Dip1 Co-opts Features of Branching Nucleation to Create Linear Actin Filaments that Activate WASP-Bound Arp2/3 Complex

Abstract

When activated by Wiskott-Aldrich syndrome proteins (WASP), Arp2/3 complex nucleates branched actin filaments important for processes like cellular motility and endocytosis [1]. WASP-mediated activation of Arp2/3 complex requires a preformed actin filament, ensuring that activation by WASP creates branched instead of linear filaments. However, this biochemical requirement also means that assembly of branched actin networks must be primed with an initial seed filament [2-4]. We recently described a class of activators called WISH/DIP/SPIN90 (WDS) proteins, which, unlike WASP, activate Arp2/3 complex without a preformed filament [4]. Although this property may allow WDS proteins to serve as seed filament generators, it is unknown whether actin filaments nucleated by WDS-activated Arp2/3 complex can activate WASP-bound Arp2/3 complex. Further, despite their potential importance as branched actin network initiators, little is known about how WDS proteins turn on Arp2/3 complex. Here, we use two-color single-molecule total internal reflection fluorescence (TIRF) microscopy to show that Dip1, the S. pombe WDS protein [5], co-opts features of branching nucleation to activate Arp2/3 complex. Specifically, it activates Arp2/3 complex to nucleate linear filaments analogous to the branch created by WASP-mediated activation. The barbed ends of Dip1-Arp2/3 nucleated filaments are free to elongate, and their pointed ends remain anchored to Dip1-bound Arp2/3 complex. The linear filaments nucleated by Dip1-bound Arp2/3 complex activate WASP-bound Arp2/3 complex as potently as spontaneously nucleated or branched actin filaments. These observations provide important insights into the regulation of Arp2/3 complex by its activators and the molecular basis for initiation of branched actin networks.

Keywords: Arp2/3 complex; Dip1; S. pombe; WASP; Wsp1; actin; endocytosis.

Figure 1:. Model of Dip1-mediated Arp2/3 complex activation and characterization of Alexa Fluor 568 labeled Dip1.

A. Cartoon of a branched filament nucleated by WASP-activated Arp2/3 complex (top) showing resulting filament polarities (barbed end, BE; pointed end, PE). Bottom half of panel shows a model of Dip1-mediated activation of Arp2/3 complex. In this model, the linear filament nucleated by Dip1-bound Arp2/3 complex is analogous to the daughter filament nucleated during branching nucleation. B. Time course of polymerization of 3 μM 15% pyrene-labeled actin in the presence of 50 nM S. pombe Arp2/3 complex (SpArp2/3 complex) and either unlabeled or Alexa Fluor 568 labeled Dip1 (568-Dip1). C. Representative image of 568-Dip1 molecules bound to a coverslip and visualized by TIRF microscopy. D. Examples of fluorescence intensities of single 568-Dip1 puncta over time. E. Quantification of events in which 568-Dip1 photobleached in one versus multiple steps. F. Plot of c(S) versus (S) for sedimentation velocity analytical ultracentrifugation of three concentrations of unlabeled Dip1. Dip1 sediments predominantly or entirely as a monomer at all three concentrations. See also Table S1.

Figure 2:. Dip1 co-opts features of branching nucleation to create linear filaments.

A. TIRF microscopy images of actin polymerization reactions containing 6 nM 568-Dip1, 1.5 μM 33% Oregon Green labeled actin and 500 nM SpArp2/3 complex. Top, middle and bottom row show three different classes of events. Blue arrows show Dip1 bound to filament ends, red arrow heads mark elongating filament ends. Scale bar: 5 μm. B. Plot of filament length versus time for free filaments or filaments with bound Dip1. A total of 3 Non-568-Dip1 bound and 3 568-Dip1 filaments were measured. C. Quantification of the percentage of pointed ends with 568-Dip1 bound in actin polymerization reactions containing 6 nM 568-Dip1 and 1.5 μM 33% Oregon Green labeled actin with or without 500 nM SpArp2/3 complex. Error bars: standard error from 4 reactions. We speculate that the single event observed in the absence of Arp2/3 complex is coincident colocalization of a filament end with a surface adsorbed molecule of 568-Dip1. D. Quantification of three classes of events in which Dip1 is observed on the ends of filaments for the conditions described in B. Error bars show standard error from 4 reactions. E. TIRF microscopy image from a reaction in which 1.5 μM 33 % Oregon Green actin filaments were sheared and flowed into the imaging chamber before adding 6 nM 568-Dip1 and 500 nM SpArp2/3 complex. The total percentage of pointed ends bound to 568-Dip1 is indicated in the upper right corner (of 362 total pointed ends observed). F. TIRF microscopy image showing the same sized field of view as F with conditions described in B, in which Dip1 activates Arp2/3 complex to nucleate linear filaments. The total percentage of pointed ends bound to 568-Dip1 is indicated in the upper right corner (of 242 total pointed ends observed). Scale bar: 5 μm. See also Videos S1–S3.

Figure 3:. Filaments nucleated by Dip1-bound Arp2/3 complex activate Wsp1-bound Arp2/3 complex to seed branched network assembly.

A, B. TIRF microscopy images of Dip1-Arp2/3 nucleated filaments seeding branching nucleation by Wsp1 and Arp2/3 complex. The reaction contained 6 nM 568-Dip1, 250 nM SpArp2/3 complex, 250 nM GST-Wsp1-VCA and 1.5 μM 33% Oregon Green actin. Blue arrows point to Dip1 molecules bound to pointed ends. Yellow arrows point to actin filament branches that grew from Dip1-Arp2/3 nucleated filaments. The red arrow points to a branch that nucleated from another branch. Panels A and B show growth of filaments from surface adsorbed Dip1 (class I event) or from a surface captured Dip1-bound filament (class II event), respectively. C. TIRF images of branches growing from spontaneously nucleated filaments used for quantification in D. The reaction contained 250 nM SpArp2/3 complex, 250 nM GST-Wsp1-VCA and 1.5 μM Oregon Green actin. White arrows point to branches. Scale bar is 1 μm in A,B and C. D. Quantification of the rate of branching on three sources of filaments: spontaneously nucleated filaments, pre-existing branches and Dip1-Arp2/3 nucleated seed filaments. Error bars: standard error from 3 separate reactions. See also Videos S4 and S5.