Figure 3.
Figure 3. Branched actin polymerization and Sing are necessary for tight apposition and contact between DLM myotubes and associated myoblasts. (A–B’) DLM preparations from WT pupae. A low magnification view (A) shows myoblasts (MBs) surrounding a multinucleated myotube (MT), and high magnification panels show myotube–myoblast interfaces before (B) and after (B’) establishment of contacts (asterisks). n, nucleus. (C–J) DLM preparations from knockdown and mutant pupae, including mef2-GAL4>UASArp2-i (C and D), mef2-GAL4>UASkette-i (E and F), wsp1/Df(3R)3450 (G and H), and mef2-GAL4>UASsing-i (I and J) pupae. Low magnification views (C, E, G, and I) reveal fusion arrest in all cases, with myoblasts congregating around a thin myotube containing few nuclei. Elongated myoblasts sending projections toward the myotube (arrows) are observed in kette knockdown (E), WASp mutant (G), and sing knockdown (I) pupae. High magnification views (D, F, H, and J) demonstrate incomplete apposition and lack of contacts between myoblast and myotube plasma membranes, similar to the WT panel (B). While kette knockdown (E), WASp mutant (H), and sing knockdown myoblasts (J) flatten their apposed surfaces, Arp2 (D) myoblasts fail to do so. (K) Bar graph showing the frequency of contact sites along apposed myotube–myoblast surfaces in different genetic backgrounds. The number of cell pairs examined and the total length of membrane surveyed for contact sites in the different genotypes were as follows. WT: 50 cell pairs, ∼200 µm; sns + hbs knockdown: 13 cell pairs, ∼20 µm; Arp2 knockdown: 18 cell pairs, ∼50 µm; WASp mutant: 21 cell pairs, ∼100 µm; kette knockdown: 14 cell pairs, ∼50 µm; and sing knockdown: 22 cell pairs, ∼100 µm. (L) Bar graph showing the distribution of myotube–myoblast intermembrane distances in different genetic backgrounds generated as in Fig. 2 H. n, number of cells analyzed in single TEM sections. WT bars are the same as in Fig. 2 H. Preparations in which the function of branched actin elements or Sing is compromised (four right bars) all display a distance distribution profile in which most (50–70%) membrane separations are of intermediate value (22–50 nm). A similar profile is characteristic of WT DLM preparations where the cells do not make contact (left bar), whereas establishment of contacts between WT cell pairs (second bar from left) is associated with a shift toward smaller distances (0–22 nm) and tight apposition. (M) Bar graph comparing the mean length of myoblast surface membrane apposed to a neighboring myotube in different genetic backgrounds. n, number of myoblast–myotube pairs analyzed. Standard deviation of the measurement range is shown. Asterisks mark bars that are distinct from the WT value in a statistically significant fashion (analysis of variance, F(6,56) = 3.79, P = 0.003). Longer appositions, matching a flattened appearance, are characteristic of both classes of WT myoblasts as well as WASp mutant (Dunnett’s test, P = 0.99), kette knockdown (P = 0.56), and sing knockdown myoblasts (P = 0.96) but are not achieved after knockdown of sns-hbs (P = 0.020), Arp2 (P = 0.011), or ELMO (P = 0.005). Bars: (A, E, G, and I) 2 µm; (B, B’, D, F, H, and J) 200 nm; (C) 5 µm.

Branched actin polymerization and Sing are necessary for tight apposition and contact between DLM myotubes and associated myoblasts. (A–B’) DLM preparations from WT pupae. A low magnification view (A) shows myoblasts (MBs) surrounding a multinucleated myotube (MT), and high magnification panels show myotube–myoblast interfaces before (B) and after (B’) establishment of contacts (asterisks). n, nucleus. (C–J) DLM preparations from knockdown and mutant pupae, including mef2-GAL4>UASArp2-i (C and D), mef2-GAL4>UASkette-i (E and F), wsp1/Df(3R)3450 (G and H), and mef2-GAL4>UASsing-i (I and J) pupae. Low magnification views (C, E, G, and I) reveal fusion arrest in all cases, with myoblasts congregating around a thin myotube containing few nuclei. Elongated myoblasts sending projections toward the myotube (arrows) are observed in kette knockdown (E), WASp mutant (G), and sing knockdown (I) pupae. High magnification views (D, F, H, and J) demonstrate incomplete apposition and lack of contacts between myoblast and myotube plasma membranes, similar to the WT panel (B). While kette knockdown (E), WASp mutant (H), and sing knockdown myoblasts (J) flatten their apposed surfaces, Arp2 (D) myoblasts fail to do so. (K) Bar graph showing the frequency of contact sites along apposed myotube–myoblast surfaces in different genetic backgrounds. The number of cell pairs examined and the total length of membrane surveyed for contact sites in the different genotypes were as follows. WT: 50 cell pairs, ∼200 µm; sns + hbs knockdown: 13 cell pairs, ∼20 µm; Arp2 knockdown: 18 cell pairs, ∼50 µm; WASp mutant: 21 cell pairs, ∼100 µm; kette knockdown: 14 cell pairs, ∼50 µm; and sing knockdown: 22 cell pairs, ∼100 µm. (L) Bar graph showing the distribution of myotube–myoblast intermembrane distances in different genetic backgrounds generated as in Fig. 2 H. n, number of cells analyzed in single TEM sections. WT bars are the same as in Fig. 2 H. Preparations in which the function of branched actin elements or Sing is compromised (four right bars) all display a distance distribution profile in which most (50–70%) membrane separations are of intermediate value (22–50 nm). A similar profile is characteristic of WT DLM preparations where the cells do not make contact (left bar), whereas establishment of contacts between WT cell pairs (second bar from left) is associated with a shift toward smaller distances (0–22 nm) and tight apposition. (M) Bar graph comparing the mean length of myoblast surface membrane apposed to a neighboring myotube in different genetic backgrounds. n, number of myoblast–myotube pairs analyzed. Standard deviation of the measurement range is shown. Asterisks mark bars that are distinct from the WT value in a statistically significant fashion (analysis of variance, F(6,56) = 3.79, P = 0.003). Longer appositions, matching a flattened appearance, are characteristic of both classes of WT myoblasts as well as WASp mutant (Dunnett’s test, P = 0.99), kette knockdown (P = 0.56), and sing knockdown myoblasts (P = 0.96) but are not achieved after knockdown of sns-hbs (P = 0.020), Arp2 (P = 0.011), or ELMO (P = 0.005). Bars: (A, E, G, and I) 2 µm; (B, B’, D, F, H, and J) 200 nm; (C) 5 µm.

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