Capping Protein Insulates Arp2/3-Assembled Actin Patches from Formins

Abstract

How actin structures of distinct identities and functions coexist within the same environment is a critical self-organization question. Fission yeast cells have a simple actin cytoskeleton made of four structures: Arp2/3 assembles actin patches around endocytic pits, and the formins For3, Cdc12, and Fus1 assemble actin cables, the cytokinetic ring during division, and the fusion focus during sexual reproduction, respectively. The focus concentrates the delivery of hydrolases by myosin V to digest the cell wall for cell fusion. We discovered that cells lacking capping protein (CP), a heterodimer that blocks barbed-end dynamics and associates with actin patches, exhibit a delay in fusion. Consistent with CP-formin competition for barbed-end binding, Fus1, F-actin, and the linear filament marker tropomyosin hyper-accumulate at the fusion focus in cells lacking CP. CP deletion also rescues the fusion defect of a mutation in the Fus1 knob region. However, myosin V and exocytic cargoes are reduced at the fusion focus and diverted to ectopic foci, which underlies the fusion defect. Remarkably, the ectopic foci coincide with Arp2/3-assembled actin patches, which now contain low levels of Fus1. We further show that CP localization to actin patches is required to prevent the formation of ectopic foci and promote efficient cell fusion. During mitotic growth, actin patches lacking CP similarly display a dual identity, as they accumulate the formins For3 and Cdc12, normally absent from patches, and are co-decorated by the linear filament-binding protein tropomyosin and the patch marker fimbrin. Thus, CP serves to protect Arp2/3-nucleated structures from formin activity.

Keywords: actin; actin homeostasis; actin patch; capping protein; cell-cell fusion; fission yeast Schizosaccharomyces pombe; formin.

Graphical abstract

Figure 1

CP Deletion Leads to Fusion Delay and Fusion Focus Persistence after Fusion (A) Fusion efficiencies 12 and 36 h after nitrogen removal in the indicated strains. (B) Fusion times in the indicated strains. (C) Time-lapse images of Myo52-tdTomato and cytosolic GFP expressed in P cells under the map3 promoter in WT and acp2Δ, from the beginning to end of the fusion process. The times shown (in h:min) are relative to the initial formation of the fusion focus. (D) Time-lapse images of strains as in (C) from the time of fusion taking place up to the disappearance of the fusion focus. The times shown (in h:min) are relative to the time of fusion. Note that the first image (at 00:00) is the same as the last image in (C). (E) Fusion focus persistence times in the indicated strains. All indicated p values are relative to WT. Scale bars represent 5 μm.

Figure 2

CP Deletion Leads to Increased Actin, Tropomyosin, and Fus1 at the Fusion Focus (A–C) Myo52-tdTomato and (A) GFP-CHD labeling F-actin, (B) Fus1-sfGFP, and (C) GFP-Cdc8 in WT and acp2Δ at fusion time. (D) Fusion focus fluorescence intensities normalized to WT at fusion time in strains as in (A)–(C). See Figures S1A–S1C for full fluorescence profile measurements. (E) Spinning-disk confocal images of Myo52-tdTomato and WT or mutant Fus1-sfGFP, as indicated, in WT and acp2Δ. Yellow arrowheads point to unfused cell pairs, with a broad Fus1 and Myo52 distribution at the contact zone. Blue arrowheads point to fusion foci. (F) Fusion efficiencies at 9 h after nitrogen removal in WT or acp2Δ strains carrying WT or mutant fus1, as indicated. All p values are relative to WT, except where indicated. Scale bars represent 5 μm. See also Figures S1 and S2.

Figure 3

CP Deletion Leads to Reduced Vesicular Markers at the Fusion Focus and Formation of Ectopic Foci (A–E) Myo52-tdTomato and (A) Exo84-GFP, (B) Exo70-GFP, (C) GFP-Ypt3, (D) Agn2-sfGFP, and (E) Eng2-sfGFP in WT and acp2Δ before fusion time. White arrows highlight ectopic foci. Note that these images, selected to show ectopic foci, stem from various time points during time-lapse imaging, for which specific timing in the fusion process and photobleaching may mask the difference in fusion focus intensity. (F) Fusion focus fluorescence intensities at fusion time normalized to WT in the strains mentioned in (A)–(E). (G) Time-lapse images of Myo52-tdTomato in WT and acp2Δ during the fusion process. White arrows show ectopic Myo52 foci. Time is in h:min. (H) Number of time frames at which a Myo52 ectopic focus was observed during the fusion process in time-lapse imaging as in (G). All p values are relative to WT. Scale bars represent 5 μm. See also Figure S3 and Video S1.

Figure 4

Myo52 Ectopic Foci Form at Actin Patches (A) Spinning-disk confocal time-lapse images of Myo52-tdTomato and cytosolic GFP expressed under the map3 promoter (shown only for the first time point) in WT and acp2Δ before fusion time. The white arrow marks an ectopic Myo52 focus that forms at 1 s and then moves toward the fusion focus. Time is in min:s. Right: number of time points, acquired at 1-s intervals during 3 min, displaying a Myo52 ectopic focus. (B–E) Spinning-disk confocal images of Myo52-tdTomato and (B) Exo70-GFP, (C) GFP-CHD, (D) Myo51-sfGFP, and (E) Fus1-sfGFP in WT and acp2Δ before fusion time. (F and G) Spinning-disk confocal images of strains expressing Fim1-mCherry and (F) Myo51-sfGFP or (G) Fus1-sfGFP in WT and acp2Δ before fusion time. The bar plots to the right of the images show the proportion of ectopic foci colocalizing with the indicated markers, of which an example is shown with a white arrow. Scale bars represent 5 μm. See also Figure S4 and Videos S2, S3, and S4.

Figure 5

CP Recruitment to Actin Patches and Barbed-End Binding Is Required to Protect against Ectopic Foci (A–G) Myo52-tdTomato- (A) Acp1-sfGFP, (B) Acp1^Δt-sfGFP, (C) Acp1-sfGFP in acp2Δ, (D) Acp2-sfGFP, (E) Acp2^Δt-sfGFP, (F) Acp2-sfGFP in acp1Δ, or (G) Acp2^R12A,Y77A, carrying mutated CPI-interacting residues, at fusion time, as indicated. Scale bars represent 5 μm. (H) Acp1-sfGFP and Acp2-sfGFP patch-to-cytosol fluorescence intensity ratios in fusing cells of strains as in (A)–(G). (I) Number of time frames at which a Myo52 ectopic focus was observed during the fusion process in time-lapse imaging of strains as in (A)–(F). (J) Ectopic foci (as in I) and fusion times for WT (acp2-sfGFP), acp2^R12A,Y77A-sfGFP, and acp2Δ strains. All p values are relative to WT. See also Figure S5.

Figure 6

CPs Insulate Actin Patches from Myo52 and Actin Cable Markers in Interphase Cells (A–F) Spinning-disk confocal images of (A) Fim1-mCherry and Myo51-sfGFP, (B) Myo52-tdTomato and Myo51-sfGFP, (C) Myo52-tdTomato and Fim1-sfGFP, (D) Fim1-mCherry and GFP-Cdc8, (E) Fim1-mCherry and For3-3GFP, and (F) Fim1-mCherry and Cdc12-3GFP in WT and acp2Δ interphase cells. In (E), bottom panels show acp2Δ treated with 500 μM CK-666 for 5 min or 200 μM latrunculin A for 5 min. White arrows highlight colocalization events in acp2Δ, which do not occur in WT cells. Yellow arrowheads point to Cdc12 spots. The proportion of colocalization at ectopic sites along the cell sides is shown with the bar plot on the right. (G) Spinning-disk confocal time-lapse images of Fim1-mCherry and For3-3GFP (green and gray) showing retrograde flow in WT (top) and acp2Δ cells (bottom two panels). Bottom: an example of retrograde flow starting at a lateral actin patch. Kymographs of the yellow dashed boxed regions are shown on the right. Green arrows point to For3-3GFP retrograde flow. Purple arrowheads in kymographs show lateral actin patches on which For3 localizes in acp2Δ but not WT cells. (H) For3 retrograde flow rate. Time is in s. Scale bars represent 5 μm. See also Figure S6 and Videos S5 and S6.