Intrinsic dynamic behavior of fascin in filopodia.
Recent studies showed that the actin cross-linking protein, fascin, undergoes rapid cycling between filopodial filaments. Here, we used an experimental and computational approach to dissect features of fascin exchange and incorporation in filopodia. Using expression of phosphomimetic fascin mutants, we determined that fascin in the phosphorylated state is primarily freely diffusing, whereas actin bundling in filopodia is accomplished by fascin dephosphorylated at serine 39. Fluorescence recovery after photobleaching analysis revealed that fascin rapidly dissociates from filopodial filaments with a kinetic off-rate of 0.12 s(-1) and that it undergoes diffusion at moderate rates with a coefficient of 6 microm(2)s(-1). This kinetic off-rate was recapitulated in vitro, indicating that dynamic behavior is intrinsic to the fascin cross-linker. A computational reaction-diffusion model showed that reversible cross-linking is required for the delivery of fascin to growing filopodial tips at sufficient rates. Analysis of fascin bundling indicated that filopodia are semiordered bundles with one bound fascin per 25-60 actin monomers.
Aratyn
Yvonne S
YS
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. y-aratyn@northwestern.edu
Schaus
Thomas E
TE
Taylor
Edwin W
EW
Borisy
Gary G
GG
D000199:Actins / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004058:Diffusion / Q000187:drug effects; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D007700:Kinetics; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism*; D011817:Rabbits
D000199:Actins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D008840:Microfilament Proteins; D010750:Phosphoproteins; C020324:fascin