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First published online 18 March 2009
doi: 10.1242/dev.031823

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RacGAP50C directs perinuclear -tubulin localization to organize the uniform microtubule array required for Drosophila myotube extension

Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane West, Piscataway, NJ 08854, USA and Joint Graduate Program in Cell and Developmental Biology, UMDNJ-Graduate School of Biomedical Sciences at RWJMS and Rutgers, The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, NJ 08854, USA.

View larger version (62K): [in this window] [in a new window]   Fig. 1. Drosophila RacGAP mutants have defects in somatic muscle patterning. Stage 16 embryos stained for Muscle myosin. Muscle patterning is shown at low (A,B) and high (D-I) magnification in wild-type (wt) (A,D,F,H) or RacGAP50CDH15 (B,E,G,I) embryos. (A) Muscle pattern as observed in the whole embryo. (B) No significant loss of muscle tissue or unfused myoblasts are observed in RacGAP50CDH15 mutants. (D,E) Arrows in D mark normal attachments of DO1. In RacGAP50CDH15, DO1 is in the incorrect position (E, arrows). (F,G) Arrowheads in F mark muscle 22 (LT2). Arrowheads in G mark an LT muscle of an abnormal shape. The arrow marks a rounded muscle that fails to migrate. (H,I) Arrowheads in H mark normal attachments for VO muscles. In RacGAP50CDH15 mutants, these muscles fail to fully extend (I, arrowheads). (C) Schematic of the wild-type muscle pattern of a single abdominal hemisegment, showing the names and numbers for muscles as referred to throughout this work. DA, dorsal acute; DO, dorsal oblique; LT, lateral transverse; VL, ventral lateral; VO, ventral oblique.

View larger version (63K): [in this window] [in a new window]   Fig. 2. RacGAPDH15 mutants can form stable muscle attachments. Wild-type (A,C) or RacGAPDH15 mutant (B,D) stage 16 Drosophila embryos. To the left is a schematic of the wild-type muscle pattern (magenta) and tendon cells (green). (A,B) Muscle myosin and β-galactosidase staining of embryos with the sr-lacZ reporter labels muscles (magenta) and tendon cells (green). Tendon cells are specified in RacGAPDH15 mutants (B), but muscles often select the wrong attachment sites. Arrowheads in A mark normal attachments for LT muscles, as compared with arrowheads in B that mark muscles making inappropriate attachments in mutants. Asterisks mark tendon cells to which muscles should be attached. (C) PS2 integrin staining in the wild-type labels muscle ends at their contact site with tendon cells. (D) RacGAPDH15 mutants make stable attachments as indicated by PS2 integrin staining.

View larger version (113K): [in this window] [in a new window]   Fig. 3. RacGAP functions cell-autonomously in muscles. Muscle myosin staining in stage 16 Drosophila embryos. Brackets mark LT muscles in two adjacent segments. (A) RacGAPDH15 homozyogotes have muscle attachment site (MAS) selection and morphology defects. Shown is a LT muscle of abnormal shape, extending past its attachment site (top right bracket). (B) G14-GAL4/+;UAS-tum::myc/+ embryo showing that overexpression of RacGAP in muscles does not cause an abnormal phenotype. (C) RacGAPDH15;69B-GAL4/UAS-tum::myc embryo showing that muscle defects are not rescued by RacGAP expression in tendon cells. (D) G14-GAL4, RacGAPDH15/+,RacGAPDH15;UAS-tum::myc/+ embryo showing that muscle defects are significantly rescued by expressing RacGAP specifically in muscles. LT muscles are now properly positioned.

View larger version (60K): [in this window] [in a new window]   Fig. 4. FC specification and myoblast fusion are not affected in RacGAP mutants. (A,B) In wild-type Drosophila embryos at stage 12, Eve is expressed in the founder cell (FC) for muscle 1 (DA1) and two pericardial cells (A). These cells are properly specified in RacGAPDH15 embryos (B). (C) Runt staining labels FCs for muscles 10 (DO2), 28 (VO3) and 15 (VO4) at stage 12. (D) In RacGAPDH15 embryos, Runt-positive FCs are properly specified, although some segments contain an extra Runt-positive muscle 10 nucleus (arrow), whereas FCs for muscles 28 and 15 are unaffected (arrowhead). (E,F) Anti-Vestigial labels a subset of FCs at stage 12 in the wild type (E), and this staining is normal in RacGAPDH15 mutants (F). (G,H) By stage 14, FCs have fused with fusion-competent myoblasts (FCMs), showing an increase in Vestigial-positive nuclei of wild-type dorsal muscles (G). Fusion also occurs in RacGAPDH15 mutants (H). (I,J) 5053A-GAL4/UAS-lacZ embryo stained for Muscle myosin to label all muscles (magenta) and for β-galactosidase to label nuclei (green) of muscle 12 (VL1). In the wild type (I), muscle 12 spans each hemisegment and contains 11 nuclei. In RacGAPDH15 mutants (J), muscle 12 is specified and contains the correct number of nuclei, but is mispositioned in 6% of hemisegments (n=52) (brackets). Asterisks mark intestinal cells that are also labeled with the 5053-GAL4 driver.

View larger version (83K): [in this window] [in a new window]   Fig. 5. Localization of RacGAP and Pavarotti in muscle fibers. Stage 16 Drosophila embryos. The schematic on the left shows interior muscles 6, 7, 12 and 13 (VL1-4), which are exposed by fillet preparation performed for embryos stained for RacGAP, Roundabout and -tubulin. (A,B,G) RacGAP (A,G) and Muscle myosin (B) staining shows RacGAP throughout all muscle fibers and in discrete cytoplasmic puncta, in contrast to RacGAP nuclear localization in the epidermis (G, arrowheads mark epidermal cells). (C,C') Merge of RacGAP (magenta) and Muscle myosin (green) in wild type showing discrete puncta of RacGAP (C', arrowheads) at the periphery of the nuclei (C', n). (D-F') MEF2-GAL4/UAS-GFP::pav embryo stained for RacGAP (D) and GFP (E) and merged (F,F'). Pav is nuclear in all muscle fibers (E-F' green), and co-staining of RacGAP (magenta) shows an increase in RacGAP concentration (F', arrowhead) at the nuclear periphery. (H) pavB200 mutants have a similar muscle phenotype to RacGAPDH15 (arrowheads mark muscles making inappropriate attachments). (I) RacGAP is mislocalized to the ends of muscle fibers in pavB200 mutants (arrowheads). (J) In RacGAPDH15 mutants, Pav (green) still localizes to the nuclei of the muscle fibers (magenta). (K,L) w1118 (K) and scraps8 (L) mutants have normal muscle patterning. (M,M') -Spectrin (magenta) and DAPI (green) staining shows that scraps8 mutants have cytokinesis defects in the epidermis. The boxed region in M is shown at high magnification in M', where arrowheads indicate binucleate cells.

View larger version (116K): [in this window] [in a new window]   Fig. 6. RacGAP mutants do not have general defects in the actin or microtubule cytoskeleton. (A-H) Phalloidin labeling showing LT (A-B',E-F') and ventral (C,D,G,H) muscles. During MAS selection, F-actin concentrates at the ends of myotubes in the wild type (A-C) and in RacGAPDH15 mutants (E-G). Magnification of LT ends shows multiple filopodia in both wild type (B,B') and RacGAPDH15 (F,F'). At stage 16, F-actin continues to accumulate at muscle ends (D). In RacGAPDH15 mutants, F-actin accumulation occurs even when muscles attach to the wrong site (H, arrowhead) or contain ectopic extensions (H, arrow). (I-L) Anti-β3-tubulin labels microtubules (MTs) in somatic muscles. At stage 13 during myoblast fusion, wild type (I) and RacGAPDH15 (K) have a similar β3-tubulin pattern. By stage 16, wild-type MTs are organized along the longitudinal axis of each muscle (J). In RacGAPDH15 mutants, MTs are present, but muscles make inappropriate attachments (L, arrowheads).

View larger version (103K): [in this window] [in a new window]   Fig. 7. RacGAP is required for uniform MT polarity. (A-I) G14-GAL4 driving UAS-nod::GFP labels MT minus ends. Brackets mark individual muscles, arrowheads mark muscle ends. As wild-type muscles extend (A,C) and make attachments (D,G), Nod::GFP accumulates towards the center of the fiber (arrows) and is notably absent from muscle ends. In RacGAPDH15 (B,C',E,H) and pavB200 (I) mutants, Nod::GFP is present throughout the entire muscle, which shows a disruption of MT polarity. C and C' are higher magnifications of stage 14/15 Nod::GFP expression in wild type and RacGAPDH15 mutants. (F) Co-expression of UAS-tum::myc and UAS-nod::GFP in RacGAPDH15 mutants results in significant rescue of muscle patterning and restoration of MT polarity, as shown by the absence of Nod::GFP at muscle ends. G and H are higher magnifications of D and E, respectively, showing Nod::GFP at the interior of muscle fibers in the wild type (G) and at the ends of muscles in RacGAPDH15 (H).

View larger version (69K): [in this window] [in a new window]   Fig. 8. RacGAP controls MT polarity through interaction with -tubulin. Yellow arrows mark segment borders, where myotubes from adjacent segments make attachments. (A,B) In wild type, -tubulin (A) and RacGAP (B) have a similar localization pattern in cytoplasmic puncta that are concentrated towards the center of the myotube. (C,C') Merge of RacGAP (red) and -tubulin (green) shows that -tubulin colocalizes with RacGAP puncta at the nuclear periphery. (D-G) Gain was increased for -tubulin imaging in D,D',E,E',F,F',G. The segment borders indicated by the yellow arrows in D, E and F are magnified in D', E' and F', respectively. D'', E'' and F'' are schematics of -tubulin localization within extending myotubes at a segment border in the wild-type, pav and RacGAP backgrounds, respectively. In wild-type mononucleated myoblasts (G), -tubulin (green) is present diffusely throughout the cytoplasm and is absent from the nucleus (magenta, labeled with anti-Mef2). In wild-type myotubes (D,D'), -tubulin is found in the cytoplasm (D), with puncta concentrated at the nuclear periphery (D', arrowheads), but is absent from the muscle ends (yellow arrow). In pavB200 mutants (E,E'), -tubulin is not as highly concentrated around nuclei (E', n), but is localized to patches at muscle ends (E', arrowhead). In RacGAPDH15 mutants (F,F'), -tubulin fails to localize to perinuclear sites and instead is diffusely localized throughout the muscle cytoplasm, similar to its pattern in mononucleated myoblasts (G).

View larger version (36K): [in this window] [in a new window]   Fig. 9. Model of RacGAP and Pav function during myotube extension in Drosophila. (A) Mononucleate myoblasts have MTs dispersed throughout the cytoplasm and diffuse cytoplasmic -tubulin localization. (B) One end of an elongating myotube. In multinucleated myotubes, the MT array must be reorganized in the longitudinal axis of the muscle to allow for elongation and extension. This organization requires MT polarity, characterized by minus ends at the interior of the myotube and plus ends at the periphery, to drive extension. RacGAP, Pavarotti (KLP, kinesin-like protein) and -tubulin are required to establish the proper MT array in migrating myotubes. Pav localizes RacGAP to discrete cytoplasmic puncta at the nuclear periphery, and RacGAP localization determines -tubulin distribution. RacGAP may transport incoming MTs to the nuclear periphery after myoblast fusion and/or promote the nucleation of new MTs in the appropriate orientation by increasing -tubulin at the nuclear periphery, thus establishing the MT array required for myotube extension.

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