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Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains

View Article: PubMed Central - PubMed

ABSTRACT

Cell surface receptors govern a multitude of signalling pathways in multicellular organisms. In plants, prominent examples are the receptor kinases FLS2 and BRI1, which activate immunity and steroid-mediated growth, respectively. Intriguingly, despite inducing distinct signalling outputs, both receptors employ common downstream signalling components, which exist in plasma membrane (PM)-localised protein complexes. An important question is thus how these receptor complexes maintain signalling specificity. Live-cell imaging revealed that FLS2 and BRI1 form PM nanoclusters. Using single-particle tracking we could discriminate both cluster populations and we observed spatiotemporal separation between immune and growth signalling platforms. This finding was confirmed by visualising FLS2 and BRI1 within distinct PM nanodomains marked by specific remorin proteins and differential co-localisation with the cytoskeleton. Our results thus suggest that signalling specificity between these pathways may be explained by the spatial separation of FLS2 and BRI1 with their associated signalling components within dedicated PM nanodomains.

Doi:: http://dx.doi.org/10.7554/eLife.25114.001

No MeSH data available.


The stability of FLS2 clusters depends on the PM lipid composition.(A, B) VAEM micrograph of FLS2-GFP (A) and a kymograph of the corresponding VAEM time series (B) after mock treatment. (C, D) VAEM micrograph of BRI1-GFP (C) and a kymograph of the corresponding VAEM time series (D) after mock treatment. (E, F) VAEM micrograph of FLS2-GFP (E) and a kymograph of the corresponding VAEM time series (F) after methyl-β-cyclodextrin (MβCD) treatment. (G, H) VAEM micrograph of BRI1-GFP (G) and a kymograph of the corresponding VAEM time series (H) after MβCD treatment. Application of MβCD, which extracts sterols from the PM, destabilised FLS2-GFP and BRI1-GFP clusters as indicated by the kymographs in (F) and (H). The VAEM micrograph and time series was acquired from a 5 day old Arabidopsis seedling cotyledon after 20 min incubation in 30 mM MβCD, dissolved in liquid MS medium, or mock solution (liquid MS medium). The white arrows in (A), (C), (E) and (G) indicate the spatial dimensions of the kymographs. The scale bars represent 5 µm.DOI:http://dx.doi.org/10.7554/eLife.25114.007
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fig2s1: The stability of FLS2 clusters depends on the PM lipid composition.(A, B) VAEM micrograph of FLS2-GFP (A) and a kymograph of the corresponding VAEM time series (B) after mock treatment. (C, D) VAEM micrograph of BRI1-GFP (C) and a kymograph of the corresponding VAEM time series (D) after mock treatment. (E, F) VAEM micrograph of FLS2-GFP (E) and a kymograph of the corresponding VAEM time series (F) after methyl-β-cyclodextrin (MβCD) treatment. (G, H) VAEM micrograph of BRI1-GFP (G) and a kymograph of the corresponding VAEM time series (H) after MβCD treatment. Application of MβCD, which extracts sterols from the PM, destabilised FLS2-GFP and BRI1-GFP clusters as indicated by the kymographs in (F) and (H). The VAEM micrograph and time series was acquired from a 5 day old Arabidopsis seedling cotyledon after 20 min incubation in 30 mM MβCD, dissolved in liquid MS medium, or mock solution (liquid MS medium). The white arrows in (A), (C), (E) and (G) indicate the spatial dimensions of the kymographs. The scale bars represent 5 µm.DOI:http://dx.doi.org/10.7554/eLife.25114.007

Mentions: Formation of PM receptor clusters is a well-known phenomenon for mammalian receptors like T-cell receptors (TCRs) or EGFR (Dinic et al., 2015; Gao et al., 2015; Su et al., 2016). The RTK EGFR constitutively undergoes clustering and EGFR clusters play important roles in cell signalling (Clayton et al., 2007; Gao et al., 2015; Paviolo et al., 2015). Using super-resolution microscopy, Gao et al. (2015) showed that EGFR clusters localise to PM nanodomains and that cluster formation relies on the integrity of the PM lipidome since sterol depletion of the PM using methyl-β-cyclodextrin (MβCD) disrupts clustering. Similar observations have recently been made for BRI1 in Arabidopsis roots (Wang et al., 2015). Wang et al. (2015) observed that BRI1 localises to FLOT1- and clathrin heavy chain (CHC)-labelled PM nanodomains and that partitioning of BRI1 into PM nanodomains as well as the PM lipidome are crucial for BR responses. These findings are in line with our observations for FLS2 and BRI1 in epidermal leaf cells since MβCD treatment also affected FLS2 clusters (Figure 2—figure supplement 1). Thus, both LRR-RKs constitutively formed PM receptor clusters that were influenced by the PM lipid-composition and localised to PM nanodomains labelled by distinct REM protein markers. However, the differential co-localisation to specific PM nanodomains allowed us to discriminate FLS2 and BRI1 receptor clusters, thus indicating their spatial separation within the PM.


Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains
The stability of FLS2 clusters depends on the PM lipid composition.(A, B) VAEM micrograph of FLS2-GFP (A) and a kymograph of the corresponding VAEM time series (B) after mock treatment. (C, D) VAEM micrograph of BRI1-GFP (C) and a kymograph of the corresponding VAEM time series (D) after mock treatment. (E, F) VAEM micrograph of FLS2-GFP (E) and a kymograph of the corresponding VAEM time series (F) after methyl-β-cyclodextrin (MβCD) treatment. (G, H) VAEM micrograph of BRI1-GFP (G) and a kymograph of the corresponding VAEM time series (H) after MβCD treatment. Application of MβCD, which extracts sterols from the PM, destabilised FLS2-GFP and BRI1-GFP clusters as indicated by the kymographs in (F) and (H). The VAEM micrograph and time series was acquired from a 5 day old Arabidopsis seedling cotyledon after 20 min incubation in 30 mM MβCD, dissolved in liquid MS medium, or mock solution (liquid MS medium). The white arrows in (A), (C), (E) and (G) indicate the spatial dimensions of the kymographs. The scale bars represent 5 µm.DOI:http://dx.doi.org/10.7554/eLife.25114.007
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fig2s1: The stability of FLS2 clusters depends on the PM lipid composition.(A, B) VAEM micrograph of FLS2-GFP (A) and a kymograph of the corresponding VAEM time series (B) after mock treatment. (C, D) VAEM micrograph of BRI1-GFP (C) and a kymograph of the corresponding VAEM time series (D) after mock treatment. (E, F) VAEM micrograph of FLS2-GFP (E) and a kymograph of the corresponding VAEM time series (F) after methyl-β-cyclodextrin (MβCD) treatment. (G, H) VAEM micrograph of BRI1-GFP (G) and a kymograph of the corresponding VAEM time series (H) after MβCD treatment. Application of MβCD, which extracts sterols from the PM, destabilised FLS2-GFP and BRI1-GFP clusters as indicated by the kymographs in (F) and (H). The VAEM micrograph and time series was acquired from a 5 day old Arabidopsis seedling cotyledon after 20 min incubation in 30 mM MβCD, dissolved in liquid MS medium, or mock solution (liquid MS medium). The white arrows in (A), (C), (E) and (G) indicate the spatial dimensions of the kymographs. The scale bars represent 5 µm.DOI:http://dx.doi.org/10.7554/eLife.25114.007
Mentions: Formation of PM receptor clusters is a well-known phenomenon for mammalian receptors like T-cell receptors (TCRs) or EGFR (Dinic et al., 2015; Gao et al., 2015; Su et al., 2016). The RTK EGFR constitutively undergoes clustering and EGFR clusters play important roles in cell signalling (Clayton et al., 2007; Gao et al., 2015; Paviolo et al., 2015). Using super-resolution microscopy, Gao et al. (2015) showed that EGFR clusters localise to PM nanodomains and that cluster formation relies on the integrity of the PM lipidome since sterol depletion of the PM using methyl-β-cyclodextrin (MβCD) disrupts clustering. Similar observations have recently been made for BRI1 in Arabidopsis roots (Wang et al., 2015). Wang et al. (2015) observed that BRI1 localises to FLOT1- and clathrin heavy chain (CHC)-labelled PM nanodomains and that partitioning of BRI1 into PM nanodomains as well as the PM lipidome are crucial for BR responses. These findings are in line with our observations for FLS2 and BRI1 in epidermal leaf cells since MβCD treatment also affected FLS2 clusters (Figure 2—figure supplement 1). Thus, both LRR-RKs constitutively formed PM receptor clusters that were influenced by the PM lipid-composition and localised to PM nanodomains labelled by distinct REM protein markers. However, the differential co-localisation to specific PM nanodomains allowed us to discriminate FLS2 and BRI1 receptor clusters, thus indicating their spatial separation within the PM.

View Article: PubMed Central - PubMed

ABSTRACT

Cell surface receptors govern a multitude of signalling pathways in multicellular organisms. In plants, prominent examples are the receptor kinases FLS2 and BRI1, which activate immunity and steroid-mediated growth, respectively. Intriguingly, despite inducing distinct signalling outputs, both receptors employ common downstream signalling components, which exist in plasma membrane (PM)-localised protein complexes. An important question is thus how these receptor complexes maintain signalling specificity. Live-cell imaging revealed that FLS2 and BRI1 form PM nanoclusters. Using single-particle tracking we could discriminate both cluster populations and we observed spatiotemporal separation between immune and growth signalling platforms. This finding was confirmed by visualising FLS2 and BRI1 within distinct PM nanodomains marked by specific remorin proteins and differential co-localisation with the cytoskeleton. Our results thus suggest that signalling specificity between these pathways may be explained by the spatial separation of FLS2 and BRI1 with their associated signalling components within dedicated PM nanodomains.

Doi:: http://dx.doi.org/10.7554/eLife.25114.001

No MeSH data available.