Formin 3 stabilizes the cytoskeleton of Drosophila tendon cells, thus enabling them to resist muscle tensile forces

Figures

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  Fig. 1.. Projection of side view and schematic drawing of direct MASs of a third-instar larva. Shown on the left is a side view (confocal YZ projection and genotype as in Fig. 6A1) of a lateral transverse muscle (dorsal end) and its attached tendon cell. The tissues were stained with phalloidin (white), an anti-Zipper (Myosin II) antibody (green) and for F-actin-associated RFP (red) in the tendon cell. Corresponding schematic drawings are shown on the right. Arrows a and b indicate apical and basal sides, respectively. Abbreviations: ap, muscle attachment plate; cu, cuticle; ep, generic epithelial cell; MTJ-ECM, extracellular matrix of MTJ; mu, muscle; tc, tendon cell.

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  Fig. 2.. formin 3 mutant third-instar larvae show detachments and aberrations in somatic transverse muscles. Shown are third-instar larvae carrying CD8-GFP-Shaker in the background of different EMS mutations, as indicated. GFP signal is shown in white. Muscles of interest are numbered, including (for reference) some unaffected somatic muscles (muscles 3, 4, 5, 10 and 12). Anterior is left, dorsal is up. Scale bars: 50 µm. Images are representative of N=22 animals (A), N=1 (B), N=12 (C), N=2 (D), N=11 (E). (A) Control transheterozygote for form3F958 and a complementing mutation from the screen show the wild-type pattern, including lateral transverse muscles 21–24, and muscle 18. (B) In a homozygous mutant F958 larva, muscle 21 and muscle 22 (yellow shapes) are shortened ventrally and dorsally, respectively, and muscles 23, 24 and 18 seem to attach to each other to form a linear string of mini-muscles (between purple arrowheads; borders between muscles corresponding to presumed ectopic muscle-to-muscle attachments are labeled with turquoise arrowheads). (C) Homozygous mutant C265 larva showing similar defects (labeled as in B), with muscle 21 additionally having a malformed dorsal tip with…

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  Fig. 3.. formin 3 gene locus, alleles and their predicted protein products. (A) form3 gene locus. Deficiencies not complementing the wing phenotypes of alleles form3F958 and form3C265 are shown as light red bars. The complementing Df(3L)BSC374 as well as the genes msl-3 and melt (complementing when mutated) are shown as black bars. (B) Gene structure of form3. Two form3 transcript variants, A and B, consist of 11 and 12 exons, respectively (open blocks represent coding sequence). An available Minos GFP protein trap (MI08774) is inserted in the sixth intron (dark green arrow). The sequence stretch containing two HA tags in our form3HA allele is added to the 3′ end of the ORF (green box). The positions and amino acids affected in the form3 alleles F958, C265 and Em31 are indicated for isoform A. Colored lines refer to the encoded protein domains shown in C. Scale bar for exons: 1000 bp (introns not to scale). (C) Form3 protein structures of isoform A (1644 amino acids) and B (1717 amino acids). The Form3 N terminus harbors a diaphanous inhibitory domain (DID, green) and a dimerization domain (DD, blue) (Hegsted et al., 2017). Formin homology (FH) domains 1 and 2 (orange and yellow,…

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  Fig. 4.. Formin 3 is present in all larval tendon cells from first to third instar. (A) Third-instar larva (L3) of form3-GFPMI08774 in trans to balancer (Bal.), stained with anti-GFP (green), anti-Shot (red) antibodies, and with phalloidin (white). Form3–GFP and Shot are co-expressed in tendon cells of both direct (muscle 18, muscles 21–24; white arrowheads) and indirect MTJs (yellow arrowheads). (B) In a homozygous (hom.) form3-GFPMI08774 larva stained as in A, tendon cells of direct MTJs (muscle 18, muscles 21–23) are stretched and the attached muscles are shortened, unlike for indirect MTJs (yellow arrowheads). In A and B, muscles of interest are numbered. Scale bars: 50 µm. Images are representative of scans from four animals. (C–D′) Stage 17 form3-GFPMI08774 embryos (e17) stained with anti-GFP (green) and anti-MHC (myosin heavy chain; red) antibodies. Form3–GFP is expressed in neurons of the CNS but not in tendon cells, including those of lateral transverse muscles (white arrowheads). Scale bar: 50 µm (C–D′). Images are representative of scans of five embryos. (E) In a first-instar larva (L1) of form3-GFPMI08774 in trans to balancer, live imaged Form3–GFP is expressed in tendon cells…

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  Fig. 5.. Loss of formin 3 results in loss of tendon cell integrity and muscle retraction at direct MTJs. Shown are third-instar larvae stained with phalloidin (F-actin; grey) for muscles and anti-βPS integrin antibodies (green), which label tendon cells and the chordotonal organ (between white asterisks in A1). Muscles 11, 12, 21–24 and 18 are numbered for reference. Anterior is left, dorsal is up. Scale bars: 50 µm. (A,A1) In controls of the indicated genotype, lateral muscles 21–24 and muscle 18 are attached at direct MTJs (white arrowheads), similar to the more ventral intrasegmentally attached muscles (between grey arrowheads). At the segment border, muscles are indirectly attached (between yellow arrowheads). (B–C1) Mutant larvae of the indicated genotype show abnormalities in almost all lateral muscles 21–24 and 18. Corresponding tendon cells are typically more stretched than crescent-shaped (compare A1 with B3 and C1, white arrowheads). Muscles are often forked at one end (e.g. muscle 22 in B2,C). Muscles frequently are shortened and either attach to a strongly elongated tendon cell (orange arrowheads) or ectopically to an opposing muscle tip (turquoise arrowheads). Muscles 24 and…

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  Fig. 6.. Effects of form3 mutations on the distributions of known cytoskeletal tendon cell markers in third-instar larvae. Shown are high-magnification views of tendon cells attached to their respective lateral transverse muscles (numbered if identified). For the controls, single tendon cells are shown (scale bars: 10 µm), whereas for the mutants, two to three tendon cells are shown at slightly lower magnification to accommodate their dramatically increased lengths (scale bars: 25 µm). Dorsal ends of LTMs are shown except for B–B2, which show ventral muscle ends. For each panel, the left-hand image shows a top-down view of tendon cells with their attached muscles (phalloidin in blue; XY scans merged in the Z dimension), and images on the right show side views of the same respective muscles (phalloidin in white; YZ re-slices merged in the X dimension; epidermis oriented to the right). Yellow dotted lines in the top-down views indicate the positions used for generating the corresponding YZ images of the side views, with the yellow brackets indicating the extent of the X-axis projection in the side views. Likewise, yellow dotted lines and brackets in the side-views indicate the positions and…

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  Fig. 7.. Localization of Form3 within the cytoskeletal belts of tendon cells at MTJs. Shown are the dorsal ends of lateral transverse muscles with their respective tendon cells from third-instar larval filets expressing endogenous Form3 fused to HA-tag sequences at the C terminus, together with other markers (as indicated on the left for each panel). Genotypes are shown within each row. Phalloidin staining is shown in blue or white (as indicated below the images). Scale bars: 10 µm. The left-hand column shows top-down views (XY scans merged in the Z dimension) and the right-hand columns show side views of the same muscles (YZ re-slices merged in the X dimension) in three different channel combinations. Yellow dotted lines and brackets are shown as explained in the legend of Fig. 6. The side views are oriented with the cuticle to the right except for C–C2, where the cuticle is towards the top. (A–A3) Simultaneous detection of Form3–HA and Form3–GFP in a larva carrying the form3HA and form3-GFPMI08774 alleles in trans. Both tagged versions of Form3 colocalize in cables in the center of the cytoskeletal belts of the attachments, but Form3–GFP is also present in basal areas close to the…