Dip1 defines a class of Arp2/3 complex activators that function without preformed actin filaments

Abstract

Background: Arp2/3 complex is a key actin cytoskeletal regulator that creates branched actin filament networks in response to cellular signals. WASP-activated Arp2/3 complex assembles branched actin networks by nucleating new filaments from the sides of pre-existing ones. WASP-mediated activation requires seed filaments, to which the WASP-bound Arp2/3 complex can bind to form branches, but the source of the first substrate filaments for branching is unknown.

Results: Here we show that Dip1, a member of the WISH/DIP/SPIN90 family of actin regulators, potently activates Arp2/3 complex without preformed filaments. Unlike other Arp2/3 complex activators, Dip1 does not bind actin monomers or filaments, and it interacts with the complex using a non-WASP-like binding mode. In addition, Dip1-activated Arp2/3 complex creates linear instead of branched actin filament networks.

Conclusions: Our data show the mechanism by which Dip1 and other WISH/DIP/SPIN90 proteins can provide seed filaments to Arp2/3 complex to serve as master switches in initiating branched actin assembly. This mechanism is distinct from other known activators of Arp2/3 complex.

Figure 1

Dip1 is a potent activator of Arp2/3 complex. (A) Domain organization of human SPIN90, budding yeast Ldb17 and fission yeast Dip1. The leucine rich domain, LRD, is conserved in all species. The LRD not have sequence homology to leucine rich repeat domains. (B) Time course of polymerization of 3 μM 15 % pyrene-actin with or without 50 nM S. pombe Arp2/3 complex and a range of concentrations of Dip1. (C) Plot of maximum polymerization rate versus Dip1 or Wsp1-VCA concentration for pyrene-actin polymerization assays described in B. (D) Plot of calculated number of barbed ends versus time for reactions described in B. Micromolar concentration of Dip1 in each reaction is indicated. (E) Pyrene actin polymerization assays with Arp2/3 complex and pyrene actin as in (B), plus 1μM Dip1 and 200 nM Wsp1-VCA as indicated. See also Figure S1.

Figure 2

Dip1-mediated activation of Arp2/3 does not require preformed actin filaments. (A) Time course of polymerization of 3 μM 15% pyrene actin with 50 nM S. pombe Arp2/3 complex, and 200 nM Wsp1-VCA or the indicated concentrations of Dip1. Arrow highlights lag in activation of Arp2/3 complex by Wsp1-VCA. (B) Plot of branch density versus time for TIRF data in panel C. Data are represented as mean +/- SEM. (C) Total internal reflection microscopy (TIRF) images of 33% Oregon green-488 actin polymerizing with 50nM S. pombe Arp2/3 complex, 150 nM Dip1 and 75 nM GST-Wsp1-VCA as indicated. Scale Bar = 2.2 μm. (D) Plot of total polymer length verses time for TIRF data in panel C. (E) Plot of filament lengths expressed in subunits of actin versus time for select single filaments in TIRF data in panel B. Dashed lines are linear fits of each filament growth. Global analysis of at least 7 filaments/reaction showed that the average growth rate in reactions with Arp2/3 alone was 9.0 ± 0.1 s⁻¹ (n=541); Dip1 alone was 9.7 ± 0.3 s⁻¹ (n=816); Arp2/3 + GST-Wsp1-VCA was 9.2 ± 0.2 s⁻¹ (n=641); and Arp2/3 + dip1 was 9.5 ± 0.2 s⁻¹ (n=775).

Figure 3

Bulk polymerization assays verify preformed filaments are not required for Dip1-mediated Arp2/3 complex activation. (A) Pyrene actin polymerization assay showing the influence of 1.5 μM Crn1 WD-CC construct (contains residues 1-410 and 594-651) on activation of 50 nM S. pombe Arp2/3 complex by 5 μM Dip1 or 200 nM GST-Wsp1-VCA. (B) Pyrene actin polymerization assay showing the influence of preformed actin filaments on Dip1- versus Wsp1-activated Arp2/3 complex. Reactions contained 50 nM S. pombe Arp2/3 complex, 1 μM Dip1, 200nM GST-Wsp1-VCA and 300 nM actin filament seeds as indicated.

Figure 4

Dip1 uses a non-WASp-like mechanism to activate Arp2/3 complex. (A) Western blot of supernatant and pelleted fractions in actin monomer pull-down assay. Actin at 1.0 μM was pulled down with 10 μM GST-Wsp1-VCA, 10 or 13.5 μM or 10 μM GST control protein. Quantified data are represented as the mean +/- SEM (n=3), asterisk represents significant difference compared to GST control p < 0.0001 (parametric two-tailed T-test) (B) Coomassie-stained native gel shift binding assay. Reactions contained indicated concentrations of each protein plus 40 μM Latrunculin B to prevent actin polymerization. Arrows indicate Dip1 (top) and actin (bottom) (C) Coomassie-stained SDS-PAGE gel of actin filament copelleting assay. Dip1 (750 nM) or cortactin (750 nM) were copelleted with a range of concentrations of polymerized actin (total actin concentration is indicated). (D) Anti-Arp3 western blot of pull-down assay containing GST-Dip1 and 1.14 μM S. pombe Arp2/3 complex. Control assays contained 11 μM GST or 11 μM GSTWsp1-VCA. Quantified data are represented as the mean +/- SEM (n=3), asterisk represents significant difference compared to GST control p < 0.05 (parametric two-tailed T-test) (E, F) Time courses of polymerization of 3 μM 15% pyrene actin with 50 nM S. pombe Arp2/3 complex, 7.5 μM profilin and 5 μM mutant or wild-type Dip1, as indicated.

Figure 5

Dip1 stimulates formation of the short pitch conformation. (A) Cartoon schematic showing relative positions of engineered cysteine residues on Arp2 and Arp3 in active or inactive conformation. (B) Anti-Arp3 western blot of crosslinking assays containing 1.0 μM S. cerevisiae Arp2/3 complex (Arp3L155C/Arp2R198C) and 25 μM BMOE, 10 μM leucine-zipper (LZ) N-WASp-VCA, 10 μM latrunculin B-bound actin, and Dip1 as indicated. Reactions were allowed to proceed for 60 s before quenching with 1.25 mM dithiolthreitol and separating by SDS-PAGE. (C) Quantification of short-pitch Arp2-Arp3 crosslinking assays as described in panel. Data are represented as mean +/- SEM. P-value calculated from parametric two-tailed t-test

Figure 6

SPIN90 and Dip1 may use the same mechanism to activate Arp2/3 complex. (A) Time course of 3 μM 15% pyrene actin polymerization showing influence of wild type and 2.2 μM GSTDip1 truncations on GST-Dip1-mediated activation of 50 nM S. pombe Arp2/3 complex. (B) Time course of 3 μM 15% pyrene actin polymerization containing SPIN90 (residues 269-722) or GSTN-WASp-VCA and 50 nM bovine Arp2/3 complex. (C) Pyrene actin polymerization assays containing 50 nM bovine Arp2/3 complex, 2 μM Crn1 WD-CC construct, 17.8 μM SPIN90, and 200 nM GST-N-WASp-VCA as indicated. (D) Pyrene actin polymerization assay showing the influence of preformed actin filaments on Spin90 versus GST-N-WASp-VCA activated bovine Arp2/3 complex. Reactions contained 50 nM bovine Arp2/3 complex, 10.6 μM Spin90, 200 nM GST-N-WASp-VCA and 300 nM actin filament seeds as indicated. (E) Total internal reflection microscopy (TIRF) images of 33% Oregon green-488 actin polymerizing with B. taurus Arp2/3 complex and 1.5 μM SPIN90 or 100 nM GST-NWASp-VCA as indicated. Reaction with 1.5 μM SPIN90 contains 25 nM Bt Arp2/3 and reaction with N-WASp-VCA contains 20 nM BtArp2/3 complex. Scale Bar = 2.2 μm. (F) Plot of maximum polymerization rate versus Spin90 concentration for pyrene-actin polymerization assays described in B.

Figure 7

Cartoon model of initiation and propagation of Arp2/3-mediated branching nucleation by Dip1 and Wsp1. See text for details.