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Guidance of subcellular tubulogenesis by actin under the control of a synaptotagmin-like protein and Moesin - PubMed

Guidance of subcellular tubulogenesis by actin under the control of a synaptotagmin-like protein and Moesin

N JayaNandanan et al. Nat Commun. 2014.

Free PMC article

Abstract

Apical membranes in many polarized epithelial cells show specialized morphological adaptations that fulfil distinct physiological functions. The air-transporting tubules of Drosophila tracheal terminal cells represent an extreme case of membrane specialization. Here we show that Bitesize (Btsz), a synaptotagmin-like protein family member, is needed for luminal membrane morphogenesis. Unlike in multicellular tubes and other epithelia, where it influences apical integrity by affecting adherens junctions, Btsz here acts at a distance from junctions. Localized at the luminal membrane through its tandem C2 domain, it recruits activated Moesin. Both proteins are needed for the integrity of the actin cytoskeleton at the luminal membrane, but not for other pools of F-actin in the cell, nor do actin-dependent processes at the outer membrane, such as filopodial activity or membrane growth depend on Btsz. Btsz and Moesin guide luminal membrane morphogenesis through organizing actin and allowing the incorporation of membrane containing the apical determinant Crumbs.

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Figures

Figure 1
Figure 1. Phenotype of terminal cells in btszK13-4 mutant larvae and subcellular localization of Btsz.

(a–d) Overview and details of control (a,c) and btszK13-4 mutant (b,d) terminal cells in third instar larvae. (a,b) The terminal cell nuclei are visualized by immunostaining with antibodies against SRF (red) and the cells are labelled with cytoplasmic GFP (green). btszK13-4 mutant terminal cells have fewer or no branches compared with control. (c,d) High magnification details of terminal cells shown in inverted colour or grey scale for better clarity. (c) In the control cell, a single lumen is present in each branch. (d) Example of a mutant cell containing many parallel, thin lumens emanating from a larger lumen. (e,f) Subcellular localization of Btsz. Single focal plane details of terminal branches expressing cytoplasmic GFP and tagged Btsz proteins, visualized by staining with antibodies against the tags. The connections of the part of the cell shown in the image to the rest of the cell is in a different focal plane. Btsz2-Glu (e) has the C2 domains and localizes to the apical membrane. Btsz2-ΔC2-HA (f) lacks the exons encoding the C2 domains and the encoded protein is distributed throughout the cytoplasm. The staining seen outside the terminal branch in e is background staining by the Anti-Glu antiserum, which is also seen in the absence of the Btsz2-Glu transgene. Images (a–d) are projections of confocal image stacks and (e,f) are single focal planes. Scale bars: (a,b) 50 μm and (c–f) 10 μm. (g) Terminal cell branching during larval development. Larvae expressing UAS-GFP btl-Gal4 were analysed at the indicated ages. The number of branches in dorsal terminal cells in tracheal segments tr2 to tr9 were counted (n>10 for each genotype and time point, error bars represent s.d.). GFP, green fluorescent protein.

Figure 2
Figure 2. Defects in terminal cell lumen formation in Btsz-depleted embryos.

Snap shots from time-lapse movies of the development of dorsal terminal cells in the embryo. (a,c) Terminal cells from control embryos expressing Dicer2 together with PLCδ-PH-GFP (a) or Ut-ABD-GFP (c). (b,d) Embryos expressing btsz-RNAi in addition to Dicer2 and the reporter constructs. The red arrowhead marks the tip of the lumen, the green arrowhead marks the leading edge of the cell. (e) Ratio of lumen-to-cell length in control, Btsz- and Talin-depleted embryos. At stage 16–17, the average tube length in control embryos was 88% (±3.5) and 88.5% (±7.7) of the length of the cell in PLCδ-PH-GFP- and Ut-ABD-GFP-expressing embryos, respectively. In Btsz-depleted cells, the average tube length was reduced to 64.4% (±16) and 64.3% (±12.4), respectively (P<0.001 in both cases by two-tailed t-test). Knockdown of Talin did not lead to a reduction in the tube-to-cell ratio (89.1%±5), (n>20 for each cross in e, error bars represent s.d.). Scale bar: (a–d) 5 μm. GFP, green fluorescent protein.

Figure 3
Figure 3. Adherens junctions and role of the MBD of Btsz in terminal cells.

(a,b) E-cadherin (E-cad) in control and in btszK13-4 mutant (c) dorsal terminal cells in third instar larvae. Junctions are seen, both in control and mutant larvae, at the site where the terminal cell is joined to the dorsal branch (red arrowheads) and between the two dorsal fusion cells (asterisk) as well as along the length of the unicellular junction in the dorsal branch cell. Compare (a) and (b) for variability in expression of E-cad in control junctions. Nuclei of the terminal cells in the low-magnification panels (a–c) are marked with green arrowheads. E-cad-stained control (d) and btszK13-4 mutant (e,f) fusion cells that connect the segmental units of the dorsal trunk in the third instar larva. 4,6-diamidino-2-phenylindole staining to visualize nuclei confirms that two cells are present (see Supplementary Movie 8). (g) Schematic of the btsz transcription unit and two of the alternative transcripts (adapted from Serrano et al.26). The wild type and the btszK13-4 genomic regions are represented as lines with the dashed region indicating the deletion in the btszK13-4 allele. Boxes below represent the 15 exons of btsz (black) and the organization of exons in btsz transcripts 1 (green) and 2 (blue). (h–j) Ability of btsz transgenes to suppress defects in btszK13-4 mutants. The indicated transgenic constructs were expressed in the tracheal system of btsz mutant animals and various aspects of the btsz mutant phenotype were scored. The colours of the bars correspond to those in the diagram above. Genotypes of crosses and number of larvae scored: red: btl-GAL4, UAS-GFP; btszK13-4/TM6B, Tb (n=17). Green: btl-GAL4, UAS-GFP; btszK13-4/TM6B, Tb X UAS-Btsz1; btszK13-4/TM6B, Tb (n=18). Blue: btl-GAL4, UAS-GFP; btszK13-4/TM6B, Tb X UAS-Btsz2; btszK13-4/TM6B, Tb (n=15). Error bars in h and i represent s.d.. Btsz2, but not Btsz1, suppresses the defects in branching (h) and the multi-lumen phenotype (i) of btszK13-4 homozygous mutants (P=5.5 × 10−7 and 5.6 × 10−5 respectively, by two-tailed t-test). (j) Ability of the same transgenes to suppress lethality of homozygous btszK13-4 adults. If lethality of btszK13-4 were completely suppressed, the expected proportion of homozygous mutant flies among the offspring of the cross would be 33% (the remaining 66% are heterozygous for the balancer chromosome; homozygous balancer animals die in early larval instars). Thus, about half of the mutants were rescued to viability by the transgene. Images (a–f) are projections of confocal image stacks. Scale bars: (a–c left) 75 μm and (a–c middle and right, d–f) 20 μm. C2, region encoding the tandem C2 domains; GFP, green fluorescent protein; MBD, region encoding the Moesin binding domain; SHD, region encoding a Synaptotagmin-like-protein homology domain.

Figure 4
Figure 4. Role of Moesin in terminal branch development.

(a–d) Distribution of activated Moesin (phosphorylated form of Moesin (P-Moe)) in control and btszK13-4 mutant larvae. (a,b) Details of terminal branches expressing cytoplasmic GFP and stained with an antibody specific for the P-Moe. (a) Control larvae and btszK13-4 mutant larvae (b). (c,d) Localization of P-Moe (red) in dorsal trunk fusion cells in control (c) and btszK13-4 (d), larvae co-stained for E-cadherin (green). The images show the two ring-shaped fusion cells that connect the tracheal segments of the dorsal trunk. The apical localization of P-Moe seen in control cells is lost in the btszK13-4 mutant cells. (e–k) Effect of modulating Moesin activity in terminal cells. (e–h) Third instar terminal cells expressing cytoplasmic GFP are shown for each genotype. moesin-RNAi or MoesinT559D was expressed in the tracheal system using btl-GAL4. Images are colour inverted. (e) Control; (f,g) moesin-RNAi; (h) MoesinT559D. (i–k) Details of branches in control cells (i) , moesin-RNAi (j) and MoesinT559D-expressing (k) cells at higher magnification. (l–n) Effect of loss of Moesin or Btsz on the actin cytoskeleton. Details of third instar terminal cells in which Ut-ABD-GFP was expressed as marker for actin together with RNAi specific for Btsz or Moesin. F-actin is enriched at the luminal membrane in control cells (l), and in discontinuous patches along the outer, basal membrane (red arrowhead). The marker is also seen in small irregular structures in the cytoplasm, and occasional large, smooth aggregates (green arrowhead). After btsz or moesin depletion, the accumulation of the marker at the luminal membrane is reduced or lost (m,n). The discontinuous patches along the outer membrane and the large aggregates are not affected (m,n). (o,p) Distribution of acetylated microtubules in terminal cells. Control (o) and Btsz-depleted (p) terminal cells in third instar larvae were stained with antibodies against acetylated tubulin. Stable acetylated microtubules extend beyond the blind end of the terminal branch lumen (red arrowhead) both in control and Btsz-RNAi cells. The RNAi constructs were co-expressed with (f,g,j,p) or without (l,m,n) Dicer2. (e–k) are of projections of confocal image stacks. l–n are single focal planes from confocal stacks. Scale bars: (e–h) 50 μm, (a,b,i–n) 10 μm and (c,d,o,p) 20 μm. GFP, green fluorescent protein.

Figure 5
Figure 5. Apical membrane markers and secretion in Btsz-depleted terminal cells.

(a,b) Terminal cells expressing PLCδ-PH-GFP (green) were stained with antibodies against Crumbs (red). Crumbs is apically enriched in control (a) but not in btsz-RNAi (b). Localization of PLCδ-PH-GFP in control (a,c), btsz-RNAi (b,d) and moesin-RNAi (e) terminal cells. Apical localization of PLCδ-PH-GFP is disrupted in btsz-RNAi (b,d) and moesin-RNAi (e) cells. Distribution of Pio (red) in control (f) and Btsz-depleted (g) cells. Localization of β-integrin (red arrowhead in h,i) to the outer membrane in control cell (h) and in Btsz-depleted cells (i). (c–e) and (h,i) are single focal planes from confocal stacks and (a,b) and (f,g) are projections of confocal image stacks. Scale bars: (a,b,f–i) 10 μm, (c–e) 5 μm.

Figure 6
Figure 6. Localization of apical and secreted proteins in terminal cells.

(a–c) Crumbs localization in control (a) and Btsz-depleted (b,c) larval terminal cells; single focal planes. (d,e) Larval terminal cells expressing cytoplasmic GFP (green) were stained with antibodies against aPKC (red). aPKC remains associated with the luminal membrane after Btsz depletion (e). (f,g) Luminal chitin deposition in control (f) and btsz-RNAi (g) dorsal terminal cells in the embryo. Embryos expressing PLCδ-PH-GFP (green) were stained with the chitin-interacting dye CBD-564. Scale bars: (a–e) 10 μm, (f,g) 5 μm.

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