Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Recovery of GFP-fascin from the cell body into filopodia is relatively slow. (A) FRAP experiment on GFP-fascin in filopodia. Entire filopodia were bleached and monitored every 5 seconds for fluorescence recovery. Scale bars: 1 μm. Time given in seconds. (B) FRAP recovery data plotted versus time and fit with a single exponential function. Recovery profile reveals fluorescence recovery to near pre-bleach intensity by 100 seconds, mimicking the slow recovery component between filopodial cross-links and the fascin pool from the cell body shown in Figure 1E.
• Supplemental Figure 2 - Neuro2a filopodia contain fascin:actin ratios of 1:27-41. (A) Western Blot of N2a cell lysate (in duplicate) and varying amounts of pure fascin protein. Comparison of band intensity reveals that N2a cells typically contain 350-500 nM fascin. (B) Percent fascin localized to filopodia was calculated using the expression, and plotted versus average intensity in cells for varying expression levels. The data was fit with a third order polynomial with y-intercept equal to the percentage of endogenous fascin that localizes to filopodia in wild type, non-expressing cells. Using this technique, we determined that approximately 29% of total fascin molecules in the cell localize to filopodia, or 100-145nM fascin. Comparison of fascin to actin concentrations in filopodia, we determined that N2a filopodia contain 1 fascin per 27-41 actin. (C) Percent fascin bound in filopodia of N2a cells was calculated as and plotted versus average cytoplasmic intensity over a range of expression levels. Both the range and mean values for percent fascin bound in filopodia were fit with a second order polynomial function, where the y-intercept yields the percent endogenous protein bound in filopodia of wild type, non-expressing cells. Using this method, we found that 94-98% of filopodial fascin is bound, with a mean value of 97%.
• Appendices