Fig. 3. CLIP-170-mDia1 complexes are recruited to MT plus-ends by EB1 and stimulate actin polymerization from the MT surface.

(A) TIRF movies show that 549-CLIP-170-1 binds MT sides, and tracks MT plus-ends only in the presence of EB1 (17). 488-mDia1 binds MT sides, with or without EB1, and does not track MT plus-ends. (B-C) 549-CLIP-170-1 and 488-mDia1 colocalize on MT sides (B), and track MT plus-ends together specifically in the presence of EB1 (C). Reactions in A-C contain 15 μM tubulin (30% AlexaFluor649 labeled), biotinylated GMP-CPP MT seeds, and variable components: 25 nM 549-CLIP-170-1, 100 pM 488-mDia1, and 500 nM EB1. (D) Percentage of molecules tracking MT plus-ends from reactions in (A–C); data averaged from 3 experiments (n > 100). Statistical differences: ns, not different from control; a, compared to CLIP-170-1 (p < 0.05); b, compared to mDia1 (p < 0.05). (E) Co-reconstitution of MTs undergoing dynamic instability and actin filaments polymerizing. Reactions contain all the same components as in (A–C) plus 1 μM G-actin (10% OG-labeled; 0.2% biotin-actin) and 5 μM profilin. (F) Kymographs of MT and actin dynamics. (G) Rate of actin filament elongation does not change significantly in the presence and absence of MTs; data averaged from 3 experiments (n = 50). (H) With the addition of unlabeled EB1 and mDia1, 549-CLIP-170-1 molecules (yellow arrows) were recruited to MT plus-ends, where they triggered assembly of actin filaments that grew at the accelerated rate. (I) Similar observations as in (H) except using 549-mDia1 (yellow arrows) and unlabeled CLIP-170-1. (J) Fraction of formin-generated actin filaments that grew from MT plus-ends in reactions as in (H & I); data from 3 experiments (n = 50). (K) Actin elongation rates from reactions as in (H & I); data from 3 experiments (n = 50). Statistical differences: ns, not different from control; a, compared to actin alone or control (p < 0.05); b, compared to EB1 control (p < 0.05). Error bars in all panels, SE.