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First published online 21 September 2005
doi: 10.1242/dev.02041

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A microtubule-binding Rho-GEF controls cell morphology during convergent extension of Xenopus laevis

Kristen M. Kwan^* and Marc W. Kirschner

Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA

View larger version (69K): [in a new window]   Fig. 1. (A) Nocodazole inhibits convergent extension, but only when explants are treated before stage 10.5. (B) Length/width ratios of explants in (A). Data are from one representative experiment of three trials, analyzing five to eight explants per sample per trial.

View larger version (143K): [in a new window]   Fig. 2. Confocal microscopic analysis of chordamesoderm explants. (A,C,E,G,I,K,M,O,Q,S) Rhodamine-actin localization. (J) eGFP-CLIP-170 fluorescence. (B,D,F,H,L,N,P,R,T) eGFP-tau fluorescence. (A,B) Control explant. (C,D) Stage 10 explant: nocodazole treatment (15 µg/ml), phase 1. Note loss of lamellipodial protrusions. (E,F) Stage-10 explant: nocodazole treatment (15 µg/ml), phase 2. Note loss of cell-cell contact. (G,H) Stage-10.5 explant: nocodazole treatment (15 µg/ml). Note maintenance of lamellipodia although microtubules are depolymerized. (I,J) Taxol treatment (20 µg/ml) of stage-10 explant. Note maintenance of lamellipodia and stabilized microtubule cytoskeleton, as evidenced by uniform eGFP-CLIP-170 binding. (K,L) dn Rho (200 pg RNA) rescues lamellipodia inhibited by nocodazole (15 µg/ml). (M,N) Y-27632 (10 µmol/l) rescues lamellipodia inhibited by nocodazole (15 µg/ml). (O,P) V12 Rac (25 pg DNA) overcomes the inhibition of lamellipodia by nocodazole (15 µg/ml). (Q,R) XLfc Y398A (2 ng RNA) rescues lamellipodia inhibited by nocodazole (15 µg/ml). (S,T) XLfc C55R (250 pg DNA) inhibits lamellipodial protrusions and cell-cell contact.

View larger version (43K): [in a new window]   Fig. 3. Velocity of microtubule growth does not change between stages 10 and 10.5. (A) eGFP-CLIP-170 marks growing ends of microtubules in explants. (B) Velocities of microtubule growth in explants, either stage 10 or 10.5. Each point represents an individual microtubule tracked for at least 20 seconds.

View larger version (142K): [in a new window]   Fig. 4. Latrunculin B inhibits convergent extension by inducing cell dissociation and preventing the explant from rounding up. (A) Morphology of explants treated with latrunculin B. Note the rectangular shape of explants, which have not healed nor undergone convergent extension, as well as the dissociation of the cells in the closeup picture. (B) Confocal microscopy of an explant treated with latrunculin B (50 nM). Note the loss of lamellipodial protrusions and cell-cell contact (rhodamine actin), although the microtubule cytoskeleton remains intact (eGFP-tau). This effect was found for latrunculin B concentrations starting at 25 nM. Lower concentrations (5 or 15 nM) resulted in a lack of actin depolymerization (as demonstrated by confocal microscopy), and, as a result, the explants healed and underwent convergent extension movements normally. Data are from one representative experiment of four trials, analyzing six to seven explants per sample in each trial.

View larger version (67K): [in a new window]   Fig. 5. Taxol and vinblastine do not inhibit convergent extension. (A) Morphology of explants treated with taxol. (B) Length/width ratios of explants in (A). (C) Tubulin staining (DM1) in vinblastine-treated explants. Note crystalline arrays of tubulin in vinblastine-treated cells. (D) Morphology of explants treated with vinblastine. (E) Length/width ratios of explants in (D). Data are from one representative experiment of three trials (for each taxol and vinblastine), analyzing six to seven explants per sample per trial.

View larger version (63K): [in a new window]   Fig. 6. Dominant-negative Rho and Rho-kinase inhibitor partially rescue the effects of nocodazole on convergent extension. (A) Morphology of explants treated with nocodazole and expressing dn Rho. (B) Length/width ratios of explants in (A). *P<0.05, as calculated by Tukey's method. (C) Morphology of explants treated with nocodazole and Rho-kinase inhibitor Y-27632. (D) Length/width ratios of explants in (C). *P<0.01, as calculated by Tukey's method. Data are from one representative experiment of five trials (dn Rho), and three trials (Y-27632), analyzing six to seven explants per sample per trial.

View larger version (52K): [in a new window]   Fig. 7. Activated Rac, but not activated Cdc42, partially overcomes the effects of nocodazole on convergent extension. (A) Morphology of explants treated with nocodazole and expressing V12 Rac. (B) Length/width ratios of explants in (A). *P<0.05, as calculated by Tukey's method. (C) Morphology of explants treated with nocodazole and expressing V12 Cdc42. (D) Length/width ratios of explants in (C). Data are from one representative experiment of three trials (V12 Rac), and two trials (V12 Cdc42), analyzing six to seven explants per sample per trial.

View larger version (57K): [in a new window]   Fig. 8. XLfc alignment and localization. (A) Alignment of XLfc with homologs GEF-H1 (human) and Lfc (mouse). Yellow marks C1/zinc-binding domain, green marks Dbl homology (DH) domain, blue marks pleckstrin homology (PH) domain, and purple marks coiled-coil (CC) region. XLfc C55, marked with a red dot, is mutated to arginine for the non-microtubule binding constitutively active mutant. XLfc Y398, also marked with a red dot, is mutated to alanine for the catalytically dead dominant negative mutant. Numbers indicate percent identity between XLfc and its homologs. (B) eGFP-XLfc localizes to microtubules in NIH3T3 cells. Cells were transfected, fixed and stained for eGFP and tubulin. Inset: magnified view of colocalization of eGFP-XLfc (green) with microtubules (red). (C) eGFP-XLfc C55R does not localize to microtubules in NIH3T3 cells. Inset: magnified view of eGFP-XLfc C55R (green) and microtubules (red). Note cytoplasmic, non-filamentous localization of eGFP-XLfc C55R.

View larger version (60K): [in a new window]   Fig. 9. XLfc nucleotide exchange activity is required for inhibition of convergent extension by nocodazole; constitutively active XLfc is sufficient to inhibit convergent extension. (A) Morphology of explants treated with nocodazole and expressing XLfc Y398A. (B) Length/width ratios of explants in (A). *P<0.05, as calculated by Tukey's method. (C) Morphology of explants expressing XLfc C55R. (D) Length/width ratios of explants in (C). (E) Whole embryos expressing XLfc C55R display convergent extension defects. Data are from one representative experiment of three trials for both XLfc Y398A and XLfc C55R, analyzing six to nine explants per sample per trial.

View larger version (45K): [in a new window]   Fig. 10. Morpholino knockdown of XLfc diminishes the ability of nocodazole to inhibit convergent extension. (A) Morphology of explants treated with nocodazole and injected with morpholino against XLfc (MoXLfc) or control morpholino (MoCon). (B) Length/width ratios of explants in (A). *P<0.05, as calculated by Tukey's method. (C) Length/width ratios of explants treated with nocodazole and injected with morpholinos and wobbled XLfc (wXLfc) rescue RNA. *P<0.01, as calculated by Tukey's method. Data are from one representative experiment of four trials (morpholino knockdown), and two trials (wXLfc rescue), analyzing six to seven explants per sample per trial.

View larger version (22K): [in a new window]   Fig. 11. Current models of XLfc action. (A) XLfc regulation by microtubules: when bound to microtubules, XLfc is inactive. Upon microtubule depolymerization, XLfc is released and becomes active for nucleotide exchange activity. Active XLfc activates Rho-family GTPases, which inhibit lamellipodial protrusions and cell-cell contact. (B) Working model for how XLfc contributes to establishment of cell polarity. Polarity cues alter local microtubule stability; local microtubule depolymerization leads to local activation of XLfc. XLfc induces local inhibition of lamellipodial protrusions.

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