(a) A portion of a wild type terminal cell is shown, with the cell shape outlined by dots (Cell, light green) – cell outline layer was made by tracing actin::RFP staining with the gain increased sufficiently to visualize basolateral actin. The terminal cell is stained with α-Wkdpep sera, revealing the lumenal membrane (Lumen, magenta) of seamless tubes. (a′) Staining (Crumbs, white) against a functional Crumbs::GFP fusion protein knocked into the endogenous crumbs locus demonstrates that the lumenal membrane of terminal cell seamless tubes is apical. White boxes in (a, a′) labeled b and b′ marquee the terminal cell soma and a branch tip, respectively, and are shown enlarged in (b, b′). Merged images of Crumbs (white) and lumenal membrane (Lumen, magenta) co-staining are shown. Subcellular localization of actin relative to the seamless tube is examined in (c – c″). In (c), a portion of a terminal cell (Cell, light green dots) containing a branched seamless tube (Lumen, magenta) is shown. In (c′), the subcellular distribution of actin (Actin, green) is shown. In (c″), a merged image is shown. The white boxes labeled (d, d′, and d″) highlight distinct domains of actin localization that are shown enlarged in (d-d″): (d) filopodial actin, (d′) basolateral actin, and (d″) apical actin. Scale bars for a, a′ in a′; for c, c′,c″ in c″ = 10 μm and in b′ and d″ for b, b′ and for d, d′, d″, respectively = 2 μm.

(a–d) Positively marked (GFP, white) mutant terminal cells were identified in mosaic animals. Cells mutant for dynein motor complex components made branched cellular extensions, but had little or no air-filled seamless tubes (arrowheads indicate the position beyond which gas-filling of tubes is not detected, a′–e′). To determine if acetylated microtubules (green) were present within terminal cells lacking air-filled tubes, wild type control (f, f′) and dlic (g, g′) mosaic animals were filleted, fixed and stained. Homozygous terminal cell clones (f, g) were marked with GFP (white). Apical membrane (Apical – α-Wkdpep – magenta) staining reveals seamless tube remnants near the terminal cell nucleus (g′) of mutant terminal cells, but not near branch tips (compare f′,g′). In (h,i), high magnification images of seamless tubes from wild type and dlic mutant terminal cells (f′, g′; marqueed area), respectively. In wild type (h), continuous seamless tube was present along the acetylated microtubule bundle. In dlic mutant cells, apical membrane was absent except for small bits of discontinuous tube that could be detected adjacent to acetylated microtubules (i, arrowhead). We found that such discontinuous bits of tube were able to organize γ-tubulin around them, but that γ-tubulin localization was mostly lost in dynein complex mutant terminal cells, with staining present diffusely throughout the cell (j, j′ compare to Figure 2a,b). As compared to wild type animals (k, k′), seamless tubes in whacked terminal cells showed excessive growth at branch tips (i, i′), resulting in a “U-turn” phenotype, in which seamless tube extended through the cytoplasm in a series of 180 degree turns. Scale bars for a-e′ in e′; for f-g′ in g′ = 10 μm; for h and i in i, for j,j′ in j′ and for k-l′ in l′ = 2 μm.

In wild type terminal cells (a–e) Whacked::mKate2 fusion protein (Whacked, magenta), is enriched apically, especially at branch tips (arrowheads). In comparison (b′,b″,d), YFP::Rab35 (white) is more broadly distributed, but also apically enriched. Whacked and Rab35 show extensive co-localization (b′), and also overlay with acetylated microtubule tracts (Acet-Tub, green). At branch tips (marqueed area in b″), Wkd (c) and Rab35 (d) are adjacent to acetylated microtubules (e), but also decorate actin-based filopodial processes (see Figure S3). In dynein motor complex mutants (Lis-1, f,f′; dlic g,g′), Whacked is dispersed throughout the cytoplasm and often detected on basolateral membrane. Although not excluded from areas with acetylated microtubule bundles, Whacked is not enriched along them (f″, g″). In (h–k), α-Wkdpep is used to stain the apical membrane of seamless tubes; in the soma surrounding the terminal cell nucleus of wild type cells (h), single seamless tubes are found growing into each cytoplasmic extension, whereas terminal cells expressing a dominant negative Rab35 isoform (i), overexpressing (O/X) wild type Whacked (j), or mutant for dynein motor complex components such as dlic (k) have ectopic seamless tubes. Scale bars in a for a, in g′ for b–g′ = 10 μm ; in e for c–e = 1 μm; and in k for h–k = 2 μm.

Terminal cell outlines (determined as in Figure 1) are indicated (Cell, white dots). (a, b) The subcellular distribution of γ-tubulin is shown in a typical wild type terminal cell. γ-tubulin (γ-Tub, green) lines the seamless tube and is enriched at the blind-end of the tube found at the tip of the terminal cell (a). Costaining of γ-tubulin and apical membrane (magenta, a′, b′) is shown (a″, b″). (c, d) The subcellular distribution of acetylated microtubules (Acet-Tub, green) is shown. Acetylated microtubule bundles run parallel the apical membrane (magenta, c′, d′). Costaining is shown (c″, d″). We note that apical membrane fragments (white arrow) discontinuous with the seamless tube are found to line acetylated microtubule tracts extending beyond the seamless tube blind end (magenta arrowhead). (e) The relationship between acetylated microtubules (white), apical membrane (Lumen, magenta) and actin rich filopodia (Actin, green) at branch tips is shown. Acetylated microtubules extend beyond (white arrowhead) the blind-end (magenta arrowhead) of the seamless tube. Actin-based filopodial projections extend beyond both the seamless tube and the stable microtubule tract. These data (and those from Figure 1) are summarized in the schematic diagrams shown in (f). In the panel on the left, microtubules are shown oriented perpendicular to the axis of the tube while in the panel on the right, microtubules are shown oriented parallel to the long axis of the tube. Either or both microtubule arrangements may be present. Scale bars for a-d are in d″ and for e, e′ and e″ are in e″ and = 2 μm.

In (a, d-f), brightfield micrographs revealing gas-filled terminal cell seamless tubes are shown. In (b, h,i, k and l), fluorescent micrographs labeling the terminal cell cytoplasm (green) and nucleus (magenta) are superimposed on a brightfield image. In (a) and (b) wild type terminal cell branch tip (a) and soma (b) are shown. In (c), a schematic illustrates the position and distribution of seamless tubes in wild type terminal cells. In wkd (d), wkd RNAi (e), or Rab35CA (f) terminal cell tips, growth of seamless tubes appears to outpace growth of cellular extensions, resulting in a tangle of gas-filled tubes coiled in the tip cytoplasm. In (g), the distal seamless tube overgrowth seen in (d – f) is illustrated in a schematic diagram. In terminal cells over-expressing wkd (h), or Rab35DN (i), ectopic gas-filled seamless tubes (*) are observed surrounding the terminal cell nucleus. This proximal overgrowth phenotype is illustrated in the schematic in (j). In (k), a terminal cell expressing an activated FGFR (λ-breathless) produces ectopic gas-filled seamless tubes (outlined with dashed white oval) surrounding the terminal cell nucleus (magenta), much like (although more extreme than) overexpression of wkd or Rab35DN. In (l), a sibling larva expressing a whacked RNAi transgene in addition to λ-Btl shows abundant ectopic tubes (white ovals) at branch tip positions rather than around the nucleus (magenta). Scale bars in f (for a, d–f), i (for b, h, and i) = 5 microns, and l (for l, k) = 50 microns.