Figure 6: MICAL1 is activated upon Rab35 binding and markedly accelerates actin filament depolymerization from both ends though oxidation.

(a) In a microfluidics set-up, ADP-actin filaments grown from surface-anchored seeds align with the flow. The fluorescent images show the typical barbed end depolymerization of individual filaments exposed to buffer alone (left) or to a solution of 600 nM FAD+120 μM NADPH (right). The kymographs correspond to the same two filaments. (b) Depolymerization velocity measured over time for ADP-actin filaments exposed either to buffer alone (black), to 600 nM FAD (green) or to 600 nM FAD+120 μM NADPH (red). Each set of data corresponds to a different population of 50 filaments observed in the microfluidics set-up. Data points are averages over the different filaments, and error bars are s.d.'s. (c) Kymographs of filaments depolymerizing in the microfluidics set-up, which is used to rapidly change the flowing solution to which the filaments are exposed. Left: an ADP-actin filament is exposed to 600 nM FAD+120 μM NADPH for 85 s, followed by buffer alone. Right: An ADP-actin filament exposed to 600 nM FAD+120 μM NADPH for 150 s was regrown from fresh (unoxidized) actin and depolymerized in buffer alone. (d) ADP-actin filaments are anchored to a surface coated with inactive myosins and are exposed to the same depolymerizing solutions as in a. (e) Recombinant GST-tagged MICAL1^879–1067 or GST alone were incubated with recombinant His-tagged MICAL1^FAD-CH-LIM and increasing amounts of active Rab35-GppNHp. Western blot anti-His₆, anti-Rab35 and Ponceau S red staining are presented. Input: 1%. (f) Depolymerization rates measured on surface-anchored ADP-actin filaments with different solutions (6–31 filaments per condition). All solutions contain 120 μM NADPH. Bars show mean±s.d. Concentrations of FAD and MICAL1 full-length were 1.2 μM. (g) Model for activation of the redox enzyme MICAL1 by Rab35-GTP (red). Actin is in green.