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First published online June 25, 2007
doi: 10.1242/10.1242/jcs.03465

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Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator

¹ Biology Department, York University, Toronto, ON, Canada
² Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
³ Department of Biophysics, Biochemistry and Molecular Biology, Iowa State University, Ames, IA, USA

View larger version (58K): [in this window] [in a new window]   Fig. 1. (A) The titin genomic region spans from the 5' end of the predicted zormin ORF through the 3' end of the predicted sallimus/titin ORF. Both of the ORFs predicted from zormin and sallimus/titin have significant homology to vertebrate titin and to the multiple C. elegans titin-like proteins. (B) The domain structure of the predicted composite titin protein, which would include 21139 residues, contains 55 immunoglobulin repeats (Ig; blue), five fibronectin type-3 repeats (FN3; silver) and two extended domains rich in Pro, Glu, Val and Lys (PEVK-1 and PEVK-2; yellow). Polyclonal antiserum was generated to three non-overlapping fragments from the composite protein (52, KZ and 56; green). The colored line above the domain structure indicates which regions of the protein are encoded by the three Celera Genomics Predicted genes: zormin (yellow), CG32307 (purple) and sallimus/titin (red). (C) Drosophila embryos stained with antisera generated to protein fragments corresponding to the zormin coding region (anti-52) and to the N-terminal region of the sallimus/titin coding region (anti-KZ) show identical patterns of accumulation, with staining in the skeletal, pharyngeal and visceral muscle, as well as in the muscle attachment points, known as apodemes (left and middle panels). Drosophila embryos stained with antiserum raised against a more central fragment of the sallimus/titin coding region show the same pattern of accumulation except that staining is not detected to high levels in the visceral muscle (right panels).

View larger version (97K): [in this window] [in a new window]   Fig. 2. Distribution of anti-D-titin antibodies in glycerinated myofibrils from crane-fly flight muscle. (A) An example of the method we used to identify Z-lines in confocal images. The same myofiber is illustrated in a phase-contrast image, a DIC image and a confocal microscope image. The brackets show the same sarcomere in the three images and the arrowheads point to Z-lines, which delimitate the sarcomere. (B-D) In general, the Z-lines (arrows), identified from corresponding DIC and phase-contrast images, are brightly stained. There is diffuse staining in between, along the length of the sarcomere. The staining patterns of all three antibodies vary with the sarcomere length, as described in text. Bars, 5 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 3. (A) Coomassie-Blue-stained agarose gel of rat muscle (Rm), locust muscle (Lm) and crane-fly muscle (CFm). The highest molecular mass components in crane-fly and locust muscle (arrows) run about half-way between rat titin and rat nebulin, similarly to the doublets of mini-titin (Nave and Weber, 1990). The arrowhead indicates the band used for mass spectrometry analysis. (B) Western blots of locust muscle probed with three anti-D-titin antibodies. All three antibodies stain the highest molecular weight bands (arrows) seen in the Coomassie-Blue-stained gels, corresponding to mini-titin, as indicated by their position relative to rat titin and rat nebulin, which act as markers (Warren et al., 2003). Myosin runs with the Bromophenol Blue dye front and is at the bottom of the lanes. The proteins were separated on agarose gels, transferred to PVDF membrane and visualized with ECL. The same membrane was used for all three anti-D-titin antibodies (after stripping and reprobing).

View larger version (75K): [in this window] [in a new window]   Fig. 4. D-titin distribution in prophase and prometaphase insect spermatocytes. (A) Distribution of three anti-D-titin antibodies in prophase crane-fly spermatocytes. These antibodies stain the nuclear membrane (arrow), the periphery of chromosomes (arrowheads) and the body of chromosomes. No staining of the periphery of the chromosomes was observed with the anti-52 antibody. Also, the staining of the nuclear membrane with anti-52 is less intense than with the other two antibodies (arrow in the central panel) and was seen in a small percentage of cells only. (B) Distribution of anti-KZ, anti-52 and anti-56 antibodies in prophase locust spermatocytes, illustrating staining of chromosomes. (C) Distribution of anti-KZ, anti-52 and anti-56 antibodies in prometaphase crane-fly spermatocytes. Left panel illustrates a cell fixed and lysed with a different protocol than our regular one: the lysis and fixation were done simultaneously, as one step, as described in the Materials and Methods. (D) Distribution of anti-KZ, anti-52 and anti-56 antibodies in prometaphase locust spermatocytes. Arrowheads in C and D indicate autosomal bivalents and open arrowheads indicate sex chromosomes. kMT indicates kinetochore microtubules. The arrows in the DIC pictures indicate the spindle poles. The DIC pictures are of lysed cells not illustrated in the fluorescent images. Bars, 5 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 5. D-titin distribution in metaphase insect spermatocytes. Distribution of anti-KZ, anti-52 and anti-56 antibodies against D-titin (green) in (A) metaphase crane-fly spermatocytes and (B) metaphase locust spermatocytes. Distribution of tubulin (red) in (C) crane-fly spermatocytes and in (D) locust spermatocytes. (E,F) Merged channels of D-titin and tubulin (yellow-orange color indicates co-localization). Bars, 5 µm.

View larger version (77K): [in this window] [in a new window]   Fig. 6. D-titin distribution in anaphase and telophase insect spermatocytes. (A,B) Distribution of anti-KZ, anti-52 and anti-56 antibodies against D-titin (green) in anaphase crane-fly spermatocytes (A) and anaphase locust spermatocytes (B). (C,D) Distribution of tubulin (red) in crane-fly spermatocytes (C) and locust spermatocytes (D). (E,F) Merged channels of D-titin and tubulin. Insert in A, left panel, is an enlarged detail of the boxed region, showing punctate D-titin connections between the separating half-bivalents. (G,H) Distribution of anti-KZ, anti-52 and anti-56 antibodies against D-titin in telophase crane-fly spermatocytes (G) and in telophase locust spermatocytes (H). D-titin is associated with mid-body microtubules, with chromatin, with the nascent nuclear membranes (arrows in G, left and right panel) and with the flagella (arrowhead in G, third panel). Bars, 5 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 7. Two-by-two combinations of anti D-titin antibody staining in metaphase insect spermatocytes. (A) One confocal slice showing the distribution of anti-56 antibody (green) and anti-52 antibody (red) staining in metaphase locust spermatocytes. The two antibodies show a punctate distribution of D-titin, which is localized in each case along the kinetochore spindle fibers (merged image) with little co-localization (yellow-orange color). (B) Enlarged region of the boxed region marked in A, showing the regular, punctate distribution of anti-56 antibody (green) and anti-52 antibody (red) staining and the co-localization of different D-titin fragments along one kinetochore spindle fiber. (C) One confocal slice showing the distribution of anti-KZ antibody (green) and anti-52 antibody (red) staining in metaphase crane-fly spermatocytes. The two antibodies show the same pattern of punctate distribution as in A and localize along the kinetochore spindle fibers (merged image), with little co-localization. (D) Enlarged details of the boxed region in C, showing the regular, punctate distribution of anti-KZ antibody (green) and anti-52 antibody (red) staining and the co-localization (yellow-orange) along one kinetochore spindle fiber. Three consecutives slices illustrate their spatial localization in the Z-axis. (E) Distribution of anti-56 antibody (green) vs anti-KZ antibody (red) in a metaphase crane-fly spermatocyte. The two antibodies show the same pattern of punctate distribution as in A and C and localize along the kinetochore spindle fibers (merged image), with little co-localization except in the chromosomes (arrowheads) and at the poles (open arrowheads). The arrows indicate a flagellum extending from a spindle pole. (F) RNase treatment of crane-fly spermatocytes during the staining protocol resulted in increased chromosomal staining with anti-KZ antibody, thus masking the spindle staining (left panel) whereas anti-56 antibody staining remained unchanged, both in its localization and intensity (right panel) (cf. Fig. 4A, untreated). Both panels depict the same cell. Arrowhead indicates a sex chromosome and arrow indicates an autosomal bivalent. Bars, 5 µm.

View larger version (71K): [in this window] [in a new window]   Fig. 8. Distribution of anti-KZ antibody compared with myosin distribution in insect spermatocytes. (A) Merged channels of D-titin (green) and myosin (red) staining in metaphase (left panel) and anaphase (right panel) crane-fly spermatocytes (Z-series), showing their co-localization (yellow-orange) in the chromosomes (arrow), at the poles (arrowheads) and along the spindle fibers. (B) Enlarged detail of the boxed region in A showing the side-by-side distribution of D-titin (green) and myosin (red) in the interzone along the connections between separating half-bivalents (open arrowheads). Arrows indicate the two sex chromosomes. (C) Merged channels of D-titin (green) and myosin (red) staining in metaphase (left panel) and anaphase (right panel) locust spermatocytes, showing their co-localization (yellow-orange) at the poles (arrowheads) and along the spindle fibers. The arrow in the left panel indicates the sex chromosome and the arrows in the right panel indicate two autosomal half bivalents. Open arrowheads indicate side-by-side distribution of D-titin and myosin staining in the interzone along the connections between separating half-bivalents. Bars, 5 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 9. Distribution of anti-KZ antibody (green) vs actin distribution (red) in insect spermatocytes. (A-C) Merged channels of D-titin and actin staining in prometaphase (A), metaphase (B) and anaphase (C) crane-fly spermatocytes (Z-series), showing their co-localization (yellow-orange) in the chromosomes (arrows), at the poles (arrowheads) and along the spindle fibers. (D,E) Merged channels of D-titin and actin staining in prophase (A) and metaphase (B) locust spermatocytes (Z-series), showing their co-localization (yellow-orange) in the chromosomes (arrow) and along the spindle fibers. Bars, 5 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 10. Skeletor distribution in crane-fly and locust spermatocytes. (A) A prophase cell illustrating staining of chromosomes, the cell cortex, the asters, the polar flagella (arrow indicates one flagellum) and the poles (asterisk indicates one pole). (B) An early anaphase cell, illustrating staining along the kinetochore and non-kinetochore spindle fibers, defining the spindle area. Asterisks indicate the two poles. (C) A telophase/cytokinesis cell illustrating staining of the mid-body region. (D) A prometaphase cell stained for skeletor (green) and tubulin (red). Skeletor and tubulin co-localize (yellow-orange) mainly along kinetochore spindle fibers and at the poles. (E) Enlarged detail of a kinetochore spindle fiber and a bivalent at metaphase (slice 24 of the same cell depicted in Fig. 12), showing the beaded distribution of skeletor along these spindle fibers and around the chromosomes. (F) A prophase locust spermatocyte illustrating skeletor staining of the chromosomes, their periphery (arrowheads) and the cell cortex. (G) An anaphase locust spermatocyte. Skeletor is seen as punctate regions in the chromosomes, along the kinetochore spindle fibers, along the interzone connections between separating half-bivalents, and in high concentration at the poles. (H) Enlarged detail of two consecutives confocal slices (merged) of the same cell as in G, illustrating the interzone connections (arrowheads) between separating half-bivalents. (I) A telophase cell. Skeletor is present in high concentration in the mid-body, in the contractile ring (arrowhead) and in the newly formed nuclear membrane (arrows). (J) Metaphase locust spermatocyte double stained for skeletor (green) and tubulin (red). Skeletor and tubulin co-localize (yellow-orange) mainly along kinetochore spindle fibers and at the poles, similar to the pattern observed in crane-fly spermatocytes. Bars, 5 µm.

View larger version (88K): [in this window] [in a new window]   Fig. 11. Megator and chromator distribution in crane-fly and locust spermatocytes. (A,D,G,J) Prophase spermatocytes illustrating megator (A,G) and chromator (D,J) staining of chromosomes. (B,E,H,K) Metaphase spermatocytes double stained for megator and tubulin (B,H) and for chromator and tubulin (E,K), illustrating spindle fiber staining. Left panels in B,E,H,K show megator or chromator staining, middle panels show tubulin staining and right panels show the merged image of the two channels (green, megator or chromator; red, tubulin; yellow-orange, co-localization). Inserts in E are enlarged images of the boxed regions and illustrate differences between chromator and tubulin: chromator has a fine granular appearance along spindle fibers, whereas tubulin has a filamentous appearance. (C,F,I,L) Telophase/cytokinesis spermatocytes illustrating megator (C,I) and chromator (F,L) staining of midbody, newly formed nuclei and contractile ring (L). Bars, 5 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 12. A prometaphase crane-fly spermatocyte stained for D-titin with anti-KZ antibody (green) and for skeletor (red). (A) D-titin and skeletor co-localize (yellow-orange) along kinetochore and non-kinetochore spindle fibers and at the poles (asterisks). Arrow indicates one autosomal bivalent. (B) Enlarged details of the boxed region in A showing D-titin vs skeletor distribution along spindle fibers in five consecutives slices to illustrate their spatial localization in the Z-axis. Arrows indicate one sex chromosome. Open arrowheads indicate D-titin vs skeletor localization along non-kinetochore microtubules. Bar, 5 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 13. Model illustrating the changing distributions of the proteins studied. D-titin (green), skeletor (blue), actin (red), myosin (yellow) and tubulin (black) in prophase (A), prometaphase (B) and in anaphase (C). Three chromosome bivalents (A,B) or three separating pairs of half-bivalents (C) are illustrated. The changing distribution patterns are described in detail in the text.

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