Figure S1. Loss of Ena/VASP proteins inhibits filopodia and neuritogenesis. (a) Bar graph of filopodia number per stage 1 control and mmvvee (Mena/ VASP/EVL-null) neurons at 40hrs in culture. Loss of all three Ena/VASP family members markedly inhibits filopodia formation (***, p<0.001, two tailed unpaired T-test with Welch s correction, numbers over bars indicate number of neurons examined). All error bars in figure are +/-SEM. (b) Stacked bar graph showing stage progression in control and mmvvee neurons. Ena/VASP deletion causes many more neurons to remain in stage 1 (no neurites), compared to controls (***p<0.001, two way ANOVA with Bonferroni post hoc comparisons). (c) Representative stage 1 and stage 2 cortical neurons transfected with EGFP-Mena and fixed at one day in culture. Mena is concentrated at the cell periphery (like VASP and EVL data not shown). The overlay image shows that EGFP-Mena concentrates at the ends of actin filament bundles in the periphery of stage 1 neurons and at the tips of filopodia in stage 2 neuron growth cones. Endogenous protein shows the same concentration at the periphery, especially at the end of peripheral actin bundles 12, 18. WWW.NATURE.COM/NATURECELLBIOLOGY 1
Figure S2. Transfection of Ena/VASP proteins rescues filopodia and neuritogenesis in mmvvee neurons. (a) Domain diagram of Ena/VASP family members. (b) Bar graph showing that Mena, VASP and EVL transfection into mmvvee neurons rescues the number of filopodia in stage 1 neurons (p values given for Kruskal-Wallis one way ANOVA with Dunn s post hoc comparisons). All error bars in figure are +/-SEM. (c) Stacked bar graph showing transfection of Mena, VASP and EVL all substantially rescue neuritogenesis in stage 1 neurons. (*p<0.05, ***p<0.001, two way ANOVA with Bonferroni post hoc comparisons). Numbers at bottom (filopodia) or top (stages) of bars indicate number of neurons examined. (d) Domain diagram of Mena with indicated regions where +, 2+ and 3+ exons are inserted. (e) Bar graphs of number of filopodia per stage 1 neuron. Each isoform rescued filopodia to similar levels (p values indicated, Kruskal-Wallis one way ANOVA with Dunn s post hoc comparisons). (f) Stacked bar graph showing efficiency to which Mena isoforms rescue neuritogenesis. Only the Mena3+2+ isoform does not rescue neuritogenesis (*p<0.05, ***p<0.001, two way ANOVA with Bonferroni post hoc comparisons). Numbers at bottom (filopodia) or top (stages) of bars indicate number of neurons examined. 2 WWW.NATURE.COM/NATURECELLBIOLOGY
S U P P L E M E N TA R Y I N F O R M AT I O N Figure S3. mdia2 is not detected in E14.5 cortex or adult cerebellum and overexpression of mdia2m1041a hyperstabilizes microtubules, causing them to form hairpin loops in filopodia. (a) A single mmvvee neuron transfected with high levels of EGFP-mDia2M1041A. Note that mdia2m1041a signal is concentrated at the tips of filopodia but is also present at high levels throughout the cytoplasm. (b) In this cell newly polymerized, dynamic microtubules (TyrMTs) extend to the tips of filopodia. (c) Older, stable microtubules (Glu-MTs) also extend well into filopodia and form hairpin loops (arrowheads in (e)). (d) Differential interference contrast (DIC) image of neuron. (e) Overlay of panels b (red) and c (green). (f) Overlay of panels a (blue), b (red) and c (green). Arrowheads indicate hairpin microtubule loops at tips of filopodia. (g) Western blot of RIPA extracts from HeLa cells, E14.5 cortex, adult cerebellum and E14.5 cortical neurons plated for 1 day in culture and labeled for mdia2. The blot was stripped and reprobed for α-tubulin to show that lanes containing neuronal extracts were overloaded compared to the HeLa cell extract. Note that there is little to no mdia2 in E14.5 cortex or when E14.5 cortical neurons are cultured for 1 day. This antibody was shown to react with RIPA extracts from mouse cell lines indicating absence of labeling is not due to lack of immunoreactivity with mouse tissue (data not shown). Standards (in middle) are 250, 150, 100, 75 and 50kd. Scale bar is 10µm. WWW.NATURE.COM/NATURECELLBIOLOGY 3
S U P P L E M E N TA R Y I N F O R M AT I O N Figure S4. Filopodia and actin-rich filopodia-like extensions that form after laminin addition contain fascin and are invaded by microtubules. (a-c) An example of a control neuron (prominent growth cone shown) cultured for 24hrs on PdL, fixed and labeled for F-actin (phalloidin) and fascin, a filopodium-enriched protein. (d-f) An example of a stage 1 mmvvee neuron cultured for 24hrs on PdL that has extended three filopodia. These filopodia are well labeled with anti-fascin antibody. (g-i) An example of a stage 1 neuron cultured on PdL and laminin (20µg/ml) that has extended several prominent filopodia enriched in fascin. (j-r ) Three examples of E14.5 mmvvee cortical neurons plated on PdL and laminin (20µg/ml) for 8 hours, fixed and labeled for F-actin (phalloidin) (j, m, p) and β3-tubulin (k, n, q). F-actin (red) and β3-tubulin (green) images are shown in overlay (l, o, r). Boxed regions in l, o and r are digitally magnified five times in l, o and r. Arrowheads indicate microtubules extending into filopodia. These images also show that neurons express a marker for differentiated neurons (β3tubulin) soon after plating. Scale bar is 10µm except 2µm for l, o and r. 4 WWW.NATURE.COM/NATURECELLBIOLOGY
Figure S5. Ena/VASP-null phenotype occurs in Ena/VASP inactivated cortical neurons. (a) Expression of EGFP-FP4-Mito blocks Ena/VASP function by depleting Ena/VASP proteins from their sites of function and sequestering them to the mitochondrial surface 12. Stacked column graph illustrating the percentage of neurons in each stage after 44hrs in culture. First bar represent untransfected neurons, second bar (AP4-Mito) represents neurons transfected with a control construct that targets the mitochondria but does not sequester Ena/VASP proteins, and the third bar (FP4-Mito) represents neurons transfected with a construct that sequesters all Ena/VASP proteins to the mitochondria, effectively inactivating them. Note the marked increase in stage 1 neurons at the expense of stage 3 neurons between AP4-Mito and FP4-Mito for stage 1 and stage 3 neurons (**p<0.01 compared to both AP4-Mito and untransfected, two way ANOVA with Bonferroni post hoc comparisons). Numbers under graph indicate number of neurons examined. All error bars in figure are +/-SEM. (b) Two cortical neurons labeled with phalloidin to label F-actin (red) and an antibody to β3-tubulin to label neuron specific microtubules (blue). The stage 1 neuron is transfected with EGFP-FP4-Mito so the mitochondria fluoresce green, while the stage 3 neuron is not transfected. Scale bar is 10µm. WWW.NATURE.COM/NATURECELLBIOLOGY 5
SUPPLEMENTAL MOVIES Movie 1. A phase contrast movie of a newly plated control cortical neuron on a poly-d-lysine substrate. Note the continued extension and retraction of filopodia. Time = 10 minutes, images captured every 5 seconds. Movie 2. A phase contrast movie a newly plated Ena/VASP-null (mmvvee) cortical neuron on a poly-d-lysine substrate. Note the lack of membrane protrusion but continued retrograde flow throughout the lamellipodium. Time = 10 minutes, images captured every 5 seconds. Movie 3. A phase contrast movie of a control neuron forming a neurite. Note that there are many filopodia in the region where the neurite forms with no one dominating to give rise to the neurite. Rather, over a period of several hours filopodial and veil protrusions give rise to a growth cone that consolidates into a neurite. Movie 4. A phase contrast movie of an mmvvee neuron forming a neurite. In contrast to the control neuron in movie 7 the mmvvee neuron is able to extend a filopodium that is stabilized over a period of several hours. This filopodium dilates to form a neurite that continues to extend, forming an axon over a period of several days. Movie 5. Another example of an mmvvee neuron forming a neurite through extension, stabilization and dilation of a single filopodium. Movie 6. A movie of a control and mmvvee neuron transfected with mcherry-β-actin and EGFP-α-tubulin. Only the control cell has extensive actin bundles that give rise to filopodia. Note the extensive microtubule invasion into the periphery of each neuron. However, in the mmvvee neuron microtubules turn parallel to the membrane and are brought back in retrograde flow. Time = 10 minutes, images captured every 5 seconds. Movie 7. A phase contrast movie of a newly plated mmvvee neuron on a poly-d-lysine and laminin substrate. Note the extensive dynamic filopodia along the segmented periphery of the cell. Time = 10 minutes, images captured every 5 seconds. Movie 8. A phase contrast movie of an mmvvee neuron plated on a poly-d-lysine and laminin substrate. Note the extensive filopodia and segmented regions around the periphery of the cell that give rise to neurites and subsequently a single axon. Time = 25 hours, images captured every 2 minutes. Movie 9. A movie of an mmvvee neuron transfected with mcherry-β-actin and EGFP-α-tubulin (not shown) and treated with blebbistatin (time of blebbistatin addition indicated on upper right of movie). Note that within 30 minutes a number of actin-rich filopodia emerge from an otherwise lamellar periphery. Time = 1:26 hrs, images captured every 20 seconds. Movie 10. A DIC movie of an Ena/VASP-inhibited neuron that was treated with blebbistatin (a specific myosin II inhibitor) at 40 minutes. A filopodium emerges from a lamellar region and continually elongates and dilates, resulting in a neurite after several hours. Time = 2:55 hrs, images captured every 20 seconds. 6 WWW.NATURE.COM/NATURECELLBIOLOGY