The F-BAR protein Syp1 negatively regulates WASp-Arp2/3 complex activity during endocytic patch formation

Abstract

Background: Actin polymerization by Arp2/3 complex must be tightly regulated to promote clathrin-mediated endocytosis. Although many Arp2/3 complex activators have been identified, mechanisms for its negative regulation have remained more elusive. To address this, we analyzed the yeast arp2-7 allele, which is biochemically unique in causing unregulated actin assembly in vitro in the absence of Arp2/3 activators.

Results: We examined endocytosis in arp2-7 mutants by live-cell imaging of Sla1-GFP, a coat marker, and Abp1-RFP, which marks the later actin phase of endocytosis. Sla1-GFP and Abp1-RFP lifetimes were accelerated in arp2-7 mutants, which is opposite to actin nucleation-impaired arp2 alleles or deletions of Arp2/3 activators. We performed a screen for multicopy suppressors of arp2-7 and identified SYP1, an FCHO1 homolog, which contains F-BAR and AP-2micro homology domains. Overexpression of SYP1 in arp2-7 cells slowed Sla1-GFP lifetimes closer to wild-type cells. Further, purified Syp1 directly inhibited Las17/WASp stimulation of Arp2/3 complex-mediated actin assembly in vitro. This activity was mapped to a fragment of Syp1 located between its F-BAR and AP-2micro homology domains and depends on sequences in Las17/WASp outside of the VCA domain.

Conclusions: Together, these data identify Syp1 as a novel negative regulator of WASp-Arp2/3 complex that helps choreograph the precise timing of actin assembly during endocytosis.

Figure 1. Endocytic dynamics in arp2 mutant cells

(A) Patch lifetimes ± standard deviation of Sla1-GFP at 25°C (white) or 37°C (gray) for ARP2 (SL5770), arp2-7 (SL5775), arp2-1 (SL5792), and arp2-2 (SL5780), respectively. * indicates p ≤ 0.0001 compared with ARP2 at comparable temperature, ^† indicates p ≤ 0.0003 compared with ARP2 at comparable temperature, ^# indicates p ≤ 0.0001 compared to same strain at 25 °C. (B) Patch lifetimes ± standard deviation of Abp1-RFP at 25°C (white) or 37°C (gray) for ARP2, arp2-7, arp2-1, and arp2-2, respectively. * Indicates p ≤ 0.0001 compared with ARP2, ^‡ indicates p ≤ 0.002 compared with ARP2 at comparable temperature, ^# indicates p ≤ 0.0001 compared to same strain at 25 °C. (C) Kymographs of Sla1-GFP and Abp1-RFP at either 25°C (upper) or 37°C (lower) illustrate the lifetimes and overlap of these factors over 240 sec. Time lapse movies were acquired with 2 sec intervals between frames, and using 250 msec exposures for both Sla1-GFP and Abp1-RFP.

Figure 2. Identification of SYP1 as a dosage suppressor of arp2-7

(A) Schematic of S. cerevisiae genomic inserts contained in library plasmids pJD11, pJD12, pJD7 and pJD9, which were isolated as high copy suppressors of arp2-7 temperature sensitive growth. (B) ARP2 (BGY134), arp2-1 (BGY136), and arp2-7 (BGY142) strains transformed with empty vector (pBG006) or pGAL1-SYP1 (pBG287) were 10-fold serially diluted, plated on selective galactose-containing medium, and grown at 25 and 37°C. (C) Previously identified high copy suppressors of pfy1Δ [34] were transformed into the arp2-7 (BGY142) strain (BGY142). Transformants were 10-fold serially diluted, plated on selective medium, and grown at 25°C and 37°C. (D) ARP2, arp2-1 and arp2-7 strains containing SYP1 or syp1Δ were 5-fold serially diluted and grown at 30°C, 35.5°C or 37°C for 48 hours. (E) Schematic of Syp1 demonstrating regions of homology to F-BAR (Green) or AP μ (Brown) domains. Also highlighted are serine (Blue) and proline (Yellow) rich regions, possible Ark/Prk phosphorylation sites (T576, T588) and an NPF motif (Orange).

Figure 3. Temporal pattern of Syp1 localization to cortical patches

(A) Syp1-GFP localization in JDY175. A Z-stack of 0.25 uM sections (600 msec exposures) was deconvolved and projected onto a single plane. (B) Patch lifetimes ± standard deviation of indicated endocytic factors were obtained from 4 min time lapse movies taken at 600 msec exposures, 2 sec between frames. Data are from SL5863 (Ede1-Cherry), SL5806 (Syp1-GFP and Abp1-RFP), SL5862 (End3-Cherry). (C) Localization of Syp1-GFP combined with Ede1-Cherry (SL5863), End3-Cherry (SL5862), or Abp1-RFP (SL5806) in a single cross-section plane. Exposures were 1 sec for GFP and Cherry fusions; 800 msec for Abp1-RFP. Note yellow in the merge shows nearly complete overlap of Ede1 and Syp1, with only occasional overlap for Syp1-GFP and End3-Cherry (arrowheads). There is essentially no overlap between Syp1-GFP and Abp1-RFP. (D) Kymographs from time lapse movies of Syp1-GFP combined with Ede1-Cherry (SL5863), End3-Cherry (SL5862), or Abp1-RFP (SL5806). Exposures were 1 sec for GFP and Cherry fusions; 800 msec for Abp1-RFP, with 3 sec between frames. Note the nearly complete overlap of Syp1-GFP and Ede1-Cherry, whereas End3-Cherry appears near the end of the fluorescent lifetime of Syp1. Syp1-GFP disappears just prior to Abp1-RFP appearance. (E) Tile views showing timing of appearance and disappearance of Syp1-GFP with End3-Cherry (SL5862) or Syp1-GFP with Abp1-RFP (SL5806). Exposures are as indicated in panel D. Beginning frames showing the early phase of Syp1 have been trimmed. Tile views were aligned based on the known timing of End3 and Abp1 [10] The black arrow in the lower tile view indicates the transition point at which Syp1 disappears but Abp1 has not yet appeared.

Figure 4. Overexpression of SYP1 slows the accelerated endocytic dynamics of arp2-7 cells

(A) Sla1-GFP lifetimes (± standard deviation) in ARP2 (SL5770) or arp2-7 (SL5775) ± SYP1 2μ shifted to 37°C. * indicates p ≤ 0.0001 comparing ± SYP1 over-expression. (B) ABP1-RFP lifetimes (± standard deviation) shifted to 37°C in the same strains as above. (C) Kymographs of Sla1-GFP and Abp1-RFP at 37°C from strains indicated above. Time lapse movies were taken with 2 sec intervals between frames, and using 250 msec exposures for both Sla1-GFP and Abp1-RFP.

Figure 5. Purified Syp1 inhibits Las17/WASp-Arp2/3-complex mediated actin assembly

(A) Coomassie stained gel of full-length 6His-Syp1 purified from E. coli. (B) 3 μM actin (5% pyrene-labeled) was polymerized in the presence of 15 nM yeast Arp2/3 complex, 15 nM yeast Las17/WASp, and a range of concentrations of 6His-Syp1. (C) Concentration-dependent effects of 6His-Syp1 on rate of actin assembly induced by 15nM Las17/WASp and 15nM Arp2/3 complex. (D) 2 μM yeast or rabbit muscle actin (5% pyrene labeled) was assembled alone and in the presence of 10nM Arp2/3 complex and 10 nM Las17/WASp with and without 2.5 μM Syp1. (E) Quantification of data in panel D. Rates of actin assembly for reactions containing yeast actin and rabbit muscle actin and lacking Syp1 were both normalized to 1 for comparative purposes. (F) Schematic of Syp1 fragments that were purified and tested for inhibition of Las17/WASp-Arp2/3 complex activity. Each Syp1 fragment (>1 μM) was tested for effects on 15nM Las17/WASp and 15nM Arp2/3 complex. (G) Concentration-dependent effects of Syp1 (aa 265-565) on rate of actin assembly induced by 15nM Las17/WASp and 15nM Arp2/3 complex.

Figure 6. Syp1 directly regulates Las17/WASp

(A) 4 μM actin (5% pyrene-labeled) was polymerized in the presence of 10 nM yeast Arp2/3 complex and 10 nM yeast Las17/WASp or 20 nM yeast Arp2/3 complex and 200 nM yeast GST-VCA in the presence or absence of 2.5 μM Syp1. (B) Quantification of data from A. Rates of actin assembly for reactions containing Las17/WASp and GST-VCA domain but lacking Syp1 were normalized to 1 for comparative purposes. (C) Control beads or beads coated with Las17/WASp were incubated with soluble Syp1, then pelleted, and the supernatants were analyzed by SDS PAGE and Coomassie staining. (D) Quantification of data in C; levels of Syp1 in the supernatants were measured by densitometry.