Essential role of the endocytic site-associated protein Ecm25 in stress-induced cell elongation

Abstract

How cells adopt a different morphology to cope with stress is not well understood. Here, we show that budding yeast Ecm25 associates with polarized endocytic sites and interacts with the polarity regulator Cdc42 and several late-stage endocytic proteins via distinct regions, including an actin filament-binding motif. Deletion of ECM25 does not affect Cdc42 activity or cause any strong defects in fluid-phase and clathrin-mediated endocytosis but completely abolishes hydroxyurea-induced cell elongation. This phenotype is accompanied by depolarization of the spatiotemporally coupled exo-endocytosis in the bud cortex while maintaining the overall mother-bud polarity. These data suggest that Ecm25 provides an essential link between the polarization signal and the endocytic machinery to enable adaptive morphogenesis under stress conditions.

Keywords: Cdc42; Ecm25; actin patches; cell shape; cell stress; endocytosis; exocytosis; hydroxyurea; yeast.

Figure 1.. Ecm25 is essential for stress-induced cell elongation regardless of the cues and cell cycle regulation

(A) Domains and motifs in Ecm25. CRAL/Sec14, CRAL-TRIO/Sec14 lipid binding domain; RhoGAP, Rho GAP domain; PXXP, PXXP motifs. N-term and C-term, the indicated N- and C-terminal fragments of Ecm25. (B) Representative cell morphologies of BY-background strains with or without HU treatment (100 mM HU at 25°C for ~13 h). Yeast strains: WT (BY4741), ecm25Δ (YEF7957), swe1Δ (YEF7958), ecm25Δ swe1Δ (YEF7961), rad53Δ (YEF8061), rad53Δ ecm25Δ (YEF8062), and rad53Δ swe1Δ (YEF8490). All experiments were performed at 25°C unless indicated otherwise. Scale bars, 4 μm for −HU and 2 μm for +HU. (C) Quantitative analysis of the bud length/width ratios for data shown in (B). About 100 cells were analyzed for each strain, and data were expressed as mean value ± standard deviation (SD). **p < 0.001, *p = 0.002. (D) ecm25Δ cells are sensitive to 200 mM HU at 30°C. Ten-fold serial dilutions of WT (BY4741) and ecm25Δ (YEF7956) cultures were spotted on synthetic complete (SC) plates in the presence or absence of HU (100 mM or 200 mM) and incubated at 25°C and 30°C, respectively, for 4 days before documentation. (E) Time-lapse analysis of cell cycle progression (Whi5-GFP and Tub1-RFP) in HU-treated WT (YEF7942) and ecm25Δ (YEF7962) cells in BY-background strains. Selected frames are chosen from time-lapse series taken with an 8-min interval. Scale bar, 4 μm. See also Figures S1B and S1C and Video S1. (F) Quantitative analysis of cell cycle progression for the data shown in (E). T-G1 (telophase to G1 phase), from appearance to disappearance of Whi5-GFP in the nucleus; S/G2, from disappearance of Whi5-GFP from the nucleus to appearance of a mitotic spindle (Tub1-FRP) of 2 μm in length; M-phase, from appearance of a 2-μm mitotic spindle at the mother side of the bud neck to start of spindle elongation; anaphase, from start of spindle elongation to its maximum length accompanied by appearance of Whi5-GFP in the nucleus. Data are expressed as mean value ± SD.

Figure 2.. Ecm25 is required for sustained polarization of exo-endocytosis at the bud tip during stress-induced cell elongation

(A) Time-lapse analysis of Exo84-GFP localization during the cell cycle (Tub1-RFP) in HU-treated WT (YEF7970) and ecm25Δ (YEF8031) cells. Interval, 6 min. Scale bar, 3 μm. See also Figure S2 and Videos S2 and S3. (B and C) Intensity profile analysis of M-phase Exo84-GFP localization in the daughter compartments of HU-treated WT (YEF7970) (B) and ecm25Δ (YEF8031) (C) cells. The distance for the x axis (in micrometers) was measured from the bud tip to the bud neck (white arrow) for the entire daughter compartment (yellow box). Scale bar, 3 μm. (D) Quantitative analysis of the duration of Exo84-GFP at the bud tip during the cell cycle in HU-treated WT and ecm25Δ cells (from the data shown in A). For WT cells, n = 22; for ecm25Δ cells, n = 20. Data were expressed as mean value ± SD. **p < 0.001. (E) Time-lapse analysis of Exo84-GFP and Abp1-RFP localization during the cell cycle in HU-treated WT (YEF7781) and ecm25Δ (YEF8217) cells. Interval, 6 min. Scale bar, 3 μm. See also Video S2. (F–I) Intensity profile analysis of Exo84-GFP and Abp1-RFP localization in the daughter compartments of HU-treated WT (YEF7781) (F and G) and ecm25Δ (YEF8217) (H and I) cells 150 min after bud emergence. The distance for the x axis (in micrometers) was measured from the bud tip to the bud neck (white arrow) for the entire daughter compartment (yellow box). Scale bar, 3 μm. (J) Quantitative analysis of Exo84 and Abp1 polarization at the bud tip during mitosis in HU-treated WT (YEF7781, n = 21) and ecm25Δ (strain YEF8217, n = 22) cells. The time-lapse data acquired in (E) were used to determine the polarization index of Exo84-GFP and Abp1-RFP by calculating the ratio of the fluorescence intensity for each protein at the bud tip versus the entire bud during mitosis. The bud tip is defined as the apical region that accounts for 32.19% of the bud area in WT or 32.71% of the bud area in ecm25Δ cells. WT cells entered mitosis 136.57 ± 11.05 min after bud emergence, whereas ecm25Δ cells entered mitosis 134.73 ± 11.52 min after bud emergence (Figures 2A and 2E). At this point, the ratio of fluorescence intensity for each protein at the defined bud-tip area versus the entire bud was calculated and plotted. Data are expressed as mean value ± SD. **p < 0.001.

Figure 3.. Association of Ecm25 with the endocytic sites is critical for its role in HU-induced cell elongation

(A) Spatial relationship between Ecm25-GFP expressed from its native promoter and the endocytic machinery (Abp1-RFP) in small- and large-budded WT cells (YEF7779). Scale bar, 2 μm. (B–D) Time-lapse analysis of the localization dynamics of Ecm25-GFP and Abp1-RFP at endocytic sites in non-HU-treated WT cells (YEF7779). Single-focal-plane images were acquired at 1-s intervals. A montage (B), kymograph (C), and relative intensity (D) of both proteins for a representative cell are shown. See also Figures S3A–S3F. (E–G) Time-lapse analysis of the localization dynamics of Ecm25-GFP and Abp1-RFP at endocytic sites in HU-treated WT cells (YEF7779). See also Figures S3G–S3L. (H and I) The effect of targeting Ecm25 to the endocytic site or the PM at the mother side on HU-induced cell elongation. Cells of strain YEF9781 carrying Sla1-GFP and Ecm25-mApple-GBP (H) and strain YEF9789 carrying Pdr5-GFP and Ecm25-mApple-GBP (I) were grown exponentially in SC medium containing 100 mM HU at 25°C for ~16 h and then harvested for morphological documentation. Some of the Ecm25-mApple-GBP molecules seemed to localize to vacuole-like structures under this condition. Scale bar, 2 μm. (J) Quantitative analysis of the bud length/width ratios for data shown in (H) and (I). Data are expressed as mean value ± SD. **p < 0.001; ns, not significant. (K) The effect of targeting Ecm25 to the PM at the mother side on endocytic site distribution. Cells of strain YEF9868 carrying Pdr5-GFP and Abp1-mRuby3 (top) and strain YEF9876 carrying Pdr5-GFP, Ecm25-GBP, and Abp1-mRuby3 (bottom) were grown and documented as described for in (H) and (I). Scale bar, 2 μm.

Figure 4.. The N-terminal Sec14-like and RhoGAP domains of Ecm25 mediate its interaction with Cdc42 and are required for stress-induced cell elongation

(A) BiFC-detected interactions between Cdc42 and FL Ecm25 (YEF7988), its N-term+RhoGAP (YEF7991), N-term (YEF8049), RhoGAP (YEF8316), or C-term (YEF8324) fragment. Negative controls include VC-Cdc42 (YEF10451), Ecm25-full-length (FL)-VN (YEF10450), and Pecm25-VN/VC-Cdc42 (YEF10426). VC, the C-terminal fragment of Venus; VN, the N-terminal fragment of Venus. Scale bar, 2 μm. See also Figure S4. (B) Localization of GFP-tagged FL Ecm25 (YEF7805) and its N-term (YEF7882), RhoGAP (YEF7813), N-term+RhoGAP (YEF7883), C-term (YEF7809), N-term+C-term (YEF7814), and RhoGAP+C-term (YEF7808) fragments expressed from a heterologous promoter on the plasmid vector pUG36 in relation to Abp1-RFP in the absence of HU. Scale bar, 2 μm. (C) The C-term on Ecm25 associates with both actin patches and actin cables. Cells of strain YEF7809 (ecm25Δ ABP1-RFP, pUG36-GFP-ECM25-C-term) were fixed and stained by Alexa Fluor 568 (red)-conjugated phalloidin to visualize filamentous actin cables and patches. Scale bar, 2 μm. (D) The patch- and cable-like structures formed by GFP-Ecm25-C-term are sensitive to LatA treatment (200 μM LatA for 10 min). Yeast strain: YEF7809 (ecm25Δ ABP1-RFP, pUG36-GFP-ECM25-C-term). Scale bar, 2 μm. (E and F) Differential contributions of different Ecm25 domains to its essential role in HU-induced cell elongation. HU-treated cells of strains expressing FL Ecm25 (BY4741) or its N-term (YEF8482), RhoGAP (YEF8487), C-term (YEF8483), N-term+RhoGAP (YEF8486), N-term+C-term (YEF8485), or RhoGAP+C-term (YEF8484) from its native promoter at the endogenous locus or containing ecm25Δ (YEF7956) were documented by differential interference contrast (DIC) microscopy (E) and quantified for the bud length/width ratios (F). Scale bar, 2 μm. Data are expressed as mean value ± SD. **p < 0.001, *p = 0.003. (G and H) The effect of targeting different Ecm25 fragments to endocytic sites on HU-induced cell elongation. Cells of the WT strain (BY4741) as well as strains carrying Sla1-GFP and Ecm25-N-term-mApple-GBP (YEF10421) or Ecm25-N-term+RhoGAP-mApple-GBP (YEF10422), Ecm25-Δ(xACT)-mApple-GBP (YEF10423), or Ecm25 FL-mApple-GBP (YEF9781) were grown exponentially in SC medium containing 100 mM HU at 25°C for ~16 h and then harvested for morphological documentation by bright-field and fluorescence microscopy (G). Some of the Ecm25-mApple-GBP molecules, regardless of the truncations, seemed to localize to vacuole-like structures under this condition. Scale bar, 2 μm. Data from (G) were used for the quantitative analysis of the bud length/width ratios (H). Data were expressed as mean value ± SD. **p < 0.001; ns, not significant.

Figure 5.. Ecm25 binds to actin filaments in budding yeast, fission yeast, and mammalian cells via the xACT motif

(A) Ecm25 binds F-actin via its C terminus (in 3 independent experiments). Top: depiction of the Ecm25 fragments and their binding capacities to F-actin. Bottom: the positive control α-actinin, the negative control BSA, and MBP fusions of the FL and different fragments of Ecm25 were used for the F-actin pelleting assay using the Non-Muscle Actin Binding Protein Spin-Down Biochem Kit. (B) The xACT motif in the C terminus of Ecm25 localizes mainly to actin cables and occasionally to actin patches. Plasmids pUG36-GFP-Ecm25(361–530aa) and pUG36-GFP-Ecm25-xACT(536–588aa) were transformed into YEF7788 (ecm25Δ::HIS3 ABP1-tagRED-Kan) to generate strains YEF7815 and YEF7854, respectively, which were then imaged by spinning-disk confocal microscopy. Cells of YEF7854 were also stained by Alexa Fluor 568 (red)-conjugated phalloidin to visualize co-localization of the Ecm25-xACT fragment and F-actin (center panel). Scale bar, 2 μm. (C) Ecm25 binds to F-actin via its xACT motif. MBP-Ecm25-xACT(536–588aa) was used in the in vitro F-actin-binding assay as described in (A). (D) Ecm25-xACT expressed in fission yeast labels actin patches, actin cables, and the actin ring. Cells of the fission yeast strain PT4374 expressing GFP-Ecm25-xACT from the thiamine-repressible promoter (Pnmt1; integrated in the chromosome) were grown in Edinburgh minimal medium (EMM) without thiamine at 25°C. Two aliquots of the culture were treated with 1% DMSO (control) and 200 μM LatA, respectively, for 15 min and then stained for F-actin. Scale bar, 5 μm. (E) Ecm25-xACT expressed in mammalian cells labels actin filaments. HeLa-Kyoto cells expressing GFP-Ecm25-xACT from the pCMV promoter carried on a plasmid were cultured in DMEM and then treated with 0.05% DMSO (control) or 10 μM LatA for 30 min. The treated cells were then stained for F-actin. Scale bar, 10 μm.

Figure 6.. Ecm25 interacts directly with the actin-patch components Sla1, Lsb3, and Ysc84

(A) Schematics showing the domains, motifs, and/or binding partners of Ecm25, Sla1(1–420aa), Lsb3, and Ysc84. For Ecm25: CRAL/Sec14, lipid binding domain; RhoGAP*, inactive (marked by an asterisk) Rho GAP domain; m1–m3, mutations introduced at each of the three PXXP motifs; xACT, F-actin-binding motif. For Sla1, Lsb3, and Ysc84: SH3, Src homology-3; W41S and W108S, mutations introduced at the first and second SH3 domains of Sla1, respectively. (B) Ecm25 interacts with Sla1, Lsb3, and Ysc84 via its C terminus (in 2 independent experiments). MBP fusions of the indicated Ecm25 fragments were examined for interactions with GST fusions of Sla1(1–420aa), Lsb3, and Ysc84 in vitro using the GST pull-down assay. (C) Ecm25 interacts with the first two SH3 domains of Sla1 via a PXXP motif-containing region (in 3 independent experiments). MBP fusions of the indicated Ecm25 fragments were examined for interactions with GST fusions of the WT and SH3-domain variants of Sla1(1–420aa). (D) Ecm25 interacts with the SH3 domains of Sla1 and the paralogs Lsb3 and Ysc84 via distinct PXXP motifs (in 3 independent experiments). MBP fusions of the WT and PXXP-motif variants (m1, m2, or m3) of the Ecm25(361–535aa) fragment were examined for interactions with GST fusions of Sla1(1–420aa), Lsb3, and Ysc84. See also Figure S6.

Figure 7.. Ecm25 does not play a major role in fluid-phase endocytosis and CME

(A) Deletion of ECM25 does not change the number of actin patches per cell in the presence or absence of HU. WT (YEF7756) and ecm25Δ (YEF7788) cells were cultured in SC medium at 25°C in the absence or presence of HU (100 mM for 8 h), and then the number of actin patches in large-budded cells was counted for each strain under each growth condition. Numbers of cells counted: WT without HU, n = 14; ecm25Δ without HU, n = 15; WT with HU, n = 12; ecm25Δ with HU, n = 15. (B) Deletion of ECM25 does not overtly affect actin patch behavior. The growth conditions were the same as described in (A), except that the actin patches were imaged with a 1-s interval. Numbers of patches quantified: WT without HU, n = 23; ecm25Δ without HU, n = 25; WT with HU, n = 40; ecm25Δ with HU, n = 42. Data are expressed as mean value ± SD. **p = 0.0011, *p = 0.0127. (C) Deletion of ECM25 causes only a mild defect in fluid-phase endocytosis. Cells of the indicated strains were grown in the presence or absence of 100 mM HU and then processed for the LY assay. Scale bar, 2 μm. (D) Quantitative analysis of data from (C). Cell numbers analyzed for each strain and each condition were as follows: WT (BY4741) without HU, n = 116; WT (BY4741) with HU, n = 227; ecm25Δ (YEF7956) without HU, n = 170; ecm25Δ (YEF7956) with HU, n = 220; ysc84Δ (YEF8181) without HU, n = 133; ysc84Δ (YEF8181) with HU, n = 172; lsb3Δ (YEF8184) without HU, n = 120; lsb3Δ (YEF8184) with HU, n = 114; sla1Δ (YEF8187) without HU, n = 102; sla1Δ (YEF8187) with HU, n = 208; ysc84Δ lsb3Δ sla1Δ (YEF8978) without HU, n = 120; ysc84Δ lsb3Δ sla1Δ (YEF8978) with HU, n = 186. (E) Ecm25 plays no apparent role in CME. Cells of the indicated strains were grown in the presence or absence of 100 mM HU and then documented by fluorescence microscopy. Scale bar, 2 μm. (F) Quantitative analysis of data from (E). Cell numbers analyzed for each strain and each condition were as follows: WT (YEF9049) without HU, n = 129; WT (YEF9049) with HU, n = 150; ecm25Δ (YEF9010) without HU, n = 171; ecm25Δ (YEF9010) with HU, n = 150; ysc84Δ (YEF9050) without HU, n = 136; ysc84Δ (YEF9050) with HU, n = 131; lsb3Δ (YEF9051) without HU, n = 155; lsb3Δ (YEF9051) with HU, n = 135; sla1Δ (YEF9052) without HU, n = 147; sla1Δ (YEF9052) with HU, n = 136; ysc84Δ lsb3Δ sla1Δ (YEF9053) without HU, n = 148; ysc84Δ lsb3Δ sla1Δ (YEF9053) with HU, n = 123. (G) Different actin patch mutants display differential defects in HU-induced cell elongation. Cells of the strains WT (BY4741), ecm25Δ (YEF7956), ysc84Δ (YEF8181), lsb3Δ (YEF8184), sla1Δ (YEF8187), and ysc84Δ lsb3Δ sla1Δ (YEF8978) were grown in the presence or absence of 100 mM HU at 25°C for ~13 h before morphology documentation by DIC microscopy. Scale bar, 2 μm. (H) Quantitative analysis of the bud length/width ratios for data shown in (G). Cell numbers analyzed for each strain and each condition were as follows: WT without HU, n = 67; WT with HU, n = 81; ecm25Δ without HU, n = 105; ecm25Δ with HU, n = 93; ysc84Δ without HU, n = 84; ysc84Δ with HU, n = 86; lsb3Δ without HU, n = 67; lsb3Δ with HU, n = 87; sla1Δ without HU, n = 80; sla1Δ with HU, n = 80; ysc84Δ lsb3Δ sla1Δ without HU, n = 91; ysc84Δ lsb3Δ sla1Δ with HU, n = 82. Data are expressed as mean value ± SD. **p < 0.001, *p = 0.005.