Regulation of actin filament cross-linking and bundle shape in Drosophila bristles.
Previous studies demonstrate that in developing Drosophila bristles, two cross-linking proteins are required sequentially to bundle the actin filaments that support elongating bristle cells. The forked protein initiates the process and facilitates subsequent cross-linking by fascin. Using cross-linker-specific antibodies, mutants, and drugs we show that fascin and actin are present in excessive amounts throughout bundle elongation. In contrast, the forked cross-linker is limited throughout bundle formation, and accordingly, regulates bundle size and shape. We also show that regulation of cross-linking by phosphorylation can affect bundle size. Specifically, inhibition of phosphorylation by staurosporine results in a failure to form large bundles if added during bundle formation, and leads to a loss of cross-linking by fascin if added after the bundles form. Interestingly, inhibition of dephosphorylation by okadaic acid results in the separation of the actin bundles from the plasma membrane. We further show by thin section electron microscopy analysis of mutant and wild-type bristles that the amount of material that connects the actin bundles to the plasma membrane is also limited throughout bristle elongation. Therefore, overall bundle shape is determined by the number of actin filaments assembled onto the limited area provided by the connector material. We conclude that assembly of actin bundles in Drosophila bristles is controlled in part by the controlled availability of a single cross-linking protein, forked, and in part by controlled phosphorylation of cross-links and membrane actin connector proteins.
Tilney
L G
LG
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Connelly
P S
PS
Vranich
K A
KA
Shaw
M K
MK
Guild
G M
GM
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003432:Cross-Linking Reagents; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000378:metabolism / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019476:Insect Proteins / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors
D000199:Actins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D019476:Insect Proteins; D008840:Microfilament Proteins; C084628:f protein, Drosophila; C020324:fascin; D019319:Okadaic Acid; D019311:Staurosporine