Mucolipidosis type IV is a poorly understood lysosomal storage disease caused by alterations in the mucolipin lysosomal Ca2+ channel. In this study, we generated mucolipin-knockout Dictyostelium cells, and observed that lysosome exocytosis was markedly increased in these cells compared with wild-type cells. In addition, mucolipin-knockout cells were more resistant to Ca2+ deprivation, and the Ca2+ concentration in their secretory lysosomes was decreased, suggesting that mucolipin transfers Ca2+ ions from the cytosol to the lumen of secretory lysosomes. We speculate that mucolipin attenuates the fusogenic effect of local cytosolic increases in Ca2+ by dissipating them into the lumen of lysosomal compartments.

In eukaryotic cells, endocytosed material is transported through early and late endosomes, and eventually reaches lysosomes, where it is degraded. In addition, in some specialized cells lysosomes can fuse with the cell surface in a regulated manner. Exocytosis of secretory lysosomes notably plays an important role in the biology of melanocytes or cytotoxic T cells (Blott and Griffiths, 2002). Fusion of lysosomes with the cell surface can be triggered by transient increases in the cytosolic free Ca2+ concentration, from the resting concentration of 0.1 μM to 1–5 μM (Rodríguez et al., 1997; Andrews, 2000), and this fusion is potentially regulated by Ca2+ transporters present at the lysosomal membrane.

Mucolipidosis type IV is a neurodegenerative lysosomal storage disorder (Berman et al., 1974), characterized by abnormally large lysosomes containing electron-dense inclusions and lipid storage bodies (Folkerth et al., 1995). It is caused by mutations in the MCOLN1 gene (Bargal et al., 2000; Bassi et al., 2000; Sun et al., 2000), which encodes a transient receptor potential (TRP) channel, named mucolipin-1 (or TRP-ML1). Mucolipin-1 is a putative Ca2+ channel (Raychowdhury et al., 2004) localized in the membrane of late endosomes and lysosomes (Manzoni et al., 2004; Pryor et al., 2006). Mammalian cells can also express two other endosomal mucolipin paralogs (TRP-ML2 and TRP-ML3) (Cheng et al., 2010).

The role of mucolipin in lysosomal biogenesis and/or function is still poorly understood. It has been proposed that loss of mucolipin might affect either lysosomal acidification or intracellular transport. An altered lysosomal pH could affect lysosomal enzyme activity and cargo degradation and thus account for the accumulation of undigested material in lysosomes (Bach et al., 1999; Soyombo et al., 2006). However, some studies concluded that lysosomal pH was decreased in knockout cells (Miedel et al., 2008), whereas others reported an increased lysosomal pH (Bach et al., 1999; Soyombo et al., 2006) and yet others an unchanged lysosomal acidification (Pryor et al., 2006). Other studies have suggested that loss of TRP-ML1 might alter intracellular transport, in particular lysosome biogenesis (Tellez-Nagel et al., 1976; Treusch et al., 2004; Martina et al., 2009) and lysosome exocytosis (LaPlante et al., 2006). TRP-ML2 and TRP-ML3 are also thought to play a role in the physiology of the endocytic pathway, although their exact function remains to be established (Cheng et al., 2010).

The amoeba Dictyostelium discoideum is a model organism widely used to study the endocytic pathway. In these cells, endocytosed material is first transported to acidic lysosomes, and subsequently transferred to less-acidic compartments, the post-lysosomes (Maniak, 2003). Post-lysosomes are functionally equivalent to mammalian secretory- and fusion-competent lysosomes, and they are constitutively exocytosed, releasing undigested material in the extracellular medium (Charette and Cosson, 2007). Similar to lysosomes, Dictyostelium post-lysosomes contain a putative copper transporter (referred to as p80) but unlike lysosomes, they are devoid of vacuolar H+-ATPase. Thus, unlike mammalian secretory lysosomes, they can be formally distinguished from other lysosomes, a key advantage for studying their biogenesis and exocytosis (Charette and Cosson, 2007). Consequently, this model organism has been used previously to characterize the function of proteins involved in the biogenesis and function of secretory lysosomes, such as the orthologs of CHS and AP-3 (defective in human Chediak–Higashi and Hermanski–Pudlak syndromes, respectively) and WASH proteins (Charette and Cosson, 2007; Charette and Cosson, 2008; Carnell et al., 2011).

In this study we analyzed the phenotype of Dictyostelium mucolipin (mcln)-knockout cells. We expected secretory post-lysosomes to function as Ca2+ stores releasing Ca2+ in the cytosol through the mucolipin channel. However, our results suggest that mucolipin allows transfer of Ca2+ from the cytosol to the lumen of post-lysosomes, thus limiting fusion of post-lysosomes with the cell surface.

TRP-ML localizes to secretory post-lysosomes

The Dictyostelium genome contains only four genes encoding putative Ca2+ channels (mcln, pkd2, tpc2 and iplA) (Wilczynska et al., 2005). Dictyostelium mucolipin (Fig. 1A) exhibits the hallmarks of the TRP (transient receptor potential) family of ion channels (Venkatachalam and Montell, 2007): six transmembrane (TM) domains, a conserved pore region (position 498–511) and a characteristic large extracellular loop between TM1 and TM2 (Fig. 1B). It shares 55% similarity with the human orthologs (TRP-ML1, TRP-ML2 and TRP-ML3) over the TRP similarity region (TM3 to TM6, pos. 385–541; Fig. 1C). In addition, the C-terminal cytosolic domain of Dictyostelium mucolipin contains a Ca2+-binding EF-hand (pos. 614–626), a feature not found in other TRP-ML channels. An EF-hand is present in the C-terminal portion of the human TRP-A1 channel, and accounts for the Ca2+-dependent activation of this channel (Doerner et al., 2007).

As observed in mammals, Drosophila and C. elegans, it is likely that Dictyostelium mucolipin is localized in the endosomal pathway. To assess this, an expression plasmid encoding a FLAG-tagged mucolipin was transfected into wild-type (WT) cells, which were stained with an antibody against the p80 protein. The p80 protein is an endosomal marker present in all endosomal compartments, but enriched in the secretory post-lysosomes (Ravanel et al., 2001). TRP-ML was detected in large p80-rich compartments typical of post-lysosomes, as well as in dot-like structures throughout the cytoplasm (Fig. 2A), which might also correspond to endocytic compartments (Charette et al., 2006). To confirm the endosomal localization of the FLAG-tagged mucolipin, phagosomes at different stages of maturation containing latex beads were purified and the presence of p80 and of FLAG-tagged mucolipin assessed (Fig. 2B). Mucolipin was detected at low levels in early phagosomes, but accumulated in later phagosomes – a profile that is similar to that of the p80 endosomal marker (Lelong et al., 2011). Together, these results indicate that mucolipin is largely present in post-lysosomes in Dictyostelium cells.

Endosomal pH is unaffected in mcln KO cells

To evaluate the role of mucolipin in the endocytic pathway of Dictyostelium, we generated mcln-knockout (KO) cells by homologous recombination (supplementary material Fig. S1), and examined several key features of the endocytic pathway in these cells. Lysosomes and post-lysosomes can be identified by immunofluorescence by the presence of two well-characterized markers, the p80 protein and vacuolar H+-ATPase (Ravanel et al., 2001). The numbers and sizes of lysosomes (p80-positive, H+-ATPase-positive) and post-lysosomes (p80-positive, H+-ATPase-negative) were indistinguishable in WT and mcln-KO cells (supplementary material Fig. S2A,B). Phagocytosis of fluorescent particles and endocytosis of the fluid phase were identical in WT and KO cells (supplementary material Fig. S2D,E). Moreover, lysosomal enzymes were normally retained inside mcln-KO cells, and secreted upon starvation (data not shown); KO cells were also able to grow normally on a large array of bacteria and to engage into multicellular development upon starvation (supplementary material Fig. S3).

We studied the pH of endosomal compartments with particular attention because conflicting results have been reported in mammalian cells. In Dictyostelium, the endosomal pH can be measured by flow cytometry after endocytosis of a mixture of pH-sensitive and pH-insensitive fluorescent dyes (Marchetti et al., 2009) (supplementary material Fig. S4). In WT cells, the fluid phase was found first in very acidic lysosomes, then after 30 minutes of chase it was gradually transferred to less-acidic post-lysosomes (Fig. 3A). Identical curves were obtained in mcln-KO cells (Fig. 3A), indicating that the pH in lysosomal compartments is unaffected by mucolipin disruption. Because this technique measures an average fluorescence value over whole individual cells, it provides an underestimated value of the pH in post-lysosomal compartments. To circumvent this limitation, we also measured the pH of post-lysosomes directly in cells loaded with fluorescent-tagged Dextrans and chased for 30 minutes. Post-lysosomes can easily be identified because they are large compartments where endocytosed fluid-phase is markedly concentrated (Neuhaus et al., 2002) (supplementary material Fig. S4D). The post-lysosomal pH was determined directly by measuring the specific extinction of the pH-sensitive fluorescence in these compartments, and was found to be 5.6 in both WT and mcln-KO cells (Fig. 3B). Thus, in Dictyostelium cells, loss of mucolipin did not result in any detectable change of endosomal pH.

Post-lysosome exocytosis is dysregulated in mcln-KO cells

Because post-lysosomes are rich in p80, their fusion with the plasma membrane leads to the transient formation of p80-rich microdomains at the cell surface, which are referred to as exocytic patches (Fig. 4A) (Charette and Cosson, 2007). In mcln-KO cells, there were approximately twice as many exocytic patches compared with WT cells (Fig. 4B), and this phenotype was complemented when cells were transfected with an expression plasmid containing the FLAG-tagged mucolipin (Fig. 4C). As the number of post-lysosomes per cell was the same in WT and KO cells, this suggested that the rate of fusion of individual post-lysosomes with the cell surface was twice as high in mcln-KO cells than in WT cells (supplementary material Fig. S2C). To evaluate directly and with a different method the frequency with which post-lysosomes fused with the cell surface, we fed cells with medium containing fluorescent-tagged Dextran, incubated them for 30 minutes to allow transfer of endocytosed Dextrans to post-lysosomes, and observed the disappearance of labeled post-lysosomes from individual cells (Fig. 4D). These observations also indicated that the rate of fusion of individual post-lysosomes with the cell surface was significantly higher in mcln-KO cells than in WT cells (Fig. 4E).

Transfer of phagocytosed material along the endocytic pathway can be assessed by following the maturation of endocytic compartments containing internalized fluorescent particles. In WT cells, particles were virtually all present in lysosomes for the first 30 minutes following their ingestion and then gradually transferred to post-lysosomes, where they were most prominently found after 90 minutes (Fig. 5A). In mcln-KO cells, the majority of particles followed a similar path, being first found in lysosomes then transferred with normal kinetics to post-lysosomes. However, in addition, a significantly higher number of ingested particles was found in post-lysosomes 15 or 30 minutes after ingestion (Fig. 5B), suggesting an aberrant maturation of lysosomes into post-lysosomes. A faster biogenesis of post-lysosomes might explain why in mcln-KO cells the faster fusion of post-lysosomes with the cell surface does not result in a decreased number of post-lysosomes when compared with WT cells.

Calcium homeostasis is affected in mcln-KO cells

To determine whether mucolipin participates in Ca2+ homeostasis, we assessed the ability of cells to grow in Ca2+-depleted medium (buffered to 1 μM CaCl2). In HL5 medium, WT cells reached a density of 106 cells/ml in 3–4 days, whereas in Ca2+-depleted medium 7–8 days were needed (Fig. 6A). Knockout cells grew at the same rate as WT cells in HL5 medium, but in Ca2+-depleted medium, they grew twice as fast as WT cells (Fig. 6A), suggesting that mucolipin activity in WT cells leads to a net loss of Ca2+ from the cells.

We also assessed directly the Ca2+ concentration in post-lysosomes in WT and mcln-KO cells, by measuring the ratio between two Dextran-coupled fluorophores, one Ca2+ insensitive and one Ca2+ sensitive. Measuring Ca2+ concentration in lysosomes was not feasible, because the calcium probes were fully quenched by the very acidic pH in these compartments. These experiments were performed with probes binding Ca2+ either with a high (Kd=0.6 μM) or a low (Kd=3 μM) affinity. With both probes, the measured fluorescence ratio was significantly lower in post-lysosomes from mcln-KO cells, indicating that the Ca2+ concentration in post-lysosomes was lower in mcln-KO cells than in WT cells (Fig. 6B). To extrapolate Ca2+ concentrations from the fluorescence signals, the minimal and maximal signals were determined in the presence of ionomycin and either EGTA or CaCl2, respectively. In WT cells, the high-affinity probe emitted a signal identical to the maximal signal, indicating a Ca2+ concentration higher than 1–2 μM (Fig. 6C). Accordingly, the Ca2+ concentration was in the range covered by the low-affinity calcium probe, at approximately 3 μM (Fig. 6D). In mcln-KO cells, both the low- and the high-affinity probes were at their minimal emission values, indicating a luminal Ca2+ concentration lower than 0.2 μM (Fig. 6C,D).

In this study we characterized mcln-KO cells to determine the role of the mucolipin channel in lysosome biology in Dictyostelium. Remarkably, the overall structure and function of the endosomal pathway was mostly unaffected in mcln-KO cells. The pH of endosomal compartments was notably unchanged by the loss of the mucolipin protein. The only noticeable difference was that a fraction of internalized latex beads were found abnormally early in late compartments (post-lysosomes), suggesting that post-lysosomes were generated at an increased rate in mcln-KO cells compared with WT cells. We did, however, observe specific alterations affecting the exocytosis of secretory lysosomes, which are discussed below.

First, we observed that fusion of post-lysosomes with the cell surface was increased in mcln-KO cells compared with WT cells. Second, mcln-KO cells grew faster than WT cells in Ca2+-depleted medium, indicating that mucolipin plays a crucial role in Ca2+ homeostasis in these conditions. Third, the Ca2+ concentration inside post-lysosomes was around 3 μM in WT cells, and much lower (below 0.2 μM) in mcln-KO cells. This converging series of results highlights the role of mucolipin in Ca2+ homeostasis, and led us to hypothesize that mucolipin actually transfers Ca2+ from the cytosol to the post-lysosomes, and in doing so, reduces the fusion of post-lysosomes with the cellular membrane (and the maturation of lysosomal compartments).

How can Ca2+ be transferred by a mucolipin channel from the normally Ca2+-depleted environment of the cytosol to an intracellular compartment? To account for our observations, we speculate that post-lysosomes sometimes encounter transiently high concentrations of cytosolic Ca2+. Indeed, high local Ca2+ concentrations (at least 10 μM) have been observed transiently in the vicinity of the plasma membrane in mammalian cells (Llinás et al., 1992; Marsault et al., 1997). To our knowledge, local transient Ca2+ increases have not been studied in vegetative Dictyostelium cells. In developing cells, external stimuli such as cAMP or folate do cause a global cytosolic Ca2+ increase, but the level of local Ca2+ increases remains to be established (Schlatterer et al., 1994; Wilczynska et al., 2005). When such local Ca2+ waves encounter a post-lysosome, the mucolipin channel might allow transfer of Ca2+ into the post-lysosomes by a mechanism similar to the efficient transfer of Ca2+ from the endoplasmic reticulum to neighboring mitochondria (Rizzuto et al., 1993). In that perspective, it might be significant that the Dictyostelium mucolipin exhibits a cytosolic EF-hand, which could allow it to open only when the local cytosolic Ca2+ level is high. This scenario could also account for the unexpected observation that loss of mucolipin increases the fusion of post-lysosomes with the cell surface: by turning post-lysosomes into calcium sinks, mucolipin activity might decrease high local cytosolic Ca2+ concentrations in the vicinity of post-lysosomes, thus limiting their fusion with the cell surface. This speculative model is depicted in Fig. 7. Although the simplest interpretation of our results proposes that mucolipin itself acts as a Ca2+ channel, we cannot exclude an indirect role of mucolipin in Ca2+ transport across the PL membrane.

Although our knowledge of mucolipin function in mammalian cells is still incomplete, the phenotype observed for Dictyostelium mcln-KO cells is more reminiscent of the phenotype of mammalian TRP-ML3-knockdown cells than to TRP-ML1 mutant cells. Indeed, although the three mammalian TRP-ML proteins have been shown to interact (Curcio-Morelli et al., 2010), they apparently play different and possibly antagonistic roles. TRP-ML1-KO cells show delayed endosomal traffic and decreased fusion of lysosomes to the cell surface (Pryor et al., 2006; Miedel et al., 2008). By contrast, TRP-ML3-knockdown cells show enhanced degradation and traffic of endocytosed material and increased vesicle fusion compared with WT cells (Kim et al., 2009; Martina et al., 2009; Lelouvier and Puertollano, 2011). So far, no simple explanation has been offered on how loss of TRP-ML3 could enhance membrane traffic. There is also no consensus on the basic pore properties of mammalian TRP-ML proteins; although results suggest the presence of inwardly rectifying currents for TRP-ML1 and TRP-ML3 channels, difficulties in measuring endogenous lysosomal currents have produced controversial results (Cheng et al., 2010). Some studies even showed outwardly rectifying currents for TRP-ML1 (Kiselyov et al., 2005). Mucolipin channels might be capable of transferring Ca2+ ions in both directions, depending on the cellular environment in which they are inserted. Our studies suggest that Dictyostelium mucolipin functions in a manner analogous to TRP-ML3, and offer a possible explanation of how loss of a Ca2+ channel could enhance membrane traffic.

Cell culture and plasmids

Dictyostelium cells were grown at 21°C in HL5 medium (Cornillon et al., 2000) and subcultured twice a week to maintain a maximal density of 106 cells/ml. In experiments where Ca2+ (CaCl2) was added, cells were kept in HL5-MES (3.6 mM KH2PO4 replaced with 6.1 mM MES). For experiments with imposed Ca2+ concentrations, we used CaCl2 and EGTA proportions defined by using the Ca-EGTA calculator program (Patton et al., 2004).

A knockout vector for mcln disruption was constructed using a blasticidin-resistance cassette flanked by two gene segments (positions 176–990 and 1771–2217 in the coding sequence; supplementary material Fig. S1A). The PvuI-digested plasmid was introduced into DH1-10 (WT) cells (Cornillon et al., 2000) by electroporation, transfected cells were selected in the presence of blasticidin (10 μg/ml), and individual clones were screened by PCR (supplementary material Fig. S1C). Three independent KO clones were used in parallel in this study, and presented identical phenotypes.

An expression vector carrying a tagged version of the mucolipin gene was constructed by introducing a C-terminal FLAG (DYKDDDDK)-tagged mucolipin cDNA into pDXA-3C (Manstein et al., 1995). This plasmid was transfected into WT and mcln-KO cells by electroporation; transfected cells were selected in the presence of G418 (10 μg/ml).

Mouse monoclonal antibodies against p80 (H161) and vacuolar H+-ATPase (221-35-2) were described previously (Neuhaus et al., 1998; Ravanel et al., 2001). Mouse monoclonal anti-FLAG antibody (clone M2) was from Sigma-Aldrich. All fluorescent probes were from Molecular Probes, Invitrogen (Eugene, OR).

Phagosome purification and protein identification by SDS-PAGE

Phagosomes at different stages of maturation containing latex beads were purified as described (Gotthardt et al., 2006). Briefly, Dictyostelium cells were incubated with 0.8 μm latex beads during a pulse of 5 or 15 minutes, then washed and chased further for 15, 45, 105 and 165 minutes. Phagosomes containing latex beads were then purified by flotation on sucrose gradient. Samples of 20 μg of phagosomal proteins or 106Dictyostelium cells were separated in 10% polyacrylamide gel, transferred to nitrocellulose membranes, and detected with primary mouse anti-FLAG and anti-p80 antibodies and secondary horseradish-peroxidase-coupled anti-mouse IgG (Bio-Rad), and revealed by chemiluminescence.

Internalization and pH measurement

The internalization of fluid phase or phagocytic particles was measured as follows: 106 cells were incubated at 21°C in HL5 medium containing 10 μg/ml Alexa-Fluor-647–Dextran (Molecular Probes, Eugene, OR) or 1 μl/ml of FITC-fluorescent 1 μm diameter latex beads (Polysciences, Warrington, PA) and aliquots of 105 cells were harvested at 30 minute intervals up to 180 minutes, washed with ice-cold HL5 containing 0.1% (w/v) sodium azide and analyzed by flow cytometry (FACS Calibur, Becton Dickinson, San Jose, CA).

Endosomal pH was measured by flow cytometry as described previously (Marchetti et al., 2009). Briefly, Dictyostelium cells were incubated for 20 minutes in the presence of two fluorescent probes: Oregon-Green–Dextran (pH-sensitive) and Alexa-Fluor-647–Dextran (pH-insensitive probe), and the fluorescence ratio of these two dyes (measured using a FACS Calibur, Becton Dickinson, San Jose, CA) was compared with a standard calibration curve (supplementary material Fig. S4C).

Fluorescence microscopy

To perform immunofluorescence, 106 cells were incubated on a glass coverslip for 3 hours at 21°C, fixed in paraformaldehyde, permeabilized with Triton X-100 (0.1%) and labeled as described previously (Charette and Cosson, 2006). Cells were visualized with an LSM510 confocal microscope (Carl Zeiss). The quantification of the number and diameter of lysosomes and post-lysosomes, kinetics of lysosome-PL maturation, and number of exocytic p80 patches was carried out as already described (Charette and Cosson, 2007).

To visualize post-lysosome exocytosis, 106 cells were incubated with 10 μg/ml Alexa-Fluor-647–Dextran for 3 hours at 21°C, washed in HL5 and allowed to attach on a glass coverslip for 30 minutes. Every 10 seconds, five pictures in successive focal planes separated by 2 μm were taken, using a LSM510 confocal microscope (Carl Zeiss) (total recording time: 200 seconds); for image analysis, all confocal planes were merged into a single image. Post-lysosomes were identified as large endosomal compartments, where fluid phase was concentrated (Fig. 4C); the percentage of post-lysosomes disappearing each minute was determined. At least 20–30 cells were analyzed for each cell line in each experiment.

To measure post-lysosomal pH directly, 106 cells were incubated in HL5 containing 10 μg/ml Alexa-Fluor-647–Dextran (pH insensitive) and 25 μg/ml of FITC–Dextran (pH sensitive) for 3 hours at 21°C, washed in HL5 and allowed to attach to a glass coverslip for 30 minutes. Pictures of live cells were taken with an LSM510 confocal microscope (Carl Zeiss), and at least 100 post-lysosomes were analyzed for each condition tested (supplementary material Fig. S4D). The fluorescence ratio was calculated and compared with a standard calibration curve [obtained by buffering cells with 40 mM NH4Cl + 0.1% (w/v) sodium azide at pH 4–7, supplementary material Fig. S4E].

Calcium probes are too sensitive to acidic pH to assess Ca2+ concentration in very acidic lysosomes, but adequate in the more neutral post-lysosomes. To measure post-lysosomal Ca2+ levels, 106 cells were incubated with 10 μg/ml Alexa-Fluor-647–Dextran (Ca2+ insensitive) and 50 μg/ml Fluo4–Dextran (Ca2+ sensitive, either in its high-affinity form, Kd=0.6 μM or low-affinity form, Kd=3 μM) for 3 hours at 21°C, washed in HL5 and allowed to attach to a glass coverslip for 30 minutes. Pictures were taken with a LSM510 confocal microscope (Carl Zeiss), and at least 100 post-lysosomes were counted for each condition. The fluorescence ratio (R) was calculated, and compared with the minimal (Rmin) and maximal (Rmax) ratios, measured after incubating cells for 15 minutes in presence of 20 μM ionomycin and 20 mM EGTA or CaCl2, respectively. The Ca2+ concentration was estimated when appropriate by using the formula [Ca2+]= Kd*(RRmin)/(RmaxR).

Sequence and phylogenetic analysis

Sequence-similarity analyses were performed using BlastP and tBlastN programs against the nucleotide or protein databases deposited at NCBI or DictyBase servers (Pruitt et al., 2005; Chisholm et al., 2006). For phylogenetic analyses, protein sequences were aligned by using the CLUSTALX 2.0 program (Larkin et al., 2007). Pair-wise genetic distances were computed by using MEGA 4.0 (Tamura et al., 2007), with the JTT (Jones–Taylor–Thornton) model of amino acid replacement, gamma-distributed rate (four categories) and inferred alpha-parameter. Maximum-likelihood (ML) trees were set up with RAxML 7.0.4 (Stamatakis, 2006), with the WAG model, and parameters for invariable sites and gamma-distributed rate heterogeneity (four categories). One thousand bootstrap replicates were executed and bootstrap values drawn up on the best-scoring ML-tree. Trees were visualized using MEGA 4.0.

Statistical analysis

Unless otherwise specified, the mean and s.e.m. of at least three independent experiments are indicated. Statistical comparisons were done with Student's t-tests (two-tailed, paired); *P<0.05; **P<0.01.

We thank Pierre Golstein, Nicolas Demaurex and Oliver Hartley for critical reading of this manuscript.

Funding

The P.C. and T.S. laboratories are supported by the Swiss National Foundation for Scientific Research [grant numbers 31003A-135789 and 31003A_132995, respectively]; the Doerenkamp-Zbinden Foundation; and the E. Naef Foundation (FENRIV).

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