Figure 7.
Nearly all fascin molecules (95–98%) in filopodia are bound. (A) Confocal slice of a B16 cell expressing GFP-fascin along with (B) schematic representation of the same cell. Average intensities in filopodia (sold line) and cytoplasm (dashed circle) were measured. The intensity in filopodia, Ifil, is proportional to total concentration of fascin, bound and unbound, whereas intensity in the lamellipodium, Icyt, is proportional to amount of unbound fascin in filopodia. (C) Thus, percentage of fascin bound in filopodia was calculated as (1 − Icyt)/Ifil and plotted versus average cytoplasmic intensity over a range of expression levels. Both the range and mean values for percentage of fascin bound in filopodia were fit with a second-order polynomial function, where the y-intercept yields the percentage of endogenous protein bound in filopodia of wild-type, nonexpressing cells. Using this method, we found that 95–98% of filopodial fascin is bound, with a mean value of 97%.