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The plakin Short Stop and the RhoA GTPase are required for E-cadherin-dependent apical surface remodeling during tracheal tube fusion

Department of Cell Biology and Center for Molecular Neuroscience, C-2210 Medical Center North,Vanderbilt University Medical Center, Nashville, TN 37232-0295, USA

View larger version (104K): [in a new window]   Fig. 1. shot mutants disrupt lumen formation at anastomosis sites. (A-D) 15 µm stacks of 1 µm confocal sections of stage 16 wild-type and shot3 null mutant embryos stained with mAb 2A12, which recognizes a lumenal antigen (Samakovlis et al., 1996a). Anastomosis sites are indicated at hemisegment boundaries in the dorsal (short arrows) and lateral (concave arrows) trunks and the dorsal midline (long arrows). Anterior, leftwards; dorsal, upwards. (A) Lateral view, wild type. The lumen branches in a stereotyped pattern and is continuous at anastomosis sites. (B) Lateral view, shot3. The lumen is discontinuous at anastomosis sites. (C) Dorsal view, wild type. The lumens of dorsal branches join together at the dorsal midline. (D) Dorsal view, shot3. Dorsal branches have migrated towards the dorsal midline, but have not joined their lumens together. The dorsal trunk lumen is discontinuous at most (short arrow), but not all (arrowhead), anastomosis sites. (E-H) 1 µm confocal sections of dorsal trunk tracheal cells. (E) Stage 14, wild type. The mAb 2A12 lumenal antigen is transiently present in vesicular structures (arrow) within tracheal cells. Dorsal trunk anastomoses are complete. (F) Stage 14, shot3. mAb 2A12 labels the lumen and vesicular structures (arrow) within tracheal cells. Vesicles are still present in cells at anastomosis sites that do not form lumen. (G) Stage 15, wild type. The Fus-5 enhancer trap labels two fusion cell nuclei (brackets) with lacZ at each dorsal trunk anastomosis site. Other tracheal nuclei are also weakly labeled. (H) Stage 15, shot3. The Fus-5 enhancer trap labels two fusion cell nuclei at each anastomosis site. shot mutant embryos also express the fusion cell specific Fus-2 and Fus-3 enhancer trap markers normally (data not shown). Scale bars: in D, 25 µm for A-D; in H, 10 µm for E-H.

View larger version (89K): [in a new window]   Fig. 2. Apical cytoskeletal polarization of tracheal cells in wild-type and shot mutant embryos. (A-L) 1 µm confocal sections of dorsal trunk tracheal cells in wild-type and shot3 mutant embryos. (A-C,G,H) Stage 15, wild type. (D-F,I,J) Stage 15, shot3. (A) Phalloidin staining of dorsal trunk tracheal cells reveals F-actin (red in C) apically concentrated around a lumen that extends through anastomosis sites. Arrowheads mark the basal surface. (B) Microtubules (green in C) detected with anti-tubulin also accumulate apically, though some cortical staining is also observed. (C) Merge of A and B. (D) F-actin accumulates apically around two lumens that end at an anastomosis site (bracket). (E) Tubulin staining is diffuse in tracheal cells, including those that form lumen. (F) Merge of D and E. (G) Actin-GFP is apically concentrated. (H) C-Shot L-GFP, a microtubule-associated protein, localizes apically. (I) Actin-GFP concentrates apically, but tracheal tubes dead end at anastomosis sites. (J) The C-Shot L-GFP distribution in tracheal cells is disorganized. (K) Stage 14, wild type. F-actin (red) accumulates apically in tracheal cells (membranes outlined in green with GAP43-GFP) and surrounds a lumen that is continuous through the fusion cells (blue). The fusion cells are compact and doughnut shaped. (L) Stage 14, shot3. F-actin (red) accumulates apically in tracheal cells (outlined in green) but the lumen does not extend through the fusion cells (blue). Arrows indicate apical sides of fusion cells. Scale bars: in F, 10 µm for A-F; in J, 10 µm for G-J; in L, 10 µm for K,L.

View larger version (80K): [in a new window]   Fig. 3. Expression of Shot proteins in tracheal cells during anastomosis. 1 µm confocal sections. (A-D) Stage 13 wild-type embryo undergoing anastomosis. (A) Anti-Shot labels a track in fusion cells (short arrow). Shot proteins (red in D) are cortically concentrated in other tracheal cells (concave arrow). The lumen (long arrow) is labeled nonspecifically (see E). (B) GAP43-GFP expression (green in D) in tracheal cells labels cell membranes. (C) esg-lacZ enhancer trap expression (blue in D) labels the fusion cells (short arrows) that bridge the two dorsal trunk branches. (D) Merge of A-C. (E-H) Stage 13, shot3. (E) Anti-Shot labels only the lumen (long arrow). (F) Tracheal cell membranes. (G) Fusion cells. (H) Merge of E-G. (I-L) Stage 15, wild type. Anastomosis is complete. (I) Shot is concentrated cortically in tracheal cells. Apically localized Shot cannot be distinguished from nonspecific lumenal staining. (J) Tracheal cell membranes. (K) Fusion cells are compact and encircle the lumen. (L) Merge of I-J. Scale bar: 10 µm.

View larger version (141K): [in a new window]   Fig. 4. Shot and F-actin form a cytoskeletal track associated with the E-cadherin (DE-cad) contacts between fusion cells. (A-T) Stage 13, wild-type embryos. (A-E) Remodeling of the fusion cell E-cadherin (red) contact (arrows) and associated Shot-GFP (green) and F-actin (blue) during anastomosis. (F) E-cadherin accumulates in a spot at the interface between the fusion cells. (G,H) Elongation of the E-cadherin spot. (I) Contraction of the E-cadherin contact into a ring. (J) The three rings of E-cadherin at the anastomosis site. (K-O) Shot L(A)-GFP is associated with the E-cadherin contact during anastomosis. (P-T) A track of F-actin forms, associated with the E-cadherin contact, that grows to span the two fusion cells (S). (T) The F-actin track becomes a ring. (U-X) C-Shot L-GFP (green in X) and F-actin (blue in X) accumulate at the E-cadherin (red in X) contact (arrow).

View larger version (93K): [in a new window]   Fig. 5. Deposition of the apical surface determinant Dlt along the Shot/F-actin track. (A-T) Stage 13, wild-type embryos. (A-E) Dlt (red) accumulates in fusion cells along a track (arrows) containing Shot-GFP (green) and F-actin (blue). (F-J) Dlt is detected along the track after the track forms (compare G with H). Dlt concentration is initially lower in fusion cells along the apical surface connecting the existing lumens (I); it then accumulates in three rings (J, inset) at the anastomosis site (arrow). (K-O) Accumulation of Shot-GFP in fusion cells. (P-T) Formation of the F-actin track in fusion cells (P-R) and its association with new apical surface (S,T). Scale bar: 10 µm (5 µm for inset).

View larger version (103K): [in a new window]   Fig. 6. Remodeling of E-cadherin (DE-cad) contacts and associated F-actin during anastomosis. (A-E) Tracheal cells (membranes outlined in green) in adjacent branches abut via the fusion cells (asterisks) and form a DE-cadherin contact (red, arrows) and associated F-actin (blue). (F-J) Remodeling of the E-cadherin contact (arrows) from a spot found in the anterior fusion cell (F,G) to one spanning both (M), and then a ring (I,J). (K-O) Membrane remodeling at the contact site. Initially (K,L), the fusion cells (asterisks) are round and their apical surfaces only face the existing lumens. As the contact matures (H), membrane is detected (arrow), spanning the fusion cells and connecting with these existing apical surfaces. This membranous compartment (arrow in N) expands as the cadherin contact becomes a ring (O). (P-T) An F-actin-rich track initiates at the site of E-cadherin contact (arrow in P) and lengthens to span the fusion cells (Q,R). As the central cadherin ring forms, the track disappears and F-actin is enriched at cadherin contacts (S,T). Scale bar: 10 µm.

View larger version (111K): [in a new window]   Fig. 7. shot mutants affect E-cadherin (DE-cad) contacts between fusion cells and F-actin associated with these contacts. (A-D) Stage 13, wild type. A E-cadherin (red in D) contact (arrow in A) between two fusion cells (asterisks in B) is associated with an F-actin (blue in D) containing track (arrow in C) spanning the two cells. Membrane (green in D)-associated with the track (arrow in B) bisects the fusion cells. (E-H) Stage 13, shot3. E-cadherin contacts (arrow in E) between fusion cells (asterisks in F) fail to form, and no F-actin rich track is detected (arrow in G). Fusion cells were identified by their position (Figs 2,3). (I-L) Stage 13, shot3. Weak E-cadherin contacts (arrows in I) form between the posterior branch fusion cell (p in J) and a tracheal cell (t) in the anterior branch. No F-actin-rich track is detected (arrow in K). (M-O) Stage 13, shot3. Fusion cells occasionally form aberrantly oriented DE-cadherin contacts (arrow in M). These contacts lack detectable associated F-actin (arrow in O).

View larger version (79K): [in a new window]   Fig. 8. The F-actin- and the microtubule-binding domains of Shot proteins redundantly promote lumen formation in fusion cells. These cells occupy anastomosis sites in the dorsal (arrows) and lateral (concave arrows) trunks and at the dorsal midline (long arrows). (A-D) Rescue activity of Shot L(A)-GFP and derivatives in stage 16 shot3 mutant embryos. Shot L(A)-GFP (schematic in A) contains an N-terminal F-actin binding domain (orange), central plakin-like (yellow) and spectrin repeat domains (black), and a C-terminal domain that binds microtubules via the GAS2 motif (blue). The gray X is used to indicate domains missing in Shot L(A)-GFP derivatives (schematics in B-D). (A) Lateral view. Tracheal expression of Shot L(A)-GFP restores lumen formation in fusion cells in the dorsal and lateral trunks. (B) Dorsal view. Tracheal expression of actin-binding defective Shot L(C)-GFP restores lumen formation in fusion cells in the dorsal trunk and at the dorsal midline. No gaps in the dorsal trunk were observed in ten rescued embryos, whereas all mutant embryos have multiple gaps. (C) Tracheal expression of a Shot L(A)-GFP derivative that lacks the microtubule binding GAS2 motif restores lumen formation in dorsal trunk and midline fusion cells. No gaps in the dorsal trunk were observed in ten rescued embryos. (D) Tracheal expression of a Shot L(A)-GFP fusion that lacks both cytoskeletal interaction domains does not restore lumen formation in dorsal trunk or midline fusion cells. (E-H) 1 µm confocal sections through the middle of the dorsal trunk of stage 15 wild-type embryos expressing Shot-GFP fusions. All Shot-GFP fusions produced green fluorescence when expressed in embryos and were expressed at comparable levels, approx. five- to tenfold higher than that of endogenous Shot proteins. GFP fusions were visualized with anti-GFP to enhance signal detection. (E) Shot L(A)-GFP accumulates apically in tracheal cells. (F) Shot L(C)-GFP accumulates apically, but is less apically concentrated than Shot L(A)-GFP. (G) Shot L(A)-GFP-GAS2 accumulates apically, but is less apically concentrated than Shot L(A)-GFP. (H) Shot L(C)-GAS2-GFP molecules lack both the F-actin- and the microtubule-binding domains, and are cortically and cytoplasmically distributed. Scale bars: in D, 25 µm for A-D; in H, 25 µm for E-H.

View larger version (94K): [in a new window]   Fig. 9. Interactions between Shot and the cytoskeleton organize the tracheal cell cytoskeleton. (A,B) Stacks of five 1 µm confocal sections through dorsal trunk cells. (A) Stage 15, shot3. Tracheal expression of Shot L(C) restores normal actin-GFP distribution in fusion cells. (B) Stage 16, shot3. Tracheal expression of Shot L(A)-GAS2 restores normal actin-GFP distribution in fusion cells. (C,D) Confocal sections (1 µm) of dorsal trunk cells. (C) Stage 15, shot3. Tracheal expression of Shot L(C) restores apical accumulation of C-Shot L-GFP, a microtubule-binding protein. (D) Stage 15, shot3. Tracheal expression of Shot L(C) restores apical accumulation of C-Shot L-GFP. (E-H) Stage 15, shot3. Dorsal trunk tracheal cells express Shot L(C)-GAS2-GFP, which lacks both cytoskeletal interaction domains. (E) Apical accumulations of F-actin (phalloidin staining) surround blind-ended lumens terminating at an anastomosis site. (F) Microtubules (detected with anti-tubulin) do not accumulate apically in tracheal cells. (G) Shot L(C)-GAS2-GFP distribution. (H) E-G merge. Scale bars: in B, 25 µm for A,B; in D, 10 µm for C,D; in H, 10 µm for E-H.

View larger version (80K): [in a new window]   Fig. 10. RhoA mutations disrupt lumen formation, the organization of apical cytoskeletal structures and apical determinant localization in tracheal cells. (A-C) Stacks of eight 1 µm confocal sections; embryos stained with mAb 2A12 to reveal the lumen. (A) Stage 16 embryo that expresses RhoAV14 in all tracheal cells (RhoAV14 embryo). Fusion cells in the dorsal and lateral trunk do not form bridging lumenal connections. (B) Stage 15 embryo that expresses RhoAN19 in all tracheal cells (RhoAN19 embryo). 2A12 antigen does not accumulate in a discrete lumenal space, and is diffusely distributed extracellularly. (C) Stage 15 embryo that expresses Cdc42N17 in all tracheal cells (Cdc42N17 embryo). The lumen is continuous through fusion cells. (D-Q) Confocal sections (1 µm) through dorsal trunk cells. (D) Stage 14, RhoAV14. F-actin surrounds blind-ended lumens at anastomosis sites. (E) Stage 14, RhoAN19. F-actin accumulates in patches in all tracheal cells. The patches tend to be in the center of the dorsal trunk, but do not form a normal subapical structure. (F) Stage 14, Cdc42N17. The F-actin distribution appears normal. (G) Stage 14, RhoAV14. In fusion cells, Dlt accumulates apically around blind-ended lumens. (H) Stage 14, RhoAN19. Dlt is more broadly distributed than in wild-type tracheal cells. (I) Stage 14, Cdc42N17. Dlt accumulates apically in all tracheal cells, as in stage 14 wild-type embryos. (J) Stage 15, RhoAV14. E-Cadherin fails to form a three-ringed structure at anastomosis sites. (K) Stage 15, RhoAN19. E-cadherin distribution is similar to wild-type (Fig. 4J). (L) Stage 15, RhoAV14. In fusion cells, Shot L(A)-GFP accumulates apically, but the lumens are blind-ended. (M) Stage 15, RhoAN19. Shot L(A)-GFP is no longer apically concentrated. (N) Stage 15, RhoAV14. Microtubules fail to accumulate apically in tracheal cells. (O) Stage 15, RhoAN19. Microtubules fail to accumulate apically in tracheal cells. (P) Stage 13, RhoAV14. No E-cadherin contact (arrow) is detectable between fusion cells. (Q) Stage 13, RhoAV14. No F-actin track (arrow) is detectable in fusion cells. Scale bars: in C, 25 µm for A-C; in O, 10 µm for D-O.

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