Fig. 5.

Wash F-actin/MT bundling and crosslinking activity is regulated by Rho1, Spire and Arp2/3. (A-H″) Stabilized F-actin (1 μM, A-H) and MTs (1 μM, A′-H′) were incubated with full-length Wash and/or Rho1, Arp2/3 and Spire, and observed by confocal microscopy: (A) no protein added; (B) SpirD only; (C) Wash and SpirD; (D) Wash, SpirD and Rho1^GTP; (E) Wash, SpirD and Rho1^GDP; (F) Wash and SpirD2; (G) Wash, SpirD2 and Rho1^GTP; (H) Wash and SpirD3. (I) Quantification of F-actin and MT bundling/crosslinking efficiency of indicated reactions (see Fig. 3; quantification of Arp2/3 reactions were not obtained owing to co-migration of Arp2/3 with actin and MT bands). (J-M″) F-actin (J-M) and microtubule bundling (J′-M′) and crosslinking assay with: (J) Arp2/3; (K) Wash and Arp2/3; (L) Wash, Arp2/3 and Rho1^GTP; (M) Wash, Arp2/3 and Rho1^GDP. All assays were performed a minimum of three times. Final protein concentrations were: Wash, 300 nM; Arp2/3, 600 nM; Rho1^GTP, 600 nM; SpirD, 300 nM; SpirD2, 300 nM; SpirD3, 300 nM. (N) Stills from a time-lapse confocal movie of branched-actin network formation by Wash and Arp2/3 in the presence of F-actin (see Movie 6 in the supplementary material). Scale bar: 10 μm.