Characterization of dip1p reveals a switch in Arp2/3-dependent actin assembly for fission yeast endocytosis

Abstract

Background: During endocytosis in yeast, a choreographed series of discrete local events at the plasma membrane lead to a rapid burst of actin polymerization and the subsequent internalization of an endocytic vesicle. What initiates Arp2/3-dependent actin polymerization in this process is not well understood.

Results: The Schizosaccharomyces pombe WISH/DIP/SPIN90 ortholog dip1p is an actin-patch protein that regulates the temporal sequence of endocytic events. dip1Δ mutants exhibit a novel phenotype in which early events such as WASp localization occur normally but arrival of Arp2/3, actin polymerization, and subsequent steps are delayed and occur with apparently random timing. In studying this mutant, we demonstrate that positive feedback loops of WASp, rapid actin assembly, and Arp2/3 contribute to switch-like behavior that initiates actin polymerization. In the absence of dip1p, a subset of patches is activated concurrently with the "touch" of a neighboring endocytic vesicle.

Conclusions: These studies reveal a switch-like mechanism responsible for the initiation of actin assembly during endocytosis. This switch may be activated in at least two ways, through a dip1p-dependent mechanism and through contact with another endocytic vesicle.

Figure 1. dip1 mutants have defects in actin organization and endocytosis

(A) AlexaFluor 488-phalloidin stained wildtype (left) and dip1Δ (right) cells. Maximum intensity projections of confocal images are shown. (B) Quantification of the average number of actin patches per cell from images as in A. (n = 60 cells for each strain). Patches within clusters were counted as individuals only if they could be visually distinguished. (C) Images of cells of indicated genotype expressing the integral membrane protein GFP-syb1. GFP-syb1 accumulates at the plasma membrane in endocytic mutants. (D) Dip1p co-localizes with the Arp2/3 marker arc5p in actin patches. Wildtype cells expressing arc5-mCherry and dip1-GFP from their endogenous promoters were imaged in a medial focal plane. White arrowheads indicate sites where dip1p and arc5p co-localize. (E) Time-lapse images of dip1-GFP and arc5-mCherry in a single patch. Arrow indicates patch internalization. GFP and mCherry images were acquired sequentially with exposure times of 2s and 0.2s respectively. (F) Normalized fluorescence intensities of dip1-GFP (green) and arc5-mCherry (red) within a single patch in WT (left). Time = 0s indicates the time of patch internalization. (G) Schematic of dip1p dynamics relative to actin patch proteins sla1p, wsp1p and arc5p. Time = 0s indicates the time of patch internalization. Boxed area indicates time at which protein concentration peaks at the patch. Scale bars = 5µm.

Figure 2. Dip1p regulates the timing of patch activation

(A) Quantification of number of patches of the indicated markers in WT and dip1Δ cells. n=20 cells each. Error bars represent standard deviations. ** Significant difference (p< 0.0001) between dip1Δ and WT strains. (B) Time-lapse images of individual patches marked by mYFP-wsp1 in WT and dip1Δ cells. The montages track the maturation of a single representative patch over time. Each interval is 2 sec. Arrows mark the time of internalization of the endocytic vesicle. In the dip1Δ cells, the lifetimes of the mYFP-wsp1 containing patch is highly variable, as depicted by the three patches shown. (C) Quantitation of the lifetimes of mYFP-wsp1 patches. Images were acquired every 2 sec for 100 sec. Histograms of patches in WT (grey bars) and dip1Δ (green bars) are shown. n = 72 patches in 5 cells in WT; N= 72 patches in 15 cells in dip1Δ. The distribution of mYFP-wsp1 in dip1Δ was fitted to a Poisson distribution (dashed histogram). Mean and variance for Poisson distribution (λ) = 1, covariance (r²) = 0.948. (D) Images were acquired every 10 sec for 500 sec to track patch behavior over a longer time scale. n = 60 patches in 6 cells. (E) Images were acquired every 1 sec for 100 sec to track patch behavior over a short time scale. n = 45 patches in 5 cells in WT, and 125 patches in 25 cells in dip1Δ. Scale bar = 5µm.

Figure 3. Dynamics of patch components in wildtype and dip1Δ mutants

(A) Time-lapse images of individual patches in WT and dip1Δ cells labeled with the indicated markers. Widefield (i) and confocal images (ii, iii) were acquired through the medial focal section of the cell at 2s intervals. Arrows denote time of patch internalization. (B) Normalized fluorescence intensities of patch proteins at a single patch over time in WT (left) and dip1Δ (right). Time = 0s indicates the time of patch internalization. Arc5-mCherry and mYFP-wsp1 intensities are normalized to mYFP-wsp1 in dip1Δ and sla1-GFP intensities are normalized to sla1-GFP in dip1Δ. (C) Left: Average number of mYFP-wsp1 molecules per patch over time in WT (n=11) and dip1Δ (n=12). Numbers were estimated based on fluorescence intensity ratios (see Supplementary Methods). Right: Numbers of mYFP-wsp1 molecules in individual patches in dip1Δ cells are each plotted in different colors.

Figure 4. A switch for patch activation requires rapid actin polymerization

(A) i) dip1Δ cells were treated with indicated concentrations of Latrunculin A (LatA) for 2 min, and then fixed and stained for F-actin with AlexaFluor 488-phalloidin. Maximum intensity projection images are shown. Note that at 1µM and 2µM doses of LatA, actin cables are disrupted, but actin patches remain. (ii) Images of mYFP-wsp1 in live dip1Δ cells treated with indicated concentrations of LatA for 2 min. Single focal plane confocal images are shown. (B) Time-lapse images of a mYFP-wsp1 patch in dip1Δ cell treated with 2µM LatA. Note that the patch does not internalize and gradually disappears. (C) Effect of LatA on the dynamic behavior of mYFP-wsp1 patches over time in dip1Δ cells. Cells were treated with indicated concentrations of LatA for 2 min and then imaged. Graphs show mean fluorescence intensities plotted over time, where t=0 at the peak of intensity. n=8 patches. (D) Effect of LatA on the rate of increase of mYFP-wsp1. Cells were treated with doses of LatA for 2 minutes and then imaged. Rates of increase of mYFP-wsp1 intensity leading up to peak at t=0 (e.g. the slopes of the upward part of the curves in C) were measured for each patch (n=8 patches at each dose). The average rates were used to fit a Hill curve using the formula f(x)= c*((C^λ/(C^λ + x^λ))) with the following parameters C (critical LatA concentration) = 1.201, c (fitting parameter) = 0.2616 and λ (Hill co-efficient) = 3.18. Error bars = SD. Scale bars = 5µm.

Figure 5. The effect of wsp1p on patch dynamics and its genetic interaction with dip1p

(A) Alexa Fluor 488 phalloidin staining of cells of indicated genotype. Maximum intensity projection confocal images are shown. (B) Single focal planes images of these mutants expressing patch markers crn1p and sla1p. (C) Time lapse images of single patches. Arrows mark patch internalization, which fails in wsp1Δ and dip1Δwsp1Δ mutants. (D) Percentage of sla1-GFP marked patches that internalized over 50s in the indicated genotypes. n=76 patches in 10 cells for each strain. (E) Spot assay for growth. Cells of indicated geneotypes were spotted at different dilutions onto agar plates and incubated for 5 days at the indicated temperature. dip1Δ and wsp1Δ show a synthetic sick genetic interaction.

Figure 6. Activation of a patch may be triggered by another endocytic vesicle

(A) Left: Time-lapse images of dip1Δ cells expressing crn1-Tomato (red) and sla1-GFP (green). The sequence shows the movement of an internalized endocytic vesicle (white arrow) moving towards and touching an immature patch marked only with sla1-GFP (yellow arrow). This touch correlates with the recruitment of crn1p to the second patch (yellow arrow with asterisk) and its subsequent internalization. Right: Graph showing position of the sla1p-marked endocytic pit or vesicle relative to the plasma membrane (marked by the dashed lines) in this sequence, and the relative timings of the touch, recruitment of crn1p, and internalization. (B) Image sequence showing the movement of an endocytic vesicle (white arrow) to a cluster of sla1-GFP marked patches at the cortex (yellow arrow and arrowhead), and then the internalization of this entire cluster in a dip1Δ cell. (C) In a wildtype cell, a similar movement of an endocytic vesicle (white arrow) to a cluster of sla1-GFP positive patches (yellow arrow and arrowhead) leading to activation of the whole cluster. (D) Schematic drawing showing the activation of a patch after being “touched” by an endocytic vesicle. Sla1p is represented in green, and actin filaments are in red. Note that the organization of actin filaments is not known, and elements are not drawn to scale.