1. Volume 13, Issue 13, Pages 1086-1095 (July 2003)
Shortstop Recruits EB1/APC1 and Promotes Microtubule Assembly at the Muscle- Tendon Junction 
Arul Subramanian, Andreas Prokop, Misato Yamamoto, Kaoru Sugimura, Tadashi Uemura, Joerg Betschinger, Juergen A. Knoblich, Talila Volk 
Current Biology 
Volume 13, Issue 13, Pages (July 2003)
DOI: /S (03)

2. Figure 1
Shot Is Detected at a Unique MT-Rich Domain that Links the Muscle-Tendon Hemiadherence Junction and the Cuticle
(A–O) Lateral transverse muscle and its specific tendon cell are shown in a flat preparation of third instar larvae labeled with (A, E, and J) Shot, (B, F, and K) Phalloidin, and (C, G, and L) Tubulin. (D) and (H) are merged images of (A)–(C) and (E)–(G), respectively (staining is as follows: Shot in green, Actin in red, and Tubulin in blue). (A)–(D) show an optical section taken at the level of the muscle-tendon junction exhibiting the Shot/MT-rich domain, while (E)–(H) are optical sections taken 0.5 μm more internally, showing the tendon cell body. The outlines of the tendon and muscle cells are indicated by a white line. An (J–M) optical cross-section marked by the white line in the (I) muscle-tendon preparation detects an intense Shot/Tubulin staining in between the muscle-tendon junction and the cuticle (marked by a white line). (N) EM analysis of a cross-section of a similar preparation shows an intense array of MTs stretched between the muscle-tendon hemiadherence junction (HAJ) and the cuticle. The area outlined by the rectangle in (N) is shown at a higher magnification below. (N) Note that the orientation of the MTs is similar to the orientation of the MFs at the muscle side, as indicated in the EM micrograph. (O) A scheme showing the orientation of the Shot/MT-rich domain, which is also marked by two arrows in (A)–(D), (I), and (M). The scale bar in (I) represents 10 μm, and the scale bar in (N) represents 0.8 μm. The arrowhead in (O) represents the plane of view in (A)–(I).
Current Biology  , DOI: ( /S (03) )

3. Figure 2 Shot Overlaps with EB1 and APC1 at the MT-Rich Domain
(A–F) Lateral transverse muscle and its specific tendon cell are shown in a flat preparation of third instar larvae labeled with (A and D) Shot (red), (B) EB1 (green), or (E) APC1 (green). (C) and (F) are the merged images of Shot/EB1 or Shot/APC1, respectively. Both EB1 and APC1 overlap Shot at the junction-associated MT-rich domain (arrows).
Current Biology  , DOI: ( /S (03) )

4. Figure 3
Basal Hemiadherens Junction-Associated Proteins Partially Overlap the Shot-Rich Domain
Lateral transverse muscle and its specific tendon cell are shown in larval flat preparations.
(A–C) (A) Shot (red), (B) PSβ-integrin (green), and (C) merged.
(D–F) (D) Shot (red), (E) Paxillin (green), and (F) merged.
(G–I) (G) Shot (red), (H) P-tyrosine (green), and (I) merged.
The Shot-rich domain (arrows) overlaps with half of the PSβ-integrin domain. Paxillin is detected in a wider domain at the muscle side and overlaps with Shot close to the muscle-tendon junction. P-tyrosine staining is restricted to the tendon side and overlaps Shot staining in a narrow stripe close to the muscle-tendon junction.
Current Biology  , DOI: ( /S (03) )

5. Figure 4
Distinct Subcellular Distribution of the Different Shot Domains in the Tendon Cell
(A–T) Tendon cells expressing (A–D, I–L) EF-GAS2-GFP (EGG), (E–H, M–P) Actin-Plakin-GFP (APG), or (Q–T) Plakin-TAP (PT) and labeled for (B, F, J, N, and R) Shot and for either (C and G) Tubulin or (K, O, and S) Phalloidin. (D), (H), (L), (P), and (T) are the respective merged images (the labeling is as follows: green represents EGG, APG and PT; blue represents Shot; red represents Tubulin in [A]–[H] and Actin in [I]–[T]). PT is labeled with rabbit IgG. The outlines of the tendon cells are marked by a white line. The EF-GAS2 domain overlapped both Tubulin and Actin staining. The Actin-Plakin domain overlapped Actin staining, while it only partially overlapped Tubulin staining. The Plakin domain did not show specific subcellular distribution. The cells in (A)–(H) are tendon cells connected to the lateral transverse muscles. The cells in (I)–(T) are tendon cells connecting the ventral oblique muscles. The muscle cells are always oriented to the right.
Current Biology  , DOI: ( /S (03) )

6. Figure 5
The Different Shot Domains Exhibit Differential Association with EB1 and MTs
(A) The EF-GAS2 domain coprecipitates with EB1. Protein extracts from Schneider cells transfected with each of the three Shot domains fused to the TAP tag (APT, PT, and EGT) were incubated with IgG bound beads. Following extensive washes, the bound beads were boiled in sample buffer and were analyzed by Western analysis with rabbit IgG (upper panels), with anti-EB1 antibody (middle panel), or with anti-Tubulin (lower panel). The left boxes represent the crude extract before precipitation. Equal levels of EB1 and Tubulin are detected in all samples. The right boxes represent the IgG-precipitated material showing no Tubulin bands and a specific EB1 band only in the EGT bound beads. The multiple bands of the Shot-TAP proteins are due to protein degradation.
(B) All domains preferentially associate with polymerized microtubules. Protein extracts from Schneider cells transfected with APT, PT, and GT were obtained, incubated in ice to depolymerize all MTs, and cleared by ultracentrifugation. The soluble fraction was allowed to polymerize in the presence of GTP and was then separated from the soluble fraction by ultracentrifugation. Western analysis with rabbit IgG detected the content of the Shot domains. EGT, APT, and to some extent PT are detected in the MT-polymerized fraction.
(C) EGT colocalizes with EB1 in transfected Schneider cells. Schneider cells transfected with Actin-Plakin-GFP (APG) (upper row) or with EF-GAS2-GFP (EGG) (lower row) fixed and labeled with anti-EB1 (red). Their corresponding merged images are shown. Note that APG does not overlap with EB1, while EGG almost completely overlaps EB1 staining.
Current Biology  , DOI: ( /S (03) )

7. Figure 6
Reduced Shot Activity in Tendon Cells Leads to Elongation of MTs, Ectopic Actin Bundles, and Altered Morphology
(A–R) Tendon cells of the lateral transverse muscles of first and second instar larvae of (A–C, J–L) wild-type or of (D–F, G–I, M–N, and P–R) mutants heterozygous for shotsf20 and expressing shot dsRNAi. (B, E, H, and Q) Tubulin staining (green) and (C, F, G, L, and O) Shot staining (red) are indicated. Actin is labeled with Phalloidin (green in [K] and [N]; red in [R]). Merged images are: A, (B + C); D, (E + F); I, (G + H); J, (K + L); M, (N + O); and P, (Q + R). The outlines of the tendon cells are marked by a white line. The muscles are at the right side. The scale bar indicates 10 μm. Note the change in the morphology of the shot mutant tendon cells (compare [D] to [A] and [M] to [J]), the split between the two Shot domains (compare [F] to [C] and [O] to [L]), the elongation of the MTs (compare [E] to [B]), and the ectopic Actin staining (compare [N] and [R] to [K]).
Current Biology  , DOI: ( /S (03) )

8. Figure 7
Reduced Shot Activity Leads to the Dissociation of EB1 and APC1 from the Muscle-Tendon Junction
Lateral transverse muscles of flat preparations of first and second instar larvae heterozygous for shotsf20 and expressing shot dsRNA.
(A–C) Larvae labeled for (B) Shot (red) and (C) EB1 (green).
(D–F) Larvae labeled for (E) Shot (red) and (F) APC1 (green).
(G–I) Larvae labeled for (H) Shot (red) and (I) PSβ-integrin (green).
(A), (D), and (G) are their merged images, respectively. While PSβ-integrin at the muscle-tendon junction is detected at both the muscle and tendon cell plasma membranes and overlaps Shot at the tendon cell side, EB1 and APC1 dissociate from the muscle-tendon junction and cuticle sides.
Current Biology  , DOI: ( /S (03) )