecollapse of the leading front should be activated by Slit-2 to reversethe soma translocation. As discussed below, this signal might be agradient of RhoA inhibition. RhoGTPases act spatially andtemporally to control cell migration by regulating cytoskeletalreorganization. Rac1 is known to promote the extension oflamellipodia, and the dominant negative mutant of Rac1 is oftenused to abolish the formation of lamellipodia. Cdc42 is known topromote the extension of filopodia. By contrast, RhoA is commonlyconsidered to regulate cell contractility at the cell body andcontributes to the retraction of the trailing end of migrating cells(Etienne-Manneville and Hall, 2002). However, increasing evidencehas shown that active RhoA distributes in the leading front. In freemigrating neurons, fibroblasts and MDCK cells, the active RhoAdisplays a polarized distribution with highest activity in the frontpart of the cell rather than in the rear (Guan et al., 2007; Kurokawaand Matsuda, 2005; Pertz et al., 2006) and might be essential for


the re-orientation and stabilization of the direction of cell migration(Palazzo et al., 2001; Wen et al., 2004). Some studies report thatSlit activates the activity of RhoA and Rac1, but inactivates Cdc42(Fan et al., 2003; Wong et al., 2001; Yang and Bashaw, 2006).However, other studies show that Slit inhibits the activity of RhoA,Rac1 and Cdc42 (Guan et al., 2007; Liu et al., 2006; Prasad et al.,2007; Tole et al., 2009; Wong et al., 2001).

RhoGTPases are known to be regulated by a large group ofGTPase activating proteins (GAPs), guanine nucleotide exchangefactors (GEFs) and guanine nucleotide dissociation inhibitors (GDIs)that selectively target different GTPases (Etienne-Manneville andHall, 2002). It is likely that different type of cells express differentcombination of GAPs, GEFs and GDIs, resulting in the differentialeffects on RhoGTPase activity and cell motility in response to Slit.In the present study, we found that RhoA was inhibited by Slit-2 incultured OECs. Because inhibition of RhoA activity was specificallyrequired for Slit-2-induced reversal of migration, but not for thecollapse of leading front, the collapse of the leading front and thereversal of soma translocation are probably two related events thatinvolve distinct intracellular signals. We found that free migratingOECs exhibited a front-high and back-low polarity of active RhoA.In response to a frontal gradient of Slit-2, this polarity of activeRhoA was gradually reversed. Moreover, a gradient of inhibition ofRhoA signaling across the cell by a gradient of Y-27632 suppressedthe soma motility, but was not sufficient to trigger the reversal ofsoma translocation. Interestingly, a gradient of Y-27632 togetherwith LA in front of migrating OECs fully mimicked the collapse andreversal of migration of OECs trigged by Slit-2. These results suggestthat a gradient of RhoA inhibition across the cell in response to aSlit-2 gradient might reverse the intrinsic polarity of the cell, but thereversal of soma migration depends on the coordination of a reversalof the cell polarity and a simultaneous collapse of the leadingprocess. These two coordinated processes are likely to allowintracellular proteins that are anchored at the original leading frontto be released and redistributed to the original tail (Fig. 9). Thiscould be a general mechanism for the repulsive guidance of themigration of different cell types, because frontal application of Y-27632 together with LA also reliably triggered the collapse andreversal of migration of another cell type, cultured Schwann cells(supplementary material Fig. S5).

Materials and MethodsPrimary culture and OEC purificationPrimary OEC cultures were prepared from the OB of adult male Sprague-Dawleyrats and purified by differential cell adhesiveness as described previously (Huang etal., 2008). Briefly, the olfactory nerve layer was peeled away from the rest of OB,

Fig. 9. Model of Slit-2-induced collapse and reversal of migrationof OECs. (A)Spontaneously migrating OEC exhibits a front–reargradient in the distribution of RhoA. (B–D) Frontal exposure of aSlit-2 gradient triggers an elevation of [Ca2+]i at the leading front andsoma, leading to the activation of cofilin, whose activity isresponsible for the disruption of F-actin and the collapse of leadingfront. Reversal of the polarity of OECs requires both the gradient ofRhoA inhibition across the cell and the collapse of the leading front.Colors indicate F-actin (red), RhoA (yellow) and Slit-2 gradient(black).

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dissociated with 0.25% trypsin (Sigma, St Louis, MO) at 37°C for 15 minutes.Tissues were triturated using a Pasteur pipette and plated on an uncoated 25 cm2

culture flask twice; each sample was then incubated for 36 hours at 37°C in 5% CO2.Non-adhesive cell suspension was collected, seeded onto 35 mm dishes (Corning,NY) coated with poly-L-lysine (0.1mg/ml, Sigma), and incubated with DMEM/F-12 (1:1, vol/vol; DF12, Gibco, Grand Island, NY) containing 10% heat-inactivatedfetal bovine serum (FBS; Hyclone, Logan, UT), 2 M forskolin (Sigma) and 10ng/ml bFGF (Sigma). The overall purity of OECs was around 95%. Primary culturesof OECs from OE of P0 Sprague-Dawley rats were prepared as described previously(Au and Roskams, 2003).

RT–PCR and western blottingFor RT-PCR, total RNA was extracted from cultures of purified OECs with Trizolreagent (Invitrogen, Gaithersburg, MD), converted to cDNA using the Revert AidFirst Strand cDNA Synthesis Kit (MBI Fermentas, Hanover, MD), and one twentiethof the product was used in 20 l PCR reactions. Primers for Robos were designedand the specificity of primers was measured by Primer Premier 5.0 software. Robo1,forward, 5�-TGCAGCAGCCGCCGAGTATG-3�, reverse, 5�-ATTATAGG -CCGTGCCTCCAG-3�; Robo2, forward, 5�-CAACACTCTGGTAACGTGGA-3�,reverse, 5�-ATCTCAGCCTTCCGAGGA-3�; and Robo3, forward, 5�-CACCAT -ACGTGGAGGAAAGC-3�, reverse, 5�-GGGCTGGGGATCTCCCTGCA-3�.

To detect changes in phosphorylated cofilin, cultures of purified OECs wereserum-starved for 8–10 hours before Slit-2 (4 ng/ml) treatments, then were lysed in0.2 ml lysis buffer containing protease inhibitor mixture set I (Calbiochem, La Jolla,CA). Proteins were separated by 12% SDS-PAGE gel electrophoresis and transferredonto the polyvinylidene difluoride (PVDF) membrane. Blotted membranes wereblocked for 3 hours at room temperature in 5% milk and incubated with anti-cofilin(1:1000; Cell Signaling Technology, Beverly, MA) or anti-p-cofilin (Ser3, 1:1000;Cell Signaling) overnight at 4°C, rinsed and incubated for 1 hour at room temperaturewith a horseradish-peroxidase-conjugated antibody against rabbit IgG (1:10,000;BioRad, Hercules, CA). Chemiluminescent detection was performed with the ECLkit (Pierce, Rockford, IL). -actin as a loading control was detected alongside theexperimental samples (1:3000; Chemicon, Temecula, CA). For detection of Robo1and Slit-2 by western blotting, 8% SDS-PAGE gel was used, with 1:200 anti-Robo1antibody (R&D, Cat. AF1749) and 1:500 anti-Slit-2 antibody (Chemicon, Cat.AB5701), respectively. For detection of the expression of mutant RhoGTPases incultured OECs after transfection, 12% SDS-PAGE gel and 1:1000 anti-GFP antibody(Molecular Probes) were used.

Pull-down assay of active RhoAFor analyzing RhoA activity in cell lysates, an activated RhoA pull-down kit wasused following protocols provided by the manufacturer (Cytoskeleton, Denver, CO).Briefly, OEC cultures were starved overnight, then stimulated by Slit-2 before beinglysed in 250 l of the supplied lysis buffer containing protease inhibitor cocktail.About 20 l of each lysate was used for protein quantification and western blottinganalysis of total RhoA. For the rest of lysates, a volume of equal protein amountsfrom each sample were incubated with Rhotekin-RBD affinity beads for 1 hour at4°C, followed by two washes in the wash buffer. Bound proteins were collected andexamined by 12% SDS-PAGE for western blotting analysis.

Single-cell migration assaysSlit-2 was purified as described previously (Guan et al., 2007) from conditionedmedium collected from a cultured HEK293 cell line stably expressing human Slit-2–myc. Single OEC migration assay and quantitative analysis were carried out asdescribed previously (Huang et al., 2008). Briefly, purified OECs were replated ontocoverslips coated with laminin (10 g/ml) at a low density (1000 cells per coverslip),and were used for experiments in serum-free Leibovitz’s L-15 medium (Gibco) 12–24 hours after plating. Experiments were carried out on a heated stage (37°C) undera phase contrast microscope (CK40, Olympus Optial, Tokyo, Japan). The micropipettetip (1 m opening) was placed at about 100 m away from the cell center. A standardpressure pulse of 3 psi was applied (2 Hz, 20 milliseconds duration) (Lohof et al.,1992). Images were recorded in a time-lapse mode (1 picture every 5 minutes) witha CCD camera (JVC TK-1381; Victor Company, Yokohama, Japan) attached to themicroscope, using Scion Image Software (Frederock, MD). For pharmacologicaltreatments, cells were pre-incubated with thapsigargin (1 M; Alamone Labs), 2-APB (100 M; Calbiochem), Y-27632 (20 M; Calbiochem), or LY-294002 (20 M;Calbiochem) for at least 30 minutes, or with toxin-B (10 ng/ml; Calbiochem)overnight. Drugs existed in the medium throughout the migration assay.

Plasmids and cell transfectionGFP-tagged cofilin-WT and cofilin-S3A were constructed by subcloning the full-length cofilin or cofilin-S3A into pEGFP vector (Clontech) between BamHI andHinDIII sites. Robo2–GFP, DN-RhoA–GFP, DN-Rac-1–GFP and DN-Cdc-42–GFPplasmids were described previously (Guan et al., 2007). The Robo1-specific siRNAsequences are: sense, 5�-GCAACAGGATGAATTAGAA-3� and antisense 5�-CGTTGTCCTACTTAATCTT-3�. For OEC transfection, we used rat AstrocyteNucleofector Kit (Amaxa, Cologne, Germany) according to the manufacturer’sinstructions (program T-20).

ImmunostainingFor brain tissue staining, embryonic brains were directly removed and fixed with 4%paraformaldehyde (PFA) at E16. Sagittal brain sections of 20 m were cut on afreezing microtome and immediately processed for immunostaining by 1 hourblocking in 5% BSA plus 0.3% Triton X-100 at room temperature, overnightincubation with primary antibodies at 4°C, and for 1 hour at room temperatureincubation with appropriate secondary antibodies (1:1000; Molecular Probes, Eugene,OR). The primary antibodies used were anti-p75 (1:500; Promega, Madison, WI),anti-Slit-2 (1:200; Chemicon) and anti-Robo1 (1:200; R&D) (Farmer et al., 2008;Marlow et al., 2008; Nguyen-Ba-Charvet et al., 2008). Sections were counterstainedfor Toto-3 (1:1000; Molecular Probes) to visualize the nucleus. For OEC staining,cultured OECs were fixed with fresh 4% PFA for 20 minutes. The primary antibodieswere anti-p-cofilin (1:200; Cell Signaling), p75 (1:500; Promega), cofilin (1:200;Santa Cruz Biotechnology), Slit-2 (1:200; Chemicon) or acetylated tubulin (1:1000;Sigma). For visualization of F-actin, cells were incubated with rhodamine-conjugatedphalloidin (1:60; Molecular Probes) at room temperature for 1 hour. Images wereacquired on an Olympus FV-1000 confocal system using a multitrack configurationand processed using Adobe Photoshop CS 8.0.

Calcium imagingCalcium imaging was performed as described previously (Guan et al., 2007). Briefly,OECs were rinsed three times before being loaded with Fluo-4 AM and Fura-RedAM (2 M, Molecular Probes, Eugene, OR), with 0.1% dimethyl sulphoxide(DMSO) in the extracellular medium (ECM), for 30 minutes at 37°C, and thenincubated for another 30 minutes after being rinsed three times with ECM. Imagingwas performed at room temperature on a Zeiss LSM-510 confocal microscope witha 40� 1.30 NA oil objective (Zeiss Plan-Neofluar). Cells were excited with a laserat 488 nm, and the intensity of the emission between 505 and 550 nm was measuredas the Fluo-4 signal; emission >635 nm was simultaneously detected as the Fura-Red signal. Images were acquired at 5-second intervals. The Ca2+ signals weremeasured as the change in the ratio of Fluo-4 to Fura-Red, relative to the baselinebefore applying Slit-2 (DR/R0), in selected regions using Zeiss LSM-510 software.The leading process and soma in OECs were defined as described in a previous work(Huang et al., 2008). To quantify the Ca2+ signal at the leading process, thefluorescence signal in the whole leading process was measured (Fig. 3A). In someexperiments, cells were incubated in ECM containing pharmacological agents for atleast 30 minutes before measurement.

FRET-based imaging of active RhoA with three-channel microscopyThe FRET probe pRaichu-RhoA (YFP–RBD-RhoA–CFP) for monitoring thesubcellular RhoA activity was kindly provided by Michiyuki Matsuda (OsakaUniversity, Osaka, Japan) (Yoshizaki et al., 2003). Cells transfected with the FRETprobe were imaged on a Nikon Ti microscope with a 40� oil lens (1.30 NA) usingthe Perfect Focus System and were illuminated by a polychrome IV monochromator(TILL Photonics). Filter sets for FRET imaging were CFP (excitation 436 nm;emission, 480/40 nm HQ, DM 455 nm), FRET (excitation 436 nm; emission, 535/30nm HQ, DM 515 nm) and YFP (excitation 510 nm; emission, 535/30 nm HQ, DM515 nm). Images of the three channels were recorded simultaneously by using theCascade 512B CCD (Roper Scientific). Background images were subtracted fromthe raw images before carrying out FRET calculation. Corrected FRET (FRETC) wascalculated on a pixel-by-pixel basis for the image using the following equation:FRETCFRET–a�YFP–b�CFP, where FRET, CFP and YFP corresponded tobackground-subtracted images, acquired through the FRET, CFP and YFP channels,respectively. a and b were the fraction of bleed-through of YFP and CFP fluorescencethrough the FRET channel, respectively, and the two values were determined byusing cells transfected with YFP or CFP alone. We used the following equation:EFRETC /(CFP+FRETC)�100% to quantify the FRET signal by using MetaFluoand NIH ImageJ software (PixFRET Plug-in) (Feige et al., 2005). To quantify theSlit-2 effect, the change in FRET signals at the leading process were measured usingImageJ software and normalized to the baseline level, presented as DE/E0 (E0 is theaverage FRET signal before Slit-2 application).

Statistical analysisAll data presented represent results from at least three independent experiments.Statistical analysis was performed using Student’s t-test with two tails, theKolmogorov–Smirnov test or ANOVA with pair-wise comparisons.

We thank C.-b. Guan and Q. Hu for technical support in imagingstudies; Yi Rao (Washington University School of Medicine, St Louis,MI) for providing the Slit-2-myc cell line; Pico Caroni (FreidrichMiescher Institute, Basel) for cofilin constructs; and Michiyuki Matsuda(Osaka University, Osaka, Japan) for pRaichu-RhoA probe. This studywas supported by National Key Basic Research Program(2006CB500702, 2006CB806600, 2007CB947100), Ministry ofScience and Technology of China (2009ZX09311-001) and NationalNatural Science Foundation (30770657, 30625023).

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Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/2/186/DC1

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cells by triggering ca2+-dependent cofilin activation and ... · the migration of oecs is guided...

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