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Long continuous actin bundles in Drosophila bristles are constructed by overlapping short filaments.

Guild GM, Connelly PS, Ruggiero L, Vranich KA, Tilney LG - J. Cell Biol. (2003)

Bottom Line: These long bundles are built from much shorter modules that graft together.Thus, bundle morphogenesis has several components: module formation, elongation, grafting, and bundle smoothing.These actin bundles are much like a rope or cable, made by overlapping elements that run a small fraction of the overall length, and stiffened by cross-linking.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA. gguild@sas.upenn.edu

ABSTRACT
The actin bundles essential for Drosophila bristle elongation are hundreds of microns long and composed of cross-linked unipolar filaments. These long bundles are built from much shorter modules that graft together. Using both confocal and electron microscopy, we demonstrate that newly synthesized modules are short (1-2 microm in length); modules elongate to approximately 3 microm by growing over the surface of longitudinally adjacent modules to form a graft; the grafted regions are initially secured by the forked protein cross-bridge and later by the fascin cross-bridge; actin bundles are smoothed by filament addition and appear continuous and without swellings; and in the absence of grafting, dramatic alterations in cell shape occur that substitutes cell width expansion for elongation. Thus, bundle morphogenesis has several components: module formation, elongation, grafting, and bundle smoothing. These actin bundles are much like a rope or cable, made by overlapping elements that run a small fraction of the overall length, and stiffened by cross-linking.

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Actin filament cross-linkers are required for module grafting. (a–d) Scanning electron micrographs of wild-type and mutant adult bristles showing the cuticular morphology of ridges and valleys. Bristle bases are at the bottom of each panel. (a) Wild-type bristle showing long parallel ridges and valleys. (b) Bristle from a singed mutant (lacking fascin) showing twisted morphology accompanied by both very short (horizontal arrowheads) and much longer (vertical arrowheads) ridges and valleys. Note that the ends of the ridges are tapered and interconnect. (c) Bristle from a forked mutant (lacking forked proteins). All ridges and valleys are short (e.g., horizontal arrowheads), whereas others run perpendicular to the bristle longitudinal axis (e.g., vertical arrowheads) and the cuticular valleys rarely fuse. (d) Bristle from a singed-forked double mutant exhibiting only very short ridges and valleys (arrowheads) that are largely oblique to the bristle longitudinal axis. The ridges rarely connect. (e–i) Confocal micrographs of wild-type mutant pupal bristles stained with fluorescent phalloidin showing internal actin bundles. (e) Wild-type bristle showing long, parallel, and continuous actin bundles. (f) Bristle from a singed mutant showing a twisted morphology. Although the bundles are shorter than the wild type, they are considerably longer than single modules (3 μm) and many interconnect (arrows). Thus, much of the F actin is cross-linked into longitudinal bundles. (g) Bristle from a forked mutant that exhibits very limited longitudinal bundling (arrow) and far more short bundles, some running perpendicular to the bristle longitudinal axis (arrowheads). forked bristles are shorter and wider than either wild-type or singed bristles. (h) Bristle from a singed-forked double mutant exhibiting vast numbers of short bundles and very few organized bundles (arrows). (i) Higher magnification of another singed-forked bristle highlighting the very short modules that are arranged in a variety of orientations (arrows). These bundles exhibit weak fluorescence and rarely fuse. Bars, 5 μm.
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fig9: Actin filament cross-linkers are required for module grafting. (a–d) Scanning electron micrographs of wild-type and mutant adult bristles showing the cuticular morphology of ridges and valleys. Bristle bases are at the bottom of each panel. (a) Wild-type bristle showing long parallel ridges and valleys. (b) Bristle from a singed mutant (lacking fascin) showing twisted morphology accompanied by both very short (horizontal arrowheads) and much longer (vertical arrowheads) ridges and valleys. Note that the ends of the ridges are tapered and interconnect. (c) Bristle from a forked mutant (lacking forked proteins). All ridges and valleys are short (e.g., horizontal arrowheads), whereas others run perpendicular to the bristle longitudinal axis (e.g., vertical arrowheads) and the cuticular valleys rarely fuse. (d) Bristle from a singed-forked double mutant exhibiting only very short ridges and valleys (arrowheads) that are largely oblique to the bristle longitudinal axis. The ridges rarely connect. (e–i) Confocal micrographs of wild-type mutant pupal bristles stained with fluorescent phalloidin showing internal actin bundles. (e) Wild-type bristle showing long, parallel, and continuous actin bundles. (f) Bristle from a singed mutant showing a twisted morphology. Although the bundles are shorter than the wild type, they are considerably longer than single modules (3 μm) and many interconnect (arrows). Thus, much of the F actin is cross-linked into longitudinal bundles. (g) Bristle from a forked mutant that exhibits very limited longitudinal bundling (arrow) and far more short bundles, some running perpendicular to the bristle longitudinal axis (arrowheads). forked bristles are shorter and wider than either wild-type or singed bristles. (h) Bristle from a singed-forked double mutant exhibiting vast numbers of short bundles and very few organized bundles (arrows). (i) Higher magnification of another singed-forked bristle highlighting the very short modules that are arranged in a variety of orientations (arrows). These bundles exhibit weak fluorescence and rarely fuse. Bars, 5 μm.

Mentions: We addressed this question by examining bristles lacking one or both of the cross-bridges, forked or fascin. Scanning electron micrographs of adult wild-type bristles reveal that each is fluted like Greek or Roman columns (Fig. 9 a). Previous work demonstrated that this fluted appearance is related to the distribution of the actin bundles formed earlier during pupal development and that run the length of the bristle (Overton, 1967; Appel et al., 1993; Tilney et al., 1995, 1996). Thus, examination of the surface of the adult bristle informs us of where the actin bundles were located during bristle elongation before the exoskeleton was deposited. In particular, continuous ridges are a measure of grafting over extended longitudinal distances.


Long continuous actin bundles in Drosophila bristles are constructed by overlapping short filaments.

Guild GM, Connelly PS, Ruggiero L, Vranich KA, Tilney LG - J. Cell Biol. (2003)

Actin filament cross-linkers are required for module grafting. (a–d) Scanning electron micrographs of wild-type and mutant adult bristles showing the cuticular morphology of ridges and valleys. Bristle bases are at the bottom of each panel. (a) Wild-type bristle showing long parallel ridges and valleys. (b) Bristle from a singed mutant (lacking fascin) showing twisted morphology accompanied by both very short (horizontal arrowheads) and much longer (vertical arrowheads) ridges and valleys. Note that the ends of the ridges are tapered and interconnect. (c) Bristle from a forked mutant (lacking forked proteins). All ridges and valleys are short (e.g., horizontal arrowheads), whereas others run perpendicular to the bristle longitudinal axis (e.g., vertical arrowheads) and the cuticular valleys rarely fuse. (d) Bristle from a singed-forked double mutant exhibiting only very short ridges and valleys (arrowheads) that are largely oblique to the bristle longitudinal axis. The ridges rarely connect. (e–i) Confocal micrographs of wild-type mutant pupal bristles stained with fluorescent phalloidin showing internal actin bundles. (e) Wild-type bristle showing long, parallel, and continuous actin bundles. (f) Bristle from a singed mutant showing a twisted morphology. Although the bundles are shorter than the wild type, they are considerably longer than single modules (3 μm) and many interconnect (arrows). Thus, much of the F actin is cross-linked into longitudinal bundles. (g) Bristle from a forked mutant that exhibits very limited longitudinal bundling (arrow) and far more short bundles, some running perpendicular to the bristle longitudinal axis (arrowheads). forked bristles are shorter and wider than either wild-type or singed bristles. (h) Bristle from a singed-forked double mutant exhibiting vast numbers of short bundles and very few organized bundles (arrows). (i) Higher magnification of another singed-forked bristle highlighting the very short modules that are arranged in a variety of orientations (arrows). These bundles exhibit weak fluorescence and rarely fuse. Bars, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172841&req=5

fig9: Actin filament cross-linkers are required for module grafting. (a–d) Scanning electron micrographs of wild-type and mutant adult bristles showing the cuticular morphology of ridges and valleys. Bristle bases are at the bottom of each panel. (a) Wild-type bristle showing long parallel ridges and valleys. (b) Bristle from a singed mutant (lacking fascin) showing twisted morphology accompanied by both very short (horizontal arrowheads) and much longer (vertical arrowheads) ridges and valleys. Note that the ends of the ridges are tapered and interconnect. (c) Bristle from a forked mutant (lacking forked proteins). All ridges and valleys are short (e.g., horizontal arrowheads), whereas others run perpendicular to the bristle longitudinal axis (e.g., vertical arrowheads) and the cuticular valleys rarely fuse. (d) Bristle from a singed-forked double mutant exhibiting only very short ridges and valleys (arrowheads) that are largely oblique to the bristle longitudinal axis. The ridges rarely connect. (e–i) Confocal micrographs of wild-type mutant pupal bristles stained with fluorescent phalloidin showing internal actin bundles. (e) Wild-type bristle showing long, parallel, and continuous actin bundles. (f) Bristle from a singed mutant showing a twisted morphology. Although the bundles are shorter than the wild type, they are considerably longer than single modules (3 μm) and many interconnect (arrows). Thus, much of the F actin is cross-linked into longitudinal bundles. (g) Bristle from a forked mutant that exhibits very limited longitudinal bundling (arrow) and far more short bundles, some running perpendicular to the bristle longitudinal axis (arrowheads). forked bristles are shorter and wider than either wild-type or singed bristles. (h) Bristle from a singed-forked double mutant exhibiting vast numbers of short bundles and very few organized bundles (arrows). (i) Higher magnification of another singed-forked bristle highlighting the very short modules that are arranged in a variety of orientations (arrows). These bundles exhibit weak fluorescence and rarely fuse. Bars, 5 μm.
Mentions: We addressed this question by examining bristles lacking one or both of the cross-bridges, forked or fascin. Scanning electron micrographs of adult wild-type bristles reveal that each is fluted like Greek or Roman columns (Fig. 9 a). Previous work demonstrated that this fluted appearance is related to the distribution of the actin bundles formed earlier during pupal development and that run the length of the bristle (Overton, 1967; Appel et al., 1993; Tilney et al., 1995, 1996). Thus, examination of the surface of the adult bristle informs us of where the actin bundles were located during bristle elongation before the exoskeleton was deposited. In particular, continuous ridges are a measure of grafting over extended longitudinal distances.

Bottom Line: These long bundles are built from much shorter modules that graft together.Thus, bundle morphogenesis has several components: module formation, elongation, grafting, and bundle smoothing.These actin bundles are much like a rope or cable, made by overlapping elements that run a small fraction of the overall length, and stiffened by cross-linking.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA. gguild@sas.upenn.edu

ABSTRACT
The actin bundles essential for Drosophila bristle elongation are hundreds of microns long and composed of cross-linked unipolar filaments. These long bundles are built from much shorter modules that graft together. Using both confocal and electron microscopy, we demonstrate that newly synthesized modules are short (1-2 microm in length); modules elongate to approximately 3 microm by growing over the surface of longitudinally adjacent modules to form a graft; the grafted regions are initially secured by the forked protein cross-bridge and later by the fascin cross-bridge; actin bundles are smoothed by filament addition and appear continuous and without swellings; and in the absence of grafting, dramatic alterations in cell shape occur that substitutes cell width expansion for elongation. Thus, bundle morphogenesis has several components: module formation, elongation, grafting, and bundle smoothing. These actin bundles are much like a rope or cable, made by overlapping elements that run a small fraction of the overall length, and stiffened by cross-linking.

Show MeSH
Related in: MedlinePlus