Why are two different cross-linkers necessary for actin bundle formation in vivo and what does each cross-link contribute?
In developing Drosophila bristles two species of cross-linker, the forked proteins and fascin, connect adjacent actin filaments into bundles. Bundles form in three phases: (a) tiny bundles appear; (b) these bundles aggregate into larger bundles; and (c) the filaments become maximally cross-linked by fascin. In mutants that completely lack forked, aggregation of the bundles does not occur so that the mature bundles consist of <50 filaments versus approximately 700 for wild type. If the forked concentration is genetically reduced to half the wild type, aggregation of the tiny bundles occurs but the filaments are poorly ordered albeit with small patches of fascin cross-linked filaments. In mutants containing an excess of forked, all the bundles tend to aggregate and the filaments are maximally crossbridged by fascin. Alternatively, if fascin is absent, phases 1 and 2 occur normally but the resultant bundles are twisted and the filaments within them are poorly ordered. By extracting fully elongated bristles with potassium iodide which removes fascin but leaves forked, the bundles change from being straight to twisted and the filaments within them become poorly ordered. From these observations we conclude that (a) forked is used early in development to aggregate the tiny bundles into larger bundles; and (b) forked facilitates fascin entry into the bundles to maximally cross-link the actin filaments into straight, compact, rigid bundles. Thus, forked aligns the filaments and then directs fascin binding so that inappropriate cross-linking does not occur.
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
L G
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Tilney
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Connelly
K A
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Vranich
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Shaw
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Guild
D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D030801:Animals, Genetically Modified; D002352:Carrier Proteins / Q000378:metabolism / Q000648:ultrastructure; D002875:Chromosomes / Q000235:genetics / Q000502:physiology; D003432:Cross-Linking Reagents; D004331:Drosophila melanogaster / Q000235:genetics* / Q000254:growth & development*; D005260:Female; D007814:Larva; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D009154:Mutation; D011679:Pupa; D011984:Sensory Receptor Cells / Q000502:physiology / Q000648:ultrastructure
D000199:Actins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D008840:Microfilament Proteins; C020324:fascin