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3.6.1.1 The receptor-like kinase FLS2 localizes to punctate membrane domains.

The introduction of hydrodynamic friction by transmembrane proteins is one hallmark of membrane sub-compartmentalization (Kusumi et al., 2012b). Hydrophobic mismatch effects and the specific miscibility characteristics of TMDs generate small areas of non-random membrane component distribution (see 2.1.1.4.1) (Marsh and Watts, 1982; Schwille et al., 1999; Lee, 2003;

Soubias and Gawrisch, 2013). Sub-compartmentalization of transmembrane proteins has been demonstrated for LINKER FOR ACTIVATION OF T-CELLs (LAT), a scaffold protein important during T-CELL RECEPTOR (TCR) mediated T-cell immune responses (Zhang et al., 1999; Sommers; Connie et al., 2004). Their composition as well as their important role during surface signal perception makes plant RLKs important candidates to study membrane sub-organization.

RLKs often interact with multiple proteins upon signal perception thereby inducing another level of membrane sub-organization. The perception of the bacterial flagellum and its elicitor epitope flg22, is one of the best characterized perception systems of pathogen-associated molecular patterns (PAMPs) (Boller and Felix, 2009). The responsible RLK recognizing the flg22 epitope, FLAGELLIN SENSITIVE 2 (FLS2), undergoes a ligand-induced complex formation with its co-receptor BAK1, which is accompanied by trans-and autophosphorylation events and ultimately the endocytosis of FLS2 (Robatzek et al., 2006; Boller and Felix, 2009). Conditional localization to raft domains was suggested for FLS2, since it co-purified with DIM fractions in a comparative proteomic study between flg22 treated and untreated A. thaliana suspension cells (Keinath et al., 2010). As discussed above, DIM fractions are not a biochemical counterpart of any membrane structure present in living cells and claims of membrane raft localization based on DIM fractionations are insufficient (see 2.2.1). Nevertheless, FRAP experiments on A. thaliana protoplasts demonstrate that lateral mobility of FLS2 decreases in the presence of its ligand indicating that the lateral organization of the protein switches upon signal perception (Ali et al., 2007). Through a collaboration with the laboratory of Dr. Cyril Zipfel, the localization of FLS2 to membrane microdomains was demonstrated in A. thaliana using TIRF microscopy. To investigate, whether the same observations can be made with our experimental setup, the subcellular localization of this key immune receptor was investigated.

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In order to investigate the putative compartmentalization of FLS2, we transiently expressed FLS2-GFP fusion constructs under expression control of the native FLS2 promoter sequence in Nicotiana benthamiana leaf epidermal cells and analysed the subcellular localization microscopically. Three days after infiltration, strong GFP fluorescence could be observed at the cell periphery confirming the PM localization of FLS2 as seen in other studies (Robatzek et al., 2006; Beck et al., 2012). Higher magnification of the upper cell plane revealed sparse but distinct domains of high fluorescence signal, surrounded by equally distributed weaker fluorescence (Figure 5). Since FLS2 undergoes endocytosis, it may be possible that the observed FLS2 membrane domains represent initiation sites of endocytosis (Robatzek et al., 2006; Beck et al., 2012). However, these structures are known to be laterally stable, whereas the observed structures in this experiments displayed a notable lateral mobility. To further describe the FLS2 labelled membrane domains, a quantitative analysis of 21 different cell surface views was conducted. FLS2 labelled membrane domains covered a mean area of 0.177 µm² (± 0.017 µm²), displayed a mean intensity value of 100 (± 7) and a circularity value of 0.8 (±0.04) in which 1 equals a perfect circle (Figure 5). FLS2 membrane domains exhibited a domain density of 0.06 domains/µm² (± 0.001 domains/µm²) making them significantly less abundant than any Remorin labelled membrane domain, or the membrane domains of FLOT1A and FLOT1B (Jarsch et al., 2014).

Figure 5: Membrane domain localization of FLS2-GFP in N. benthamiana leaf epidermal cells.

(A) Confocal image of the upper PM plane of N. benthamiana leaf epidermal cell expressing FLS2:GFP under endogenous promoter control. Sparse but distinct bright accumulations of fluorescence signal indicate the proteins localize to membrane domains (arows). (B) Segmentation of ‘A’ to quantify membrane domains labeled by FLS2:GFP.

(C) A quantitative analysis of 21 different cells enabled us to describe the domains with the parameters given below.

Scale bar indicates 5 µm

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173 3.6.1.2 Relocalization of membrane proteins to non-native membrane domains

One way to show the biological role of punctate membrane domains would be by altering a protein’s membrane domain localization, without the removal of the protein from the PM.

Mislocalization could be achieved via mutation of known membrane localization motifs or by forced interactions with proteins that localize to different membrane domains. Forced interaction can be conferred by antibodies derived from members of the Camelidae family. They differ from conventional mammalian antibodies because they lack light chain subunits. Therefore, these

“nanobodies” are structurally less complex than conventional antibodies, facilitating their heterologous expression in diverse cell types. A special GFP-binding nanobody (GBP), suitable for expression and localization experiments in vivo has been developed (Rothbauer et al., 2006) that enables these antibodies to be used in a variety of biotechnological approaches (Muyldermans, 2001; Conrad and Sonnewald, 2003).

To investigate the effects of membrane domain mislocalization, FLS2 was chosen as a target protein.

We generated GBP:RFP fusions of Remorin proteins At3g57540 and At4g36970 and transiently expressed these constructs in N. benthamiana leaf epidermal cells. Overall cells varied in expression strength. In highly expressing cells domain localization was not clear. This observation is due to the reported accumulation of protein that is not bound to a domain within the inter-domain space (Otto and Nichols, 2011; Jarsch et al., 2014). For this reason, only weak expressing cells were considered. In those cells, fluorescence signals derived from the two GBP:RFP:Remorin constructs

Figure 6: Localization of GBP:RFP labelled Remorins At3g57540 and At4g36970.

Confocal microscopy of GBP:RFP labelled Remorins At3g57540 and At4g36970 expressed under constitutive expression in N. benthamiana. At3g57540 (A) as well as At4g36970 (B) labelled distinct membrane domains in the upper PM plane of leaf epidermal cells. Images of at least 17 individual cells were used to quantitatively describe these domains (C). Scale bars indicate 5 µm

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were observed in distinct membrane domains (Figure 6 A & B). The domains displayed no notable lateral mobility, but were stable over 1 h.

Even though the added GBP tag is small compared to conventional antibodies, it adds a considerable size to the fusion protein. Moreover, the binding capacity of GBP might interfere with native localization to membrane domains of Remorin proteins. Therefore, 17 cells expressing GBP:RFP:At3g57540 and 19 cells expressing GBP:RFP:At4g36970 were used for quantification of the domain parameters size, intensity, circularity and density. As Figure 6 C shows, for both constructs, all values except for the mean intensity value differed significantly from the published domain parameters. The increase of domain size to 0.265 µm² (±0.023 µm²) for At3g57540 and 0.307 µm² (±0.026 µm²) for AtRem 6.4 as well as the increase of domain density to 4.10 domains/µm² (±0.66 domains/µm²) for At3g57540 and 1.96 domains/µm² (±0.29 domains/µm²)

Figure 7: Relocalization of FLS2:GFP due to co-expression with GBP:RFP labelled Remorins.

Co-localization analysis of cells co-expressing FLS2:GFP and either GBP:RFP:At3g57540 (A) or GBP:RFP:At4g36970 (D). Of every image, only areas showing signal (indicated by white masks) were considered for the calculation of Pearson’s correlation coefficient (Rr) and the squared overlap coefficient (R²). Likewise, the correlation coefficients were calculated on a simulated randomized image samples that were generated by flipping the image of RFP fluorescence horizontally (B & D). The correlation coefficients are indicated in C and F. Scale bars indicate 5 µm. ***=

students t-test p-value> 0.001.

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175 for At4g36970 suggest that the protein abundance is higher and domain localization might be altered due to overexpression of the protein.

To test whether the GBP:RFP:Remorin constructs are able to successfully bind and mislocalize FLS2:GFP, we co-expressed FLS2:GFP either with GBPRFP:At3g57540 or GBP:RFP:At4g36970 in N. benthamiana leaf epidermal cells. The localization pattern was investigated microscopically (Figure 7A and D). To quantify the co-localization coefficients, at least 8 images were acquired.

Similar to Jarsch et al. 2014, the Pearson correlation coefficient Rr (Manders et al., 1993) and the squared overlap coefficient R² (Manders et al., 1993) were calculated (Figure 7 C and F). The strength of calculating correlations using the Pearson correlation coefficient is its insensitivity to background noise (Adler and Parmryd, 2014). Its values theoretically range from -1 to 1 where 1 describes perfect co-localization and -1 exclusion of signal (Manders et al., 1993). However, negative and low Rr values are difficult to interpret and often vary significantly because of intensity differences in the two observed channels (Zinchuk et al., 2007). The squared overlap coefficient R² varies between 0 and 1, where 1 is defined as a perfect co-localization event. It is less prone to calculation artifacts due to intensity variations, but therefore also considerably error prone to image noise (Adler and Parmryd, 2014). Since R² is also strongly influenced by the amount of pixels considered in both channels, the pixel ratio from both channels was calculated (N channel 1/N channel 2).

Only images with a ratio, close to 1 were considered. To simulate a random distribution of proteins, one channel of each image pair was flipped horizontally and the co-localization of signal containing regions of interest in this new “simulated randomized” image pair was calculated (Figure 7 B and E). In case of the co-expression of FLS2-GFP and GBP:RFP:At3g57540, fluorescence signal of both fluorophores showed a dense, irregular distribution over the whole membrane that was interrupted by filamentous structures (Figure 7 A ). The high Pearson correlation coefficient of 0.757 and equally high Manders correlation coefficient of 0.943 (Figure 7 C) demonstrate the almost perfect localization of the two fluorescence signals. The fluorescence pattern of the co-localization of FLS2:GFP and GBP:RFP:At4g36970 exhibited a more scattered, network-like structure, that was disrupted by filamentous structures (Figure 7 D). Again, the Pearson and Manders correlation coefficients yield high values of 0.796 and 0.936 respectively, demonstrating that the GBP binding tag is able to actively alter the localization of FLS2-GFP in N. benthamiana.

To summarize, these experiments show that co-expression of GBP:RFP tagged Remorins and FLS2:GFP induces significant alterations of both proteins lateral distribution within the PM. With further improvement of experimental conditions, the introduced system is a promising tool to investigate the role of FLS2 localization to membrane domains.

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