Differential sorting of lysosomal enzymes in mannose 6-phosphate receptor-deficient fibroblasts.

In higher eukaryotes, the transport of soluble lysosomal enzymes involves the recognition of their mannose 6-phosphate signal by two receptors: the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR). It is not known why these two different proteins are present in most cell types. To investigate their relative function in lysosomal enzyme targeting, we created cell lines that lack either or both MPRs. This was accomplished by mating CD-MPR-deficient mice with Thp mice that carry a CI-MPR deleted allele. Fibroblasts prepared from embryos that lack the two receptors exhibit a massive missorting of multiple lysosomal enzymes and accumulate undigested material in their endocytic compartments. Fibroblasts that lack the CI-MPR, like those lacking the CD-MPR, exhibit a milder phenotype and are only partially impaired in sorting. This demonstrates that both receptors are required for efficient intracellular targeting of lysosomal enzymes. More importantly, comparison of the phosphorylated proteins secreted by the different cell types indicates that the two receptors may interact in vivo with different subgroups of hydrolases. This observation may provide a rational explanation for the existence of two distinct mannose 6-phosphate binding proteins in mammalian cells.     

Neither type of mannose 6-phosphate receptor is sufficient for targeting of lysosomal enzymes along intracellular routes

Mouse embryonic fibroblasts that are deficient in the two mannose 6- phosphate receptors (MPRs) MPR 46 and MPR 300 missort the majority (> or = 85%) of soluble lysosomal proteins into the medium. Human MPR 46 and MPR 300 were expressed in these cells to test whether overexpression of a single type of MPR can restore transport of lysosomal proteins to lysosomes. Only a partial correction of the missorting was observed after overexpression of MPR 46. Even at MPR 46 levels that are five times higher than the wild-type level, more than one third of the newly synthesized lysosomal proteins accumulates in the secretions. Two-fold overexpression of MPR 300 completely corrects the missorting of lysosomal enzymes. However, at least one fourth of the lysosomal enzymes are transported along a secretion-recapture pathway that is sensitive to mannose 6-phosphate in medium. In control fibroblasts that express both types of MPR, the secretion-recapture pathway is of minor importance. These results imply that neither overexpression of MPR 46 nor MPR 300 is sufficient for targeting of lysosomal proteins along intracellular routes.     

Molecular cloning, expression and functional characterization of the chicken cation dependent mannose 6-phosphate receptor protein

Mammalian mannose 6-phosphate (M6P) receptors function in transport of lysosomal enzymes. To understand the structural and functional significance of the chicken cation dependent mannose 6-phosphate receptor (MPR) (Mr 46kDa), a full-length cDNA for the chicken protein was cloned and expressed in mpr((-/-)) MEF cells devoid of both the receptors. The stably transfected cells express the receptor that could be affinity purified by phosphomannan chromatography. The authenticity of the receptor was confirmed by its immuno-reactivity with mammalian MPR 46 antibodies and its ability to sort cathepsin D in transfected cells (92.3%) as compared to mock transfected cells (50.2%), establishing a functional role for the chicken receptor.     

The lysosomal trafficking of acid sphingomyelinase is mediated by sortilin and mannose 6-phosphate receptor

Acid sphingomyelinase (ASM), a member of the saposin-like protein (SAPLIP) family, is a lysosomal hydrolase that converts sphingomyelin to ceramide. Deficiency of ASM causes a variant form of Niemann-Pick disease. The mechanism of lysosomal targeting of ASM is poorly known. Previous studies suggest that ASM could use in part the mannose 6-phosphate receptor (M6P-Rc). Sortilin, a type I transmembrane glycoprotein that belongs to a novel family of receptor proteins, presents structural features of receptors involved in lysosomal targeting. In this study we examined the hypothesis that sortilin may be implicated in the trafficking of ASM to the lysosomes. Using a dominant-negative sortilin construct lacking the cytoplasmic tail, which is essential to recruit adaptor proteins and clathrin, we demonstrated that sortilin is also involved in the lysosomal targeting of ASM. Confocal microscopy revealed that truncated sortilin partially inhibited the lysosomal trafficking of ASM in COS-7 cells and abolished the lysosomal targeting of ASM in I-cells. Pulse-chase experiments corroborated that sortilin is involved in normal sorting of newly synthesized ASM. Furthermore, over-expression of truncated sortilin accelerated and enhanced the secretion of ASM from COS-7 cells and I-cells. Co-immunoprecipitation assays confirmed the interaction between sortilin and ASM. In conclusion, ASM uses sortilin as an alternative receptor to be targeted to the lysosomes.     

Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor

Disruption of latent TGF-beta binding protein (LTBP)-4 expression in the mouse leads to abnormal lung development and colorectal cancer. Lung fibroblasts from these mice produced decreased amounts of active TGF-beta, whereas secretion of latent TGF-beta was significantly increased. Expression and secretion of TGF-beta2 and -beta3 increased considerably. These results suggested that TGF-beta activation but not secretion would be severely impaired in LTBP-4 -/- fibroblasts. Microarrays revealed increased expression of bone morphogenic protein (BMP)-4 and decreased expression of its inhibitor gremlin. This finding was accompanied by enhanced expression of BMP-4 target genes, inhibitors of differentiation 1 and 2, and increased deposition of fibronectin-rich extracellular matrix. Accordingly, increased expression of BMP-4 and decreased expression of gremlin were observed in mouse lung. Transfection of LTBP-4 rescued the -/- fibroblast phenotype, while LTBP-1 was inefficient. Treatment with active TGF-beta1 rescued BMP-4 and gremlin expression to wild-type levels. Our results indicate that the lack of LTBP-4-mediated targeting and activation of TGF-beta1 leads to enhanced BMP-4 signaling in mouse lung.     

Mr 46,000 mannose 6-phosphate specific receptor: its role in targeting of lysosomal enzymes.

Antibodies that block the ligand binding site of the cation-dependent mannose 6-phosphate specific receptor (Mr 46,000 MPR) were used to probe the function of the receptor in transport of lysosomal enzymes. Addition of the antibodies to the medium of Morris hepatoma 7777 cells, which express only the Mr 46,000 MPR, resulted in a decreased intracellular retention and increased secretion of newly synthesized lysosomal enzymes. In fibroblasts and HepG2 cells that express the cation-independent mannose 6-phosphate specific receptor (Mr 215,000 MPR) in addition to the Mr 46,000 MPR, antibodies against the Mr 46,000 MPR inhibited the intracellular retention of newly synthesized lysosomal enzymes only when added to the medium together with antibodies against the Mr 215,000 MPR. Morris hepatoma (M.H.) 7777 did not endocytose lysosomal enzymes, while U937 monocytes, which express both types of MPR, internalized lysosomal enzymes. The uptake was inhibited by antibodies against the Mr 215,000 MPR, but not by antibodies against the Mr 46,000 MPR. These observations suggest that Mr 46,000 MPR mediates transport of endogenous but not endocytosis of exogenous lysosomal enzymes. Internalization of receptor antibodies indicated that the failure to mediate endocytosis of lysosomal enzymes is due to an inability of surface Mr 46,000 MPR to bind ligands rather than its exclusion from the plasma membrane or from internalization.     

The interaction of phosphorylated oligosaccharides and lysosomal enzymes with bovine liver cation-dependent mannose 6-phosphate receptor

We have analyzed the interaction of phosphorylated oligosaccharides and lysosomal enzymes with immobilized bovine liver cation-dependent mannose-6-P receptor. Oligosaccharides with phosphomonoesters were the only species that interacted with the receptor, and molecules with two phosphomonoesters showed the best binding. Lysosomal enzymes with several oligosaccharides containing only one phosphomonoester had a higher affinity for the receptor than did the isolated oligosaccharides, indicating the possible importance of multivalent interactions between weakly binding ligands and the receptor. The binding of a mixture of phosphorylated lysosomal enzymes to the cation-dependent Man-6-P receptor was markedly influenced by pH. At pH 6.3, almost all of the lysosomal enzymes bound to the receptor; whereas at pH 7.0-7.5, approximately one-third of the material passed through the column, one-third interacted weakly, and one-third bound tightly. The distribution of individual lysosomal enzyme activities was similar to that of the total material. The species of phosphorylated oligosaccharides present on the lysosomal enzymes which interacted poorly with the receptor were similar to those found on the tightly bound material and included species of oligosaccharides with two phosphomonoester groups. Isolated oligosaccharides of this type bound to the receptor over the entire pH range tested. These findings indicate that at neutral pH the phosphorylated oligosaccharides on some lysosomal enzyme molecules are oriented in a manner which makes them inaccessible to the binding site of the cation-dependent Man-6-P receptor. Since the same enzymes bind to the cation-independent Man-6-P receptor at neutral pH, at least a portion of the phosphomannosyl residues must be exposed. We conclude that small variations in the pH of the Golgi compartment where lysosomal enzymes bind to the receptors could potentially modulate the extent of binding to the two receptors.     

Role for dynamin in late endosome dynamics and trafficking of the cation-independent mannose 6-phosphate receptor

It is well established that dynamin is involved in clathrin-dependent endocytosis, but relatively little is known about possible intracellular functions of this GTPase. Using confocal imaging, we found that endogenous dynamin was associated with the plasma membrane, the trans-Golgi network, and a perinuclear cluster of cation-independent mannose 6-phosphate receptor (CI-MPR)-containing structures. By electron microscopy (EM), it was shown that these structures were late endosomes and that the endogenous dynamin was preferentially localized to tubulo-vesicular appendices on these late endosomes. Upon induction of the dominant-negative dynK44A mutant, confocal microscopy demonstrated a redistribution of the CI-MPR in mutant-expressing cells. Quantitative EM analysis of the ratio of CI-MPR to lysosome-associated membrane protein-1 in endosome profiles revealed a higher colocalization of the two markers in dynK44A-expressing cells than in control cells. Western blot analysis showed that dynK44A-expressing cells had an increased cellular procathepsin D content. Finally, EM revealed that in dynK44A-expressing cells, endosomal tubules containing CI-MPR were formed. These results are in contrast to recent reports that dynamin-2 is exclusively associated with endocytic structures at the plasma membrane. They suggest instead that endogenous dynamin also plays an important role in the molecular machinery behind the recycling of the CI-MPR from endosomes to the trans-Golgi network, and we propose that dynamin is required for the final scission of vesicles budding from endosome tubules.     

Biochemical and functional characterization of cation dependent (Mr 46,000) goat mannose 6-phosphate receptor

The Mannose 6-phosphate receptor (MPR’s) proteins are important for transporting lysosomal enzymes from trans-golgi to the pre-lysosomal compartment. These are conserved in the vertebrates from fish to mammals. We have cloned the full length cDNA for the goat MPR 46 protein and compared its sequences to the other known vertebrate MPR 46 proteins. In the present study the full-length cDNA for the goat MPR 46 protein was expressed in MPR deficient cells. The expressed protein was purified on the multivalent phosphomannan gel in the presence of divalent metal ions. The apparent molecular mass of the expressed protein was found to be ∼46 kDa and also exhibits oligomeric nature as observed in the other species, by using an MSC1 antibody (that recognizes the MPR 46 from molluscs to mammals) as well as with a peptide specific antibody corresponding to amino acid residues (218–237) of the cytoplasmic tail of human MPR 46 protein. Furthermore the distribution of the expressed protein was visualized by immunofluorescence using MSC1 and LAMP1 antibody. Additionally in the goat MPR 46 expressing cells, the sorting function of the expressed protein to sort cathepsin D to lysosomes was studied by confocal microscopy using cathepsin D antiserum and LAMP1 antibody. The binding of goat MPR 46 to cathepsin D was shown in far Western blotting and the mannose 6-phosphate dependent binding was shown by co-immunoprecipitation.     

Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis

The cation-independent mannose 6-phosphate receptor (Cl-MPR) sorts newly synthesized lysosomal enzymes in the Golgi and endocytoses extracellular lysosomal enzymes. To determine the role of the 163 amino acid cytoplasmic domain of the Cl-MPR in these functions, receptor-deficient mouse L cells were transfected with normal bovine Cl-MPR cDNA or cDNAs mutated in the cytoplasmic domain. The normal Cl-MPR functioned in sorting and endocytosis. Mutant receptors with 40 and 89 residues deleted from the carboxyl terminus of the cytoplasmic tail functioned normally in endocytosis, but were partially impaired in sorting. Mutant receptors with larger deletions leaving only 7 and 20 residues of the cytoplasmic tail were defective in endocytosis and sorting. A mutant receptor containing alanine instead of tyrosine residues at positions 24 and 26 was defective in endocytosis, and partially impaired in sorting. Receptors deficient in endocytosis accumulated at the cell surface. These results indicate that the cytoplasmic domain of the Cl-MPR contains different signals for rapid endocytosis and efficient lysosomal enzyme sorting.     

Mannose 6-phosphate receptor dependent secretion of lysosomal enzymes.

BHK and mouse L cells transfected with the cDNA for the human 46 kd mannose 6-phosphate receptor (MPR 46) secrete excessive amounts of newly synthesized mannose 6-phosphate containing polypeptides. The secretion is dependent on the amount, the recycling and the affinity for ligands of MPR 46. Incubation of transfected cells with antibodies blocking the binding site of MPR 46 reduces the secretion, and cotransfection with the cDNA for the human 300 kd mannose 6-phosphate (MPR 300) restores it to normal values. These results indicate that the two mannose 6-phosphate receptors compete for binding of newly synthesized ligands. In contrast to ligands bound to MPR 300, those bound to the MPR 46 are transported to and released at a site, e.g. early endosomes or plasma membrane, from where they can exit into the medium. Since antibodies blocking the binding site of MPR 46 reduce secretion also in non-transfected BHK and mouse L cells, at least part of the basal secretion of M6P-containing polypeptides is mediated by the endogenous MPR 46.     

Targeted disruption of the mouse cation-dependent mannose 6-phosphate receptor results in partial missorting of multiple lysosomal enzymes.

In mammalian cells two mannose 6-phosphate receptors (MPRs) are involved in lysosomal enzyme transport. To understand the precise function of the cation-dependent mannose 6-phosphate receptor (CD-MPR), one allele of the corresponding gene has been disrupted in mouse embryonic stem cells and homozygous mice lacking this receptor have been generated. The homozygous mice appear normal, suggesting that other targeting mechanisms can partially compensate for the loss of the CD-MPR in vivo. However, homozygous receptor-deficient cells and animals clearly exhibit defects in targeting of multiple lysosomal enzymes when compared with wild-types. Increased levels of phosphorylated lysosomal enzymes were present in body fluids of homozygous animals. In thymocytes from homozygous mice or in primary cultures of fibroblasts from homozygous embryos, there is a marked increase in the amount of phosphorylated lysosomal enzymes that are secreted into the extracellular medium. The cultured fibroblasts have decreased intracellular levels of multiple lysosomal enzymes and accumulate macromolecules within their endosomal/lysosomal system. Taken together, these results clearly indicate that the CD-MPR is required for efficient intracellular targeting of multiple lysosomal enzymes.     

Characterization of the endosomal sorting signal of the cation-dependent mannose 6-phosphate receptor

Intracellular cycling of the cation-dependent mannose 6-phosphate receptor (CD-MPR) between different compartments is directed by signals localized in its cytoplasmic tail. A di-aromatic motif (Phe18-Trp19 with Trp19 as the key residue) in its cytoplasmic tail is required for the sorting of the receptor from late endosomes back to the Golgi apparatus. However, the cation-independent mannose 6-phosphate receptor (CI-MPR) lacks such a di-aromatic motif. Therefore the ability of amino acids other than aromatic residues to replace Trp19 in the CD-MPR cytoplasmic tail was tested. Mutant constructs with bulky hydrophobic residues (valine, isoleucine, or leucine) instead of Trp19 exhibited 30-60% decreases in binding to the tail interacting protein of 47 kDa (Tip47), a protein mediating this transport step, and partially prevented receptor delivery to lysosomes. Decreasing hydrophobicity of residues at position 19 resulted in further impairment of Tip47 binding and an increase of receptor accumulation in lysosomes. Intriguingly, mutants mislocalized to lysosomes did not completely co-localize with a lysosomal membrane protein, which might suggest the presence of subdomains within lysosomes. These data indicate that sorting of the CD-MPR in late endosomes requires a distinct di-aromatic motif with only limited possibilities for variations, in contrast to the CI-MPR, which seems to require a putative loop (Pro49-Pro-Ala-Pro-Arg-Pro-Gly55) along with additional hydrophobic residues in the cytoplasmic tail. This raises the possibility of two separate binding sites on Tip47 because both receptors require binding to Tip47 for endosomal sorting.     

Domain 5 of the cation-independent mannose 6-phosphate receptor preferentially binds phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester)

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46 kDa cation-dependent MPR (CD-MPR) are key components of the lysosomal enzyme targeting system that bind newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and divert them from the secretory pathway. Previous studies have mapped two high-affinity Man-6-P binding sites of the CI-MPR to domains 1-3 and 9 and one low-affinity site to domain 5 within its 15-domain extracytoplasmic region. A structure-based sequence alignment predicts that domain 5 contains the four conserved residues (Gln, Arg, Glu, Tyr) identified as essential for Man-6-P binding by the CD-MPR and domains 1-3 and 9 of the CI-MPR. Here we show by surface plasmon resonance (SPR) analyses of constructs containing single amino acid substitutions that these conserved residues (Gln-644, Arg-687, Glu-709, Tyr-714) are critical for carbohydrate recognition by domain 5. Furthermore, the N-glycosylation site at position 711 of domain 5, which is predicted to be located near the binding pocket, has no influence on the carbohydrate binding affinity. Endogenous ligands for the MPRs that contain solely phosphomonoesters (Man-6-P) or phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester, Man-P-GlcNAc) were generated by treating the lysosomal enzyme acid alpha-glucosidase (GAA) with recombinant GlcNAc-phosphotransferase and uncovering enzyme (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase). SPR analyses using these modified GAAs demonstrate that, unlike the CD-MPR or domain 9 of the CI-MPR, domain 5 exhibits a 14-18-fold higher affinity for Man-P-GlcNAc than Man-6-P, implicating this region of the receptor in targeting phosphodiester-containing lysosomal enzymes to the lysosome.     

Plasmalemmal vesicles are involved in transendothelial transport of albumin, lysosomal enzymes and mannose 6-phosphate receptor fragments in capillary endothelium

With the use of immunoelectron microscopy we have demonstrated the presence of lysosomal enzymes (acid alpha-glucosidase and glucocerebrosidase) and fragments of the 270 kDa receptor for mannose 6-phosphate and insulin-like growth factor II in blood plasma, plasmalemmal vesicles of endothelial cells and pericapillary spaces in human skeletal muscle tissue. At these locations, the three proteins colocalized with albumin known to be transported from the capillaries into the pericapillary spaces. Immunoblot analysis of plasma revealed the presence of relatively high molecular weight polypeptides in this material. These observations strongly suggest that high molecular weight species of lysosomal enzymes can pass the endothelial barrier in skeletal muscle tissue.     

Drug-induced phospholipidosis is caused by blockade of mannose 6-phosphate receptor-mediated targeting of lysosomal enzymes

Cationic amphiphilic drugs (CADs) cause massive intracellular accumulation of phospholipids, thereby resulting in phospholipidosis (PLD); however, the molecular mechanism underlying CAD-induced PLD remains to be resolved. Here, we found that treatment of normal rat kidney cells with CADs known to induce PLD caused redistribution of a mannose 6-phosphate/IGF-II receptor (MPR300) from the TGN to endosomes and concomitantly increased the secretion of lysosomal enzymes, resulting in a decline of intracellular lysosomal enzyme levels. These results enable the interpretation of why CADs cause excessive accumulation of undegraded substrates, including phospholipids in lysosomes, and led to the conclusion that the impaired MPR300-mediated sorting system of lysosomal enzymes reflects the general mechanism of CAD-induced PLD. In addition, our findings suggest that the measurement of lysosomal enzyme activity secreted into culture medium is useful as a rapid and convenient in vitro early screening system to predict drugs that can induce PLD.     

Biosynthesis and endocytosis of lysosomal enzymes in human colon carcinoma SW 1116 cells: impaired internalization of plasma membrane-associated cation-independent mannose 6-phosphate receptor.

The human colon adenocarcinoma cell lines SW 948, SW 1116, and SW 1222 were tested for their ability to sort and internalize lysosomal enzymes. The biosynthesis of the lysosomal enzymes cathepsin B, arylsulfatase A, and beta-hexosaminidase in these cell lines exhibits no significant differences to that in human fibroblasts. The intracellular targeting of newly synthesized hydrolases to the lysosomes relies in colon carcinoma cells on the mannose 6-phosphate receptor system. Both the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor are expressed in all colon carcinoma cell lines investigated. Endocytosis of lysosomal enzymes via mannose 6-phosphate receptors is reduced in colon carcinoma cells as compared with human fibroblasts. SW 1116 cells were shown to be deficient in receptor-mediated endocytosis of mannose 6-phosphate containing ligands. Ligands of other endocytic receptors as well as the fluid-phase marker horseradish peroxidase were internalized at normal rates. While antibodies against CI-MPR bind to the surface of SW 1116 cells, these antibodies cannot be internalized. These data suggest that the cycling of CI-MPR is specifically impaired in SW 1116 cells.     

The cytoplasmic tail of the mannose 6-phosphate/insulin-like growth factor-II receptor has two signals for lysosomal enzyme sorting in the Golgi

The mannose 6-phosphate/insulin-like growth factor-II (Man-6-P/IGF-II) receptor is known to cycle between the Golgi, endosomes, and the plasma membrane. In the Golgi the receptor binds newly synthesized lysosomal enzymes and transports them directly to an endosomal (prelysosomal) compartment without traversing the plasma membrane. Deletion of the carboxyl-terminal Leu-Leu-His-Val residues of the 163 amino acid cytoplasmic tail of the bovine Man-6-P/IGF-II receptor partially impaired this function, resulting in the diversion of a portion of the receptor-ligand complexes to the cell surface, where they were endocytosed. The same phenotype was observed when 134 residues of the cytoplasmic tail were deleted from the carboxyl terminus. Disruption of the Tyr24-Lys-Tyr-Ser-Lys-Val29 plasma membrane internalization signal alone had little effect on Golgi sorting, but when combined with either deletion resulted in a complete loss of this function. The mutant receptors retained the ability to recycle to the Golgi and bind cathepsin D. These results indicate that the cytoplasmic tail of the Man-6-P/IGF-II receptor contains two signals that contribute to Golgi sorting, presumably by interacting with the Golgi clathrin-coated pit adaptor proteins. The Leu-Leu-containing sequence represents a novel motif for mediating interaction with Golgi adaptor proteins.     

Characterization of the mannose 6-phosphate receptor (Mr 300 kDa) protein dependent pathway of lysosomal enzyme targeting in Biomphalaria glabrata mollusc cells

Mammalian mannose 6-phosphate receptors (MPR 300 and 46) are involved in the targeting of newly synthesized lysosomal enzymes and only MPR 300 also participates in the endocytosis of various exogenous ligands. The present study describes for the first time the MPR 300 dependent pathway of lysosomal enzyme sorting in the Biomphalaria glabrata embryonic (Bge) cells. Lysosomal enzymes (arylsulfatase A, β-hexosaminidase and α-fucosidase) were identified by their enzymatic activities and by immunoprecipitation with specific antisera. Exposure of Bge cells to unio MPR 300 antiserum resulted in a dramatic loss of MPR 300 protein with a shortened half life of ∼20 min as compared to control cells exposed to preimmune serum in which the half life of MPR 300 was of ∼13 h. Loss of receptor proteins resulted in a significant misrouting of newly synthesized lysosomal enzymes and their secretion in cell culture medium as demonstrated by immunoprecipitation. The ability of Bge cells to uptake and internalize labeled arylsulfatase A, β-hexosaminidase and α-fucosidase enzymes contained in cell secretion products also indicated the role of B. glabrata MPR 300 (CIMPR) protein in internalization and targeting of lysosomal enzymes. M6P dependent binding of lysosomal enzymes to MPR 300 was shown by confocal microscopy and coimmunoprecipitation experiments.