Author response: Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains (2017)

Article Figures and data Abstract eLife digest Introduction Results Discussion Methods and materials References Decision letter Author response Article and author information Metrics 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. https://doi.org/10.7554/eLife.25114.001 eLife digest Unlike most animals, plants cannot move away if their environment changes for the worse. Instead, a plant must sense these changes and respond appropriately, for example by changing how much it grows. Disease-causing microbes in the immediate environment represent another potential threat to plants. To detect these microbes, plant cells have proteins called “pattern recognition receptors” in their surface membranes that sense certain molecules from the microbes (similar receptors are found in animals too). When a receptor protein recognises one such microbial molecule, it becomes activated and forms a complex with other proteins referred to as co-receptors. The protein complex then sends a signal into the cell to trigger an immune response. Plants also use similar receptor proteins to sense their own signalling molecules and regulate their growth and development. These growth-related receptors rely on many of the same co-receptors and signalling components as the immunity-related receptors. This posed the question: how can plant cells use the same proteins to trigger different responses to different signals? Bücherl et al. have now used high-resolution microscopy and the model plant Arabidopsis thaliana to show that the plant’s immune receptors and growth receptors are found in separate clusters at the plant cell’s surface membrane. These clusters are only a few hundred nanometres wide, and they also contained other signalling components that are needed to quickly relay the signals into the plant cell. Bücherl et al. suggest that, by organizing their receptors into these physically distinct clusters, plant cells can use similar proteins to sense different signals and respond in then different ways. This idea will need to be tested in future studies. Further work is also needed to understand how these clusters of signalling proteins are assembled and inserted at specific locations within the surface membrane of a plant cell. https://doi.org/10.7554/eLife.25114.002 Introduction Multicellular organisms employ cell-surface receptors for surveying the environment and adjusting to changing physiological conditions. In plants, the repertoire of cell surface receptors has been considerably expanded and receptor kinases (RKs) form one of the largest protein families with over 600 members in Arabidopsis thaliana (hereafter, Arabidopsis) (Shiu and Bleecker, 2001). The schematic architecture of plant RKs is similar to that of animal receptor tyrosine kinases (RTKs); comprising an extracellular ligand binding domain, a single transmembrane helix, and an intracellular kinase domain (Shiu and Bleecker, 2001). Prominent examples of plant RKs are the immune receptor FLAGELLIN SENSING 2 (FLS2) (Gómez-Gómez and Boller, 2000) and the growth receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) (Clouse et al., 1996; Li and Chory, 1997). FLS2 is a pattern recognition receptor (PRR) that perceives the pathogen-associated molecular pattern (PAMP) flg22, an immunogenic epitope of bacterial flagellin, to initiate PAMP-triggered immunity (PTI) (Felix et al., 1999; Zipfel et al., 2004; Chinchilla et al., 2006; Boller and Felix, 2009). BRI1 binds brassinosteroids (BRs), a class of phytohormones involved in various aspects of plant growth and development (Kinoshita et al., 2005; Kim and Wang, 2010; Singh and Savaldi-Goldstein, 2015). Despite their different biological functions, FLS2- and BRI1-mediated signalling pathways share several similarities, in particular at or close to the plasma membrane (PM). The PM is the cellular compartment, where both receptors localise to (Robatzek et al., 2006; Friedrichsen et al., 2000), where they bind their respective ligands flg22 or BRs (Gómez-Gómez et al., 2001; Bauer et al., 2001; Kinoshita et al., 2005), and where presumably their main signalling activity is executed (Smith et al., 2014; Irani et al., 2012). Although FLS2 and BRI1 are competent for ligand binding via their extracellular leucine-rich repeat (LRR) domains, they rely on SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) co-receptors for signalling initiation (Nam and Li, 2002; Li et al., 2002; Chinchilla et al., 2007; Heese et al., 2007; Roux et al., 2011; Gou et al., 2012), which are also LRR-RKs (Aan den Toorn et al., 2015). Structural and biochemical analysis of FLS2- and BRI1-SERK hetero-oligomers revealed that flg22 and BRs act as ‘molecular glues’ that stabilise or induce receptor complexes (Sun et al., 2013; She et al., 2011; Hothorn et al., 2011). Ligand binding additionally triggers auto- and trans-phosphorylation events within the receptor complexes (Schulze et al., 2010; Wang et al., 2008) and, in the case of BRI1, also the release of inhibitory mechanisms (Wang and Chory, 2006; Jaillais et al., 2011). After gaining their full kinase activities, FLS2 and BRI1 receptor complexes initiate phosphorylation cascades that culminate in flg22- or BR-responsive transcriptional regulation (Guo et al., 2013; Li et al., 2016). The relay of phosphorylation signals from the PM to the nucleus involves receptor-like cytoplasmic kinases (RLCKs) that can associate to the PM and that are direct substrates of the ligand-binding receptor complexes (Lin et al., 2013; Belkhadir and Jaillais, 2015; Couto and Zipfel, 2016). Similar to the SERK co-receptors, the RLCKs BRASSINOSTEROID SIGNALING KINASE 1 (BSK1) and BOTRYTIS-INDUCED KINASE 1 (BIK1) are common signalling components in both pathways. Whereas BSK1 is a positive regulator for both signalling routes (Tang et al., 2008; Shi et al., 2013), BIK1 is a positive regulator for PTI responses (Lu et al., 2010; Zhang et al., 2010), but a negative regulator for BR signalling (Lin et al., 2013). Even though FLS2- and BRI1-mediated signalling pathways have been extensively studied genetically and biochemically, little is known about how FLS2 and BRI1 are organised within the PM and how these two receptors fulfil their sensory activity at the cell periphery. In contrast to the original fluid mosaic model (Singer and Nicolson, 1972), which considered the PM as a two-dimensional liquid composed of a lipid bilayer that is interspersed by integral or associated proteins, it is nowadays accepted that the PM is a highly structured and dynamic cellular compartment organised at three hierarchic levels (Kusumi et al., 2011; Nicolson, 2014). The first level of PM organisation is characterised by the interaction of the lipid bilayer with the underlying cortical cytoskeleton, the second level by protein-lipid interactions with the PM, and the third level of PM organisation is the result of protein-protein interactions that lead to formation of PM-associated or -integral homo- and hetero-oligomers (Kusumi et al., 2011), e.g. FLS2- or BRI1-SERK3/BAK1 complexes. In plants, the cell wall has additional influence on the PM organisation and dynamics (Martinière et al., 2012). As a consequence, lateral mobility and distribution of lipids and proteins within the PM is highly heterogeneous leading to the formation of dynamic protein clusters and PM sub-compartments with different shapes and sizes (Jaqaman and Grinstein, 2012; Jarsch et al., 2014; Ziomkiewicz et al., 2015). Each compartment or domain provides specific biophysical and biochemical environments for its residents and thus directly influences associated signalling activities (Kusumi et al., 2012; Saka et al., 2014; Garcia-Parajo et al., 2014; Tapken and Murphy, 2015). With regard to PM signalling, specialised PM areas often referred to as PM nanodomains have attracted particular attention. For example, EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR), a mammalian RTK, forms clusters that co-localise with PM nanodomains, and EGFR cluster formation depends on the integrity of the PM lipid composition (Gao et al., 2015). A recent report showed that BRI1 also localises to PM nano- or micro-domains in Arabidopsis roots and that partitioning of BRI1 into different PM microdomains is crucial for BR signalling (Wang et al., 2015). Due to the numerous similarities between BRI1 and FLS2 signalling initiation, we were interested in elucidating the localisation patterns of these two LRR-RKs in a comparative fashion within the highly structured PM of plant cells. Here, we investigated the steady-state PM organisation of FLS2 and BRI1 receptors using live-cell imaging, single-particle tracking and quantitative co-localisation analysis in the PM of leaf epidermal cells, a cell file, in which both receptors are similarly expressed. Our results showed that FLS2 and BRI1 are heterogeneously distributed and that both receptors form transient receptor clusters. Although the spatial characteristics of FLS2 and BRI1 receptor clusters were similar, we observed differences in their dynamic behaviour with FLS2 clusters being more stable. Moreover, we detected only a limited overlap between the two receptor populations. This finding was confirmed by visualising FLS2 and BRI1 clusters within distinct remorin-labelled PM nanodomains. We additionally investigated the PM localisation patterns of BSK1 and BIK1 in complex with the two ligand-binding LRR-RKs. Imaging of FLS2 and BRI1 signalling complexes revealed a confined PM localisation and cluster formation. Importantly, BIK1 signalling complexes localised differentially, whereby BRI1-BIK1, but not FLS2-BIK1, complexes associated with cortical microtubules. Together, our data suggest that the distinct spatiotemporal localisation of FLS2 and BRI1 within specialised PM nanodomains may contribute to signalling specificity between immune and growth signalling mediated by these receptors. Results FLS2 and BRI1 form receptor clusters within the PM To obtain an overview of the PM distribution of FLS2 and BRI1, we investigated two stable transgenic Arabidopsis lines that express the C-terminally GFP-tagged receptors under their native promoters (Göhre et al., 2008; Geldner et al., 2007). Live-cell imaging using confocal laser scanning microscopy (CLSM) revealed that FLS2 and BRI1 were heterogeneously distributed within the PM (Figure 1A and B). Both receptors formed dispersed punctate structures with increased fluorescence intensities. This local concentration of FLS2 and BRI1 subpopulations within the PM indicates the formation of receptor clusters, a phenomenon known from mammalian transmembrane receptors or helper proteins, such as EGFR (Clayton et al., 2007) or LINKER OF ACTIVATED T CELLS (LAT) (Su et al., 2016). To emphasis our observation of receptor clusters, we processed the presented images using a spot-enhancing filter described by Sage et al. (2005) and applied the ‘fire’ lookup table (Figure 1—figure supplement 1). Importantly, we observed similar cluster formation of FLS2 and BRI1 whether upon native expression in Arabidopsis or after transient expression in Nicotiana benthamiana (N. benthamiana) (Figure 1C and D). This feature enabled us to perform further co-localisation analysis in this heterologous system. Figure 1 with 2 supplements see all Download asset Open asset FLS2 and BRI1 form receptor clusters within the plasma membrane. (A, B) Plasma membrane localisation of FLS2-GFP (A) and BRI1-GFP (B) in epidermal cells of Arabidopsis seedling cotyledons. (C, D) Plasma membrane localisation of FLS2-GFP (C) and BRI1-GFP (D) after transient expression in epidermal leaf cells of N. benthamiana. (E) Quantification of FLS2-GFP and BRI1-GFP plasma membrane receptor cluster diameters in epidermal cells of Arabidopsis cotyledons and after transient expression in N. benthamiana leaves. The coloured data points represent the technical replicates of 3 independent experiments. No statistical differences were observed based on two-tailed heteroscedastic t-tests and a Bonferroni multiple hypothesis correction. (F) Quantification of FLS2-GFP and BRI1-GFP plasma membrane receptor cluster densities in epidermal cells of Arabidopsis seedlings and after heterologous expression in N. benthamiana. The coloured data points represent the technical replicates of 3 independent experiments. No statistical differences were observed based on two-tailed heteroscedastic t-tests and a Bonferroni multiple hypothesis correction. The presented images were acquired using confocal laser scanning microscopy (CLSM). Scale bars represent 5 µm. The colour bar represents the colour code for fluorescence intensities. Black dots represent outliers. Red arrowheads indicate endosomal compartments of BRI1-GFP. Endosomal compartments were omitted for quantitative analysis. https://doi.org/10.7554/eLife.25114.003 Besides residing within the PM, BRI1-GFP additionally localised to mobile endomembrane compartments with high fluorescence intensities (highlighted by red arrowheads in Figure 1B and D). These intracellular compartments most likely resembled trans-Golgi network/early endosomes and late endosomes/multi-vesicular bodies (Geldner et al., 2007; Irani et al., 2012). In contrast to the highly mobile endomembrane compartments, the visualised FLS2- and BRI1-GFP clusters appeared rather immobile and seemed to follow a certain spatial organisation. FLS2 and BRI1 clusters often aligned in pearl chain-like structures and interconnected lines with low fluorescence intensities frequently separated by PM areas containing several receptor clusters, in particular for FLS2-GFP (Figure 1—figure supplement 2). It is known that the cytoskeleton influences the hierarchic PM organisation (Plowman et al., 2005; Kusumi et al., 2011) and therefore we visualised FLS2 and BRI1 in the presence of actin and microtubule markers. As shown in Figure 1—figure supplement 2, pearl chain-like aligned FLS2 and BRI1 clusters often coincided with the underlying actin cytoskeleton, whereas the interconnected lines with low fluorescence intensities overlapped with cortical microtubule filaments, both in Arabidopsis and N. benthamiana. Next, we quantified the size and density of individual receptor clusters. The mean values (± standard deviation) for FLS2 receptor cluster diameters were 356 ± 49 nm and 387 ± 55 nm in Arabidopsis and N. benthamiana, respectively, compared to 372 ± 38 nm in Arabidopsis and 392 ± 37 nm in N. benthamiana for BRI1 (Figure 1E and Supplementary file 1). Although there was a slight trend towards increased receptor cluster sizes in N. benthamiana, no statistical difference was observed. On average, we observed 2.21 ± 0.33 FLS2 clusters in Arabidopsis and 2.23 ± 0.26 in N. benthamiana per µm2, whereas the densities of BRI1 clusters were 2.02 ± 0.27 and 2.43 ± 0.39 per µm2 in Arabidopsis and N. benthamiana, respectively (Figure 1F and Supplementary file 1). Thus, based on our confocal micrograph analysis, FLS2 and BRI1 showed similar spatial features. Both LRR-RKs were heterogeneously distributed within the PM and formed receptor clusters of comparable size and density. Only a difference with regard to the exclusion of receptor clusters from PM areas by cortical microtubules was observed, with FLS2 being more strongly affected. In addition to confocal microscopy, we applied variable angle epifluorescence microscopy (VAEM) to further characterise the dynamic behaviour of PM-localised FLS2 and BRI1 receptors. VAEM is a technique related to total internal reflection microscopy (TIRF) (Vizcay-Barrena et al., 2011). In comparison to CLSM, VAEM has the advantages of improved z-resolution and fast image acquisition (Vizcay-Barrena et al., 2011). Therefore VAEM is ideal for studying protein localisation and protein dynamics within or close to the PM (Wan et al., 2011; Vizcay-Barrena et al., 2011). As shown in Figure 2A–D, single frame VAEM images resembled our observations made using CLSM (Figure 1A–D). We repeatedly observed dispersed FLS2- and BRI1-GFP clusters in the PM of Arabidopsis or N. benthamiana leaf epidermal cells. However, using VAEM we were able to acquire image time series (Video 1 and 2). Micrograph analysis using kymograph representations revealed increased stability and reduced mobility for FLS2-GFP compared to BRI1-GFP clusters, both in Arabidopsis and N. benthamiana (Figure 2E). These observations were confirmed using single-particle tracking. BRI1-GFP showed a trend towards increased lateral cluster displacement (Figure 2F) and FLS2-GFP showed an increased population of long-lived receptor clusters (Figure 2G). Additionally, we noticed that some FLS2 and BRI1 clusters suddenly appeared or disappeared from the PM. These observations most likely reflect constitutive exo- and endocytosis processes (Beck et al., 2012; Martins et al., 2015; Wang et al., 2015). Figure 2 with 1 supplement see all Download asset Open asset FLS2 receptor clusters are more stable than BRI1 clusters. (A, B) Plasma membrane localisation of FLS2-GFP (A) and BRI-GFP (B) in epidermal cells of Arabidopsis seedling cotyledons. (C, D) Plasma membrane localisation of FLS2-GFP (C) and BRI1-GFP (D) after transient expression in epidermal leaf cells of N. benthamiana. (E) Kymograph analysis of FLS2-GFP and BRI1-GFP plasma membrane receptor clusters in epidermal cells of Arabidopsis seedling cotyledons and after transient expression in N. benthamiana. Kymographs were obtained from VAEM time series with a temporal resolution of 0.5 s over 250 frames along the indicated arrows in micrographs (A) to (D). (F) Quantification of FLS2-GFP and BRI1-GFP receptor cluster displacements in epidermal cells of Arabidopsis seedling cotyledons obtained from VAEM time series with a temporal resolution of 0.5 s over 250 frames. The coloured data points represent the technical replicates of 4 independent experiments. No statistical differences were observed based on two-tailed heteroscedastic t-tests and a Bonferroni multiple hypothesis correction. (G) Quantification of FLS2-GFP and BRI1-GFP receptor cluster lifetimes in epidermal cells of Arabidopsis seedling cotyledons obtained from VAEM time series with a temporal resolution of 0.5 s over 250 frames. The coloured data points represent the technical replicates of 4 independent experiments. The indicated p-values were obtained using a two-tailed heteroscedastic t-test and a Bonferroni multiple hypothesis correction. The presented images were acquired using variable angle epi-fluorescence microscopy (VAEM). Scale bars represent 5 µm. The colour bar represents the colour code for fluorescence intensities. Black dots represent outliers. https://doi.org/10.7554/eLife.25114.006 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Dynamics of FLS2 receptor clusters within the plasma membrane. The presented time series was acquired from an epidermal cell of an Arabidopsis seedling cotyledon expressing FLS2-GFP under its native promoter using variable angle epi-fluorescence microscopy (VAEM). The acquisition time was 0.5 s per frame over 250 frames in total. The scale bar represents 5 µm. https://doi.org/10.7554/eLife.25114.008 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Dynamics of BRI1 receptor clusters within the plasma membrane. The presented time series was acquired from an epidermal cell of an Arabidopsis seedling cotyledon expressing BRI1-GFP under its native promoter using variable angle epi-fluorescence microscopy (VAEM). The acquisition time was 0.5 s per frame over 250 frames in total. The scale bar represents 5 µm. https://doi.org/10.7554/eLife.25114.009 Taken together, we observed that FLS2 and BRI1 formed non-randomly distributed receptor clusters across the PM. These receptor clusters were comparable in size and density; however, showed differences with regard to dynamic features. Receptor activation reduces the lateral receptor cluster mobility Using our non-treated conditions, we visualised both inactive FLS2 populations and partially active BRI1 receptors given the presence of endogenous BRs. This experimental setup most likely best reflects the steady-state configuration of both receptors in a natural situation prior to pathogen attack. Still, we were also interested to test how ligand availability influences the dynamic behaviour of FLS2 and BRI1 receptor clusters. First, we deprived seedlings from endogenous BRs by cultivating them for 2 in liquid containing 5 an of et al., As shown in Figure of endogenous BRs in increased BRI1 cluster mobility (Figure In the lateral displacement of FLS2 clusters was not (Figure The cluster lifetimes for FLS2 and BRI1 showed a similar distribution as under non-treated with a slight trend towards increased stability for BRI1 clusters upon (Figure we activated BRI1-mediated signalling by of In to no on FLS2 clusters but the lateral displacement of BRI1 receptor clusters within a time frame of (Figure Figure 3 Download asset Open asset BRs BRI1 cluster displacement within the plasma membrane. (A) Quantification of FLS2-GFP and BRI1-GFP receptor cluster displacements in epidermal cells of Arabidopsis seedling cotyledons after 2 in liquid containing 5 (B) Quantification of FLS2-GFP and BRI1-GFP receptor cluster lifetimes in epidermal cells of Arabidopsis seedling cotyledons after 2 in liquid containing 5 (C) of FLS2-GFP and BRI1-GFP receptor cluster displacements in epidermal cells of Arabidopsis seedling cotyledons after and of (D) of FLS2-GFP and BRI1-GFP receptor cluster lifetimes in epidermal cells of Arabidopsis seedling cotyledons after and of (E) of FLS2-GFP and BRI1-GFP receptor cluster displacements in epidermal cells of Arabidopsis seedling cotyledons after and of (F) of FLS2-GFP and BRI1-GFP receptor cluster lifetimes in epidermal cells of Arabidopsis seedling cotyledons after and of (G) of FLS2-GFP and BRI1-GFP receptor cluster displacements in epidermal cells of Arabidopsis seedling cotyledons after and of of FLS2-GFP and BRI1-GFP receptor cluster lifetimes in epidermal cells of Arabidopsis seedling cotyledons after and of The presented data points were obtained from VAEM time series with a temporal resolution of 0.5 s over 250 frames. The coloured data points represent the technical replicates of 3 independent experiments. The indicated p-values were obtained using a heteroscedastic t-test and a Bonferroni multiple hypothesis correction. we observed a in lateral cluster displacement for FLS2 receptors after seedlings were to flg22, whereas BRI1 was (Figure there was no quantitative on the receptor cluster lifetimes of both receptors (Figure Figure 4 Download asset Open asset of FLS2 results in reduced lateral receptor cluster (A) of FLS2-GFP and BRI1-GFP receptor cluster displacements in epidermal cells of Arabidopsis seedling cotyledons after of (B) of FLS2-GFP and BRI1-GFP receptor cluster lifetimes in epidermal cells of Arabidopsis seedling cotyledons after of (C) of FLS2-GFP and BRI1-GFP receptor cluster displacements in epidermal cells of Arabidopsis seedling cotyledons after of (D) of FLS2-GFP and BRI1-GFP receptor cluster lifetimes in epidermal cells of Arabidopsis seedling cotyledons after of (E) of FLS2-GFP and BRI1-GFP receptor cluster displacements in epidermal cells of Arabidopsis seedling cotyledons after of (F) of FLS2-GFP and BRI1-GFP receptor cluster lifetimes in epidermal cells of Arabidopsis seedling cotyledons after of The presented data points were obtained from VAEM time series with a temporal resolution of 0.5 s over 250 frames. The coloured data points represent the technical replicates of 3 independent experiments. The indicated p-values were obtained using a heteroscedastic t-test and a Bonferroni multiple hypothesis correction. Our that receptor activation to reduced lateral mobility within the PM are to Wang et al. increased BRI1 after ligand in Arabidopsis but in with observations made for the plant receptors FLS2 and et al., 2007; et al., 2011) as as the mammalian cell surface receptor EGFR et al., 2011). Thus, FLS2 and BRI1 comparable behaviour in response to their respective the dynamic of both cluster populations still from FLS2 and BRI1 show distinct plasma membrane localisation patterns To whether FLS2 and BRI1 clusters or are separated within the PM, we co-localisation studies. As positive we first the overlap of two FLS2 receptor populations. We FLS2-GFP and et al., in leaf epidermal cells of N. benthamiana and, as shown in Figure both FLS2 populations showed similar PM localisation patterns and also (Figure and D). on quantitative co-localisation analysis, we to high for FLS2-GFP and fluorescence signals (Figure and Figure supplement 1). Figure 5 with 1 supplement see all Download asset Open asset FLS2 and BRI1 show distinct plasma membrane localisation micrographs of FLS2-GFP (A) and (B) plasma membrane localisation after transient in epidermal leaf cells of N. benthamiana as as the image (C) and an image (D). micrographs of BRI1-GFP (E) and (F) plasma membrane localisation after transient in epidermal leaf cells of N. benthamiana as as the image (G) and an image co-localisation analysis for FLS2-GFP or respectively, with after transient in epidermal leaf cells of N. benthamiana. The coloured data points represent the technical replicates of independent experiments. The indicated p-values were obtained using a two-tailed heteroscedastic t-test and a Bonferroni multiple hypothesis correction. co-localisation analysis for FLS2-GFP or respectively, with after transient in epidermal leaf cells of N. benthamiana and The coloured data points represent the technical replicates of 2 independent experiments. The indicated p-values were obtained using a two-tailed heteroscedastic t-test and a Bonferroni multiple hypothesis correction. The presented images were acquired using confocal laser scanning microscopy (CLSM). Scale