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Mutation of SIMPLE in Charcot–Marie–Tooth 1C alters production of exosomes

Mutations in the protein SIMPLE account for the rare autosomal-dominant demyelination in type 1C CMT patients (CMT1C). SIMPLE plays a role in the production of exosomes. Dysregulated endosomal trafficking and changes in exosome-mediated intercellular communications might account for CMT1C molecular pathogenesis.


INTRODUCTION
Charcot-Marie-Tooth (CMT) disease is a common inherited neurological disorder of the peripheral nervous system (Boerkoel et al., 2002;Shy, 2004). CMT1C is a subclass of the CMT1 demyelinating family (Latour et al., 2006;Szigeti and Lupski, 2009). Although CMT1C is a rare form of neuropathy, its distinctive autosomal-dominant trait is unique among the CMT1 family of diseases, as other forms of CMT1 are mainly recessive in nature and affect the level of myelin expression. Mutations in the small integral membrane protein of the lysosome/late endosome (SIMPLE; also known as LITAF, EET1, and PIG7; Myokai et al., 1999;Zhu et al., 1999;Moriwaki et al., 2001;Xie et al., 2002) account for the demyelination found in CMT1C patients (Street et al., 2003;Bennett et al., 2004;Saifi et al., 2005;Latour et al., 2006;Gerding et al., 2009). The molecular basis of these SIMPLE mutations in causing autosomal-dominant demyelination in CMT1C, however, is elusive.
The lack of knowledge on the role of SIMPLE further complicates the understanding of SIMPLE in CMT1C demyelination. Despite the proposal of SIMPLE as a transcription factor (Myokai et al., 1999;Bolcato-Bellemin et al., 2004;Tang et al., 2005), a transcriptional 2010; Rauch and Martin-Serrano, 2011). Thus we first ascertained localization of exogenous SIMPLE in transfected cells and found, as reported (Moriwaki et al., 2001;Eaton et al., 2011;Lee et al., 2011), punctated staining pattern with partial colocalization of SIMPLE with LAMP2 and Rab7 in lysosomes and late endosomes, respectively (Figure 1, B and C). Among the punctated staining pattern, we also found partial colocalization of SIMPLE and lysobisphosphatidic acid (LBPA), a specific lipid present in late endosomes/ MVBs ( Figure 1D). Minimal colocalizations in the punctae, however, were detected by SIMPLE and early endosome antigen-1 (EEA1) ( Figure 1E). Taken together, these data indicate that SIMPLE is present in late endosomes/MVBs and lysosomes.
We further performed immuno-electron microscopy to establish the intracellular localization of SIMPLE. As expected, SIMPLE was found in endosome-like tubular structures and lysosome-like, electron-dense granules ( Figure 1F). Surprisingly, we also found membrane-bound SIMPLE on intraluminal vesicles inside MVBs ( Figure  1F). These data confirm that SIMPLE is present in late endosomes and lysosomes. Unexpectedly, SIMPLE is also localized in intraluminal vesicles inside MVBs.

Localization of SIMPLE inside exosomes
Recent studies indicated that, upon fusion with plasma membrane, intraluminal vesicles inside MVBs can be secreted extracellularly as exosomes (Simons and Raposo, 2009;Huotari and Helenius, 2011). Exosomes, however, are different from microvesicles, which are budded directly from the plasma membranes. Given that SIMPLE localizes in intraluminal vesicles of MVBs, we hypothesized that SIM-PLE is secreted via the exosome pathway. To test this hypothesis, we performed differential ultracentrifugation on conditioned media to prepare exosome fractions for detection of endogenous SIMPLE. Conditioned media from NIH 3T3 mouse fibroblasts and primary mouse Schwann cells were examined ( Figure 2A). We found that endogenous SIMPLE was present in pelleted exosome fraction, but not in the supernatant, after ultracentrifugation at 100,000 × g ( Figure 2A). We also confirmed that flotillin, previously detected in exosomes (de Gassart et al., 2003), was present in pelleted exosome fraction after ultracentrifugation at 100,000 × g (Figure 2A). Because SIMPLE is widely expressed, we also detected SIMPLE in exosomes in rat primary Schwann cells, HepG2 liver carcinoma cells, MCF7 breast epithelial cancer cells, and COS monkey kidney cells (data not shown). Similarly, exogenously expressed SIMPLE (tagged with either FLAG or hemagglutinin [HA] epitope) and SIMPLE fused to enhanced green fluorescent protein (EGFP) were also targeted to exosomes ( Figure 2B), whereas EGFP alone was mainly found in the remaining supernatant after ultracentrifugation at 100,000 × g (data not shown). These data indicate that SIMPLE is secreted in exosomes in multiple different cell types.
We further performed sucrose gradient floatation assays to isolate exosomes based on their density ( Figure 2C). Sucrose gradient floatation assays indicated that endogenous SIMPLE was enriched in density ∼1.13 g/cm 3 , within the biochemical characteristic of exosomes ( Figure 2C). In addition, Alix, CD63 (LAMP3), and flotillin-1, previously known proteins secreted in exosomes (de Gassart et al., 2003), were also copurified with SIMPLE at the specific density of exosome fractions ( Figure 2C). More important, immuno-electron microscopy indicated that SIMPLE was associated with purified exosomes ( Figure 2D). These data indicate that SIMPLE is membrane bound and secreted in exosomes.
Our ultrastructural analysis indicated that SIMPLE seems to be localized inside the intraluminal vesicles ( Figure 1F). If so, SIMPLE would be protected and encircled inside secreted exosomes. To test capability of SIMPLE has not been reproduced (Moriwaki et al., 2001;Huang and Bennett, 2007;Lee et al., 2011;and data not shown). Indeed, punctated localization of SIMPLE in cells implies a role in endosomal regulation and vesicular trafficking.
The purpose of this study is to elucidate the role of SIMPLE and its molecular pathology in CMT1C. We report that SIMPLE resides within the intraluminal vesicles of MVBs and inside exosomes. We show that SIMPLE increases the production of exosomes, whereas CMT1C mutation causes improper formation of MVBs, accumulation of lysosomes, and reduced secretion of exosomes. We propose that SIMPLE is a previously unrecognized player in regulating the production of exosomes. Elevated intracellular stress or ineffective extracellular communications could account for the pathological consequences of CMT1C mutation.

Intracellular locations of SIMPLE
Previous studies indicated localization of SIMPLE in endosomal trafficking compartments (Moriwaki et al., 2001;Eaton et al., 2011;Lee et al., 2011). Indeed, signature motifs for endocytic functions, including di-Leu (LL) and YKRL motifs, are found on SIMPLE ( Figure  1A). In addition, SIMPLE encodes a PTAP motif for binding of ESCRT1 protein Tsg101 (Saifi et al., 2005;Shirk et al., 2005), a key component of multiprotein ESCRT complexes for the formation of MVBs (Falguières et al., 2009;Hurley, 2010;Henne et al., 2011;Rusten et al., 2012). SIMPLE also encodes PPxY motifs for binding of Nedd4 type E3 ubiquitin ligases (Jolliffe et al., 2000;Shirk et al., 2005;Eaton et al., 2011), key regulatory molecules in ubiquitinmediated signaling and viral budding (Timmins et al., 2003;Chen and Matesic, 2007;Zwang and Yarden, 2009 Quantification of exosome secretion using the C2 phosphatidylserine-binding domain of lactadherin The presence of signature motifs required for endocytic trafficking ( Figure 3) and the unique intraluminal position in MVBs and inside exosomes (Figures 1 and 2) suggested that SIMPLE might play a role in the formation of these nanovesicles. To determine whether SIMPLE modulates exosome production in transiently transfected this possibility, we performed immunoprecipitation assays ( Figure  2E). Purified exosomes were immunoprecipitated in either lysis buffer to disrupt exosomes or in phosphate-buffered saline (PBS) to keep exosomes intact. Immunoprecipitation assays indicated that SIMPLE could only be immunoprecipitated after lysis ( Figure 2E). We further performed limited trypsin digestion on purified exosomes to ascertain internal localization of endogenous SIMPLE ( Figure 2F). We found that endogenous SIMPLE located within exosomes was more resistant to limited trypsin digest as compared with other surface-bound exosome proteins, such as Hsp70 and Hsc70 ( Figure 2F). Indeed, SIMPLE present inside intact exosomes was more resistant to trypsin digestion than solubilized SIMPLE present in cell lysate ( Figure 2G). These data support the notion that SIMPLE is localized inside the exosomes.
Next we examined the role of signature binding motifs proposed for endocytic trafficking ( Figure 1A) in secretion of SIMPLE via exosomes ( Figure 3). Ala replacement of the di-Leu (LL) [LL 154,155 AA] and YKRL [YL 158,161 AA] motifs abolished secretion of SIMPLE ( Figure  3A). Similarly, Ala replacement of the PTAP motif [PP 17,20 AA], to abolish binding of ESCRT1 protein Tsg101, also reduced secretion of SIMPLE ( Figure 3B). SIMPLE also encodes two PPxY motifs for recruitment of Nedd4 type E3 ubiquitin ligases, such as Nedd4 and ITCH (Shirk et al., 2005;Eaton et al., 2011). Unexpectedly, Ala replacement of the PPxY motifs [YY 23,61 AA] potentiated the secretion of SIMPLE ( Figure 3B). These data indicate that signature motifs to quantify the amount of secreted exosomes (Supplemental Figure S1 and Figure  4). The rationale of using LactC2-fluorescence reporter in quantifying exosome release in transiently transfected cells is in parallel to the common use of promoter-luciferase reporter cassette in gene transcription assays.
Sucrose gradient floatation assays indicated that fluorescence signal derived from the LactC2-RFP was enriched in density where exosomes were localized, whereas only background fluorescence signal was detected in other fractions (Supplemental Figure S1A). These data indicate that LactC2-RFP is present in expected exosome fractions. In addition, fluorescence signal derived from the LactC2-RFP reporter was increased in media with higher amounts of transfected DNA plasmids (Supplemental Figure S1B), indicating dose dependence of the fluorescence reporter. More important, administration of the calcium ionophore ionomycin, which facilitates exosome release (Kramer-Albers et al., 2007), increased LactC2-RFP fluorescence signal in conditioned media, confirming the robustness of this transient assay (Supplemental Figure  S1C). Hence the fluorescence intensity of LactC2-RFP in conditioned media is responsive to stimuli that elicit exosome release.
We also examined whether LactC2-RFP affects secretion of known exosome proteins. Expression of LactC2-GFP in cells did not affect secretion of Alix while modestly reducing the level of flotillin in exosome fractions (Supplemental Figure S1D). The latter findings might be due to a squelching effect, which is known in using the promoterluciferase reporter cassette in transient transcription assays (Natesan et al., 1997). Thus recruitment of endogenous flotillin to the limiting membrane of exosomes might be competed by the overexpressed, exosometargeted LactC2-GFP reporter proteins. Indeed, LactC2-GFP was enriched in exosomes as compared with EGFP (Supplemental Figure S1D). Taken together, these data indicate that LactC2-RFP is a practical fluorescence reporter to quantify exosome production in transient transfection assays.

Increased formation of exosomes by SIMPLE
On cotransfection with varying amounts of exogenous SIMPLE in COS cells, we found increased LactC2-RFP fluorescence signal in conditioned media in a dose-dependent manner ( Figure 4A). Of note, administration of ionomycin provided a synergy effect and further augmented LactC2-RFP fluorescence signal in conditioned media harvested from cells expressing exogenous SIMPLE ( Figure 4A). Expression of cells, we used the C2 phosphatidylserine-binding domain isolated from lactadherin (LactC2; Yeung et al., 2008) in a fluorescence reporter assay (LactC2-red fluorescent protein [RFP] or LactC2-GFP) FIGURE 2: Localization of SIMPLE inside exosomes. (A) Conditioned media from NIH 3T3 fibroblasts and primary mouse Schwann cells were subjected to differential centrifugation to isolate exosome pellets. The presence of endogenous SIMPLE and flotillin was detected by immunoblotting. Five percent of supernatant (S) and 10% of SDS-solubilized pellets (P) were examined after each step of centrifugation. (B) COS cells were transiently transfected with different versions of epitope-tagged SIMPLE. Conditioned media from transfected cells were subjected to differential centrifugation to isolate exosome pellets. The presence of endogenous and exogenous SIMPLE in exosome fractions and in cell lysates was detected by immunoblotting. Forty percent of exosome fraction and 4% of cell lysate were examined. (C) Exosome pellets purified from conditioned media of rat primary Schwann cells were subjected to sucrose density gradient ultracentrifugation. Ten fractions were collected and the presence of endogenous SIMPLE, Alix, CD63, and flotillin were determined by immunoblotting. (D) Immuno-electron microscopy was performed to determine the localization of SIMPLE (arrowheads) in purified exosomes isolated from transfected COS cells. Scale bar, 100 nm. (E) Exosome pellets purified from conditioned media of HA-SIMPLE-transfected COS cells were subjected to immunoprecipitations. The presence of HA-SIMPLE in precipitates and remaining supernatant was determined by immunoblotting using antibody against the HA epitope. (F) Exosome pellets purified from conditioned media of COS cells were subjected to limited trypsin digestion. The presence of remaining endogenous SIMPLE, Hsp70, and Hsc70 was determined by immunoblotting. (G) Exosome pellets purified from conditioned media of COS cells or solubilized cell lysate were subjected to trypsin digestion. Levels of SIMPLE and ESCRT-0 protein Tom1 was determined by immunoblotting. different tagged versions of exogenous SIMPLE, as well as the Caenorhabditis elegans orthologue, also increased LactC2-RFP fluorescence reporter in conditioned media ( Figure 4B). We also confirmed increased secretion of LactC2 domain after the expression of exogenous SIMPLE, using immunoblotting to detect LactC2-GFP in exosomes ( Figure 4C). To quantify the amount of secreted exosomes in the presence and absence of exogenous SIMPLE, we further performed nanoparticle tracking analysis ( Figure 4D). Conditioned media harvested from cells expressing exogenous SIMPLE showed increased concentration of exosomes as compared with mock-transfected cells ( Figure 4D). In addition, CD63 and Alix, proteins previously found in exosomes, were enriched in exosomes isolated from cells expressing exogenous SIMPLE ( Figure 4E). The level of flotillin in exosomes, however, seems unaffected ( Figure 4E). Taken together, these data indicate that expression of SIMPLE increases the number of exosomes and secretion of specific exosome proteins.

Genetic models of SIMPLE
We sought to determine whether this newly established role for SIMPLE in exosome production might feature in the onset or progression of CMT1C. Thus we examined the effect of different CMT1C genotypes on exosome formation (Street et al., 2003;Saifi et al., 2005;Latour et al., 2006;Gerding et al., 2009). We introduced by gene targeting a single point mutation in floxed exon 3 of the Simple gene to convert the codon Thr115 (ACC) to that for Asn (AaC; T115N) and model CMT1C disease ( Figure 5, A and B). This knock-in approach was specifically selected to exclude the complications encountered with an overexpression model, particularly as the basis of dominant inheritance in CMT1C is unknown. Removal of floxed exon 3 was carried out by Cre-mediated recombination to generate SIMPLE-null mice (Simple −/− ). PCR analysis indicated successful creation of Simple T115N/+ , Simple T115N/T115N , and Simple +/− genotypes ( Figure 5, C-E). Immunoblotting analysis revealed similar levels of SIMPLE protein in primary mouse embryonic fibroblasts (MEFs) with Simple +/+ , Simple T115N/+ , and Simple T115N/T115N genotypes ( Figure 5F). In Simple −/− MEFs, expression of SIMPLE protein was not detected ( Figure 5F). With a physiological model that expresses mutated SIMPLE at endogenous levels, we asked whether the localization of SIMPLE in late endosomes/MVBs/lysosomes was affected upon CMT1C mutation ( Figure 6). We found similar punctated staining with partial colocalization of endogenous SIMPLE and LAMP1 in Simple +/+ , Simple T115N/+ , and Simple T115N/T115N MEFs ( Figure 6, A and B). Punctated staining of SIMPLE was not detected in Simple −/− MEFs ( Figure  6A), confirming the effective of ablation in Simple −/− ( Figure 5F). We further separated cytosolic and membrane fractions to ascertain the localization of endogenous SIMPLE upon CMT1C mutation ( Figure  6C). As expected, endogenous SIMPLE expressed in Simple +/+ and Simple T115N/+ MEFs was detected in membrane fraction along with known membrane-associated protein LAMP1 ( Figure 6C). Soluble proteins, such as Hsp70 and tubulin, were found exclusively in cytosolic fractions where no SIMPLE was detected ( Figure 6C). Small GTPases Rab5 and Rab7 were detected in both membrane and cytosolic fractions, as reported previously ( Figure 6C; Felberbaum-Corti et al., 2005;Wang et al., 2011). Taken together, these data indicate that neither the expression level of SIMPLE ( Figure 5F) nor its subcellular distribution ( Figure 6) is affected by CMT1C mutation.

CMT1C mutation impairs exosome localization of SIMPLE
Despite similar levels of endogenous SIMPLE in the cell lysates, we found that the amount of secreted SIMPLE present in exosomes secretion of CMT1C-mutated SIMPLE ( Figure 7E). These data indicate that the role of Nedd4 type E3 ligases dominates over CMT1C mutation in regulating exosome secretion of SIMPLE. Perhaps modulating Nedd4 type E3 ubiquitin ligases is beneficial for CMT1C.

Changes in the secretion of exosomes upon CMT1C mutation
Next we performed scanning electron microscopy to visualize secreted exosomes from Simple +/+ and Simple T115N/+ MEFs ( Figure 8A). We found that exosomes were readily detected in Simple +/+ MEFs. As expected, the size of exosomes isolated from Simple +/+ MEFs were ∼100 nm in diameter ( Figure 8A). Exosomes isolated from Simple T115N/+ MEFs, however, were smaller, and many of them were <40 nm in diameter ( Figure 8A). In addition to the smaller size, there were fewer exosomes in Simple T115N/+ MEFs ( Figure 8A). To quantify secreted exosomes from Simple +/+ and Simple T115N/+ MEFs, we carried out morphometric determination to count the number and determine the size of exosomes in multiple electron micrographs ( Figure 8B). We also performed nanoparticle tracking analysis to was reduced for Simple T115N/+ and Simple T115N/T115N MEFs versus Simple +/+ MEFs ( Figure 7A). Relative to the exogenous wild-type SIMPLE, similar reduction of secreted SIMPLE in exosomes was found in cells exogenously expressing other CMT1C isoforms [A 111 G, G 112 S, T 115 N, W 116 G, P 135 S, P 135 T and V 144 M] (Figure 7, B and C). Substitutions in SIMPLE, T 49 M and L 122 V, however, did not affect secretion of SIMPLE ( Figure 7B), suggesting that these changes might represent rare polymorphisms. More important, secretion of exogenously expressed, CMT1C-mutated SIMPLE was also greatly impaired as compared with its wild-type counterpart in rat primary Schwann cells ( Figure 7D). These data indicate that SIMPLE mutations strongly associated with CMT1C abrogate exosomal localization of SIMPLE.
We also tested whether removal of Nedd4-binding motif [YY 23,61 AA], which increased the presence of SIMPLE in exosomes (Figure 3)  (D) COS cells were mock transfected (Control) or transiently transfected with HA-SIMPLE. Nanoparticle tracking analysis was performed to quantify the amount of secreted exosomes present in conditioned media. *p < 0.05; # p < 0.005. (E) COS cells were transiently transfected with HA-SIMPLE and vector control. The levels of CD63, Alix, and flotillin in isolated exosomes were determined by immunoblotting. Expression levels of tubulin were used as controls. Ten percent of exosome fraction and 1% of cell lysate were examined.
prepared from Simple T115N/+ mice as compared with Simple +/+ mouse plasma ( Figure 8D). These data indicate that secretion of exosomes is compromised upon CMT1C mutation of endogenous SIMPLE. These data also support a role of SIMPLE in regulating exosome production ( Figure 4).
We also examined the level of exosome proteins secreted by Simple +/+ and Simple T115N/+ MEFs. We found that the level ascertain the changes in overall exosome profile of Simple +/+ and Simple T115N/+ MEFs ( Figure 8C). Morphometric determination ascertained that the bulk of exosomes secreted from Simple T115N/+ MEFs were smaller ( Figure 8B). In addition, morphometric determination and nanoparticle tracking analysis confirmed that fewer exosomes were secreted from Simple T115N/+ MEFs (Figure 8, B and C). Decreased exosome concentration was also found in mouse plasma were evident ( Figure 9A). These data indicate that CMT1C mutation of SIMPLE generates a vacuolated appearance in MVBs.
We also examined the cellular ultrastructure in Simple −/− and Simple T115N/T115N MEFs ( Figure 9A). Similar to Simple T115N/+ MEFs, typical multivesicular appearances of MVBs were rarely identified in Simple −/− and Simple T115N/T115N MEFs (Figure 9, A and B). Indeed, reduced exosome secretion and decreased levels of exosome proteins were found in Simple −/− and Simple T115N/T115N MEFs (Supplemental Figure S2). In Simple −/− MEFs, however, vacuolated appearance was not as evident as in Simple T115N/+ MEFs (Figure 9, A and B). Instead, the presence of multilamellar cisternal compartments and accumulation of electron-dense granules in Simple −/-MEFs were more apparent than with Simple T115N/+ MEFs (Figure 9, A and B).
In Simple T115N/T115N MEFs, vacuolated appearances in MVBs were manifested as in Simple T115N/+ MEFs (Figure 9, A and B). The presence of multilamellar cisternal compartments and accumulation of electron-dense granules in Simple T115N/T115N MEFs, however, were more evident than in Simple T115N/+ MEFs (Figure 9, A and B). Indeed, the appearances of multilamellar cisternal compartments and accumulation of electron-dense granules in Simple T115N/T115N MEFs were comparable to those in Simple −/-MEFs ( Figure 9B).
Taken together, these data demonstrate a role of SIMPLE in MVBs. In particular, all three genetic models (Simple T115N/+ , of known exosome proteins, including Alix, flotillin, and Hsp70, in Simple T115N/+ exosomes was reduced ( Figure 8E). The level of clathrin heavy chain in exosomes, however, was similar for both genotypes ( Figure 8E). These data indicate that SIMPLE regulates exosome production and also suggest that mutation of SIMPLE likely affects a subset of exosomes.

Defects in endosomal/lysosomal compartments upon CMT1C mutation
Exosomes are derived, in part, from the extracellular release of intraluminal vesicles upon fusion of MVBs with plasma membrane (Simons and Raposo, 2009;Huotari and Helenius, 2011). Given that SIMPLE resides inside intraluminal vesicles of MVBs, changes in the landscape of exosome profile upon CMT1C mutation might affect the formation of MVBs. Indeed, electron microscopy showed that typical multivesicular appearances of MVBs were rarely identified in Simple T115N/+ MEFs ( Figure 9A). Instead, in Simple T115N/+ MEFs, we found either the presence of empty vacuoles without obvious intraluminal vesicles or enlarged vacuoles containing electron-dense granules inside ( Figure 9A). Multilamellar cisternal compartments near the vacuoles, hallmarks of vps mutants with defective MVBs (Raymond et al., 1992;Rieder et al., 1996), were also found in Simple T115N/+ MEFs ( Figure 9A). In Simple +/+ MEFs, as expected, compartments containing intraluminal vesicles, indicative of MVBs, Arrows point to colocalization of SIMPLE and LAMP1. (C) Simple +/+ and Simple T115N/+ MEFs were extracted with hypotonic buffer to prepare cytosolic extract (S). Membrane fraction (P) was also collected after centrifugation at 100,000 × g. Localization of SIMPLE, LAMP1, Hsp70, tubulin, Rab5, and Rab7 in cytosol (S) and in membrane fraction (P) was determined by immunoblotting. electron microscopy ( Figure 9) and immunofluorescence assays ( Figure 10) indicate that CMT1C mutation elicits changes in structure and appearance in endosomes, MVBs, and lysosomes without disturbing the expression and localization of SIMPLE (Figures 5 and 6).

Reduced exosomal trafficking and aberrant endosomal/ lysosomal compartments in CMT1C patient B cells
Next we examined secretion of SIMPLE in exosomes from CMT1C patient B cells (Shirk et al., 2005) to compare it with results from CMT1C mouse cells. Analogous to the CMT1C MEFs ( Figure 5F), endogenous levels of SIMPLE protein were similar in control and CMT1C B cell lysates ( Figure 11A). As expected, control B cells showed secretion of SIMPLE via exosomes, which was further increased by ionomycin ( Figure 11A). The levels of secreted SIMPLE in exosomes, however, were reduced in CMT1C patient B cells, even in the presence of ionomycin ( Figure 11A). Scanning electron microscopy and morphometric determination, to count the number and determine the size of exosomes, further revealed fewer and smaller exosomes in CMT1C patient B cells (Figure 11, B and C). In addition, nanoparticle tracking analysis ascertained that the concentration of exosomes secreted by CMT1C patient B cells was reduced as compared with their control counterparts ( Figure 11D). Furthermore, the level of known exosome proteins, such as Alix and CD63, Simple −/− , and Simple T115N/T115N MEFs) show defects in exosome production ( Figure 8 and Supplemental Figure S2). Furthermore, these data indicate that CMT1C mutation of SIMPLE (Simple T115N/+ and Simple T115N/T115N ) leads to different ultrastructural changes as compared with the loss of expression of SIMPLE (Simple −/− ), suggesting the possibility that CMT1C-associated mutation may impart a partial toxic gain of function.
We also performed immunofluorescence analysis to examine the appearances of other endosomal compartments in Simple +/+ and Simple T115N/+ MEFs. Confocal microscopy indicated that LAMP1stained ( Figure 5H) or LAMP2-stained ( Figure 10, A and B) lysosomal compartments were more apparent and appeared enlarged in Simple T115N/+ MEFs as compared with Simple +/+ MEFs. Confocal microscopy also indicated more apparent staining of Rab7 in late endosomes in Simple T115N/+ MEFs ( Figure 10C). Rab7-stained, late endosomal compartments also appeared enlarged in Simple T115N/+ MEFs ( Figure 10C). LBPA-stained MVB compartments, however, seemed indistinguishable between Simple +/+ and Simple T115N/+ MEFs ( Figure  10, C and D). Early endosomes, as indicated by staining of EEA1, appeared diffused, with smaller punctae in Simple T115N/+ MEFs (Figure 10, B and D). Costaining of EEA1 with either LBPA or LAMP2 confirmed distinct compartmentalization of early endosomes, late endosomes, and lysosomes ( Figure 10, B and D). Taken together, Taken together, these data indicate compromised appearances in endosomes, MVBs, and lysosomes of CMT1C patient B cells.
We further ascertained morphological changes in endosomes/ MVBs in CMT1C patient B cells by electron microscopy. Representative structures of typical MVBs containing intraluminal vesicles were readily detected in control B cells ( Figure 12E). Ultrastructural analysis, however, revealed the presence of enlarged empty vacuoles without obvious intraluminal vesicles in CMT1C patient B cells (Figure 12, E and F). These data confirm that CMT1C mutation elicits defects in production of intraluminal vesicles and generates vacuolated appearances in MVBs.

Defects in endosomal/lysosomal compartments upon CMT1C mutation in primary Schwann cells
CMT1C neuropathy is a peripheral demyelinating disease in patients. Indeed, we observed that some mice harboring CMT1C mutations exhibit paralysis at old age (data not shown; also see Discussion). Defects in Schwann cells were suggested to account for the CMT1C neuropathy. To test whether CMT1C mutation also leads to defects in endosomal/lysosomal compartments in Schwann cells, was reduced in the exosomes of CMT1C patient B cells ( Figure 11E). Similar to Simple +/+ and Simple T115N/+ MEFs, the level of clathrin heavy chain in exosomes isolated from control and CMT1C patient B cells was similar ( Figure 11E). These data confirm that, in both mouse and human cells, CMT1C mutation reduces the localization of endogenous SIMPLE in exosomes without obvious effect on its expression. CMT1C mutation also causes production of smaller exosomes and reduced concentration of secreted exosomes.
CMT1C patient B cells also displayed changes in appearances in endosomal/lysosomal compartments, although localization of endogenous CMT1C-mutated SIMPLE was indistinguishable from wildtype SIMPLE in control cells, as suggested previously ( Figure 12A; Shirk et al., 2005). LAMP2-stained lysosomal compartments seemed more intense in CMT1C patient B cells as compared with control B cells ( Figure 12A). Staining of Rab7 in late endosomes was manifested in CMT1C patient B cells ( Figure 12B), whereas staining of LBPA in MVBs appeared similar (Figure 12, B and C). Indeed, increased Rab7stained puntae were evident in CMT1C patient B cells ( Figure 12D). Staining of EEA1 in early endosomes, however, showed discrete punctae with reduced intensity in CMT1C patient B cells ( Figure 12C).  we further used our mice harboring mutation of SIMPLE at the endogenous levels and examined ultrastructures of isolated primary Schwann cells by electron microscopy. Similar to Simple T115N/+ MEFs and CMT1C patient B cells, electron microscopy analysis revealed the presence of enlarged empty vacuoles without obvious intraluminal vesicles in Simple T115N/+ Schwann cells ( Figure 13A). Electrondense granules and multilamellar cisternal compartments in Simple T115N/+ Schwann cells were also evident ( Figure 13, A and B).
As expected, typical MVBs containing intraluminal vesicles were readily detected in Simple +/+ , but not in Simple T115N/+ , Schwann cells. Taken together, these data indicate that CMT1C mutation generates vacuolated appearances in MVBs, which contribute to defects in exosome secretion, in multiple cell types.

DISCUSSION
In this article, we show that SIMPLE regulates the production of exosomes. We also show that a single point mutation of endogenous SIMPLE in CMT1C mouse primary cells (MEFs and Schwann cells) and patient B cells alters exosome production. Improper formation of MVBs and accumulation of lysosomes, in part, contribute to the reduced production of exosomes by CMT1C mutation. These findings therefore demonstrate a role of SIMPLE in vesicular trafficking and for the first time a possible mechanistic understanding of CMT1C.

SIMPLE in vesicular trafficking
Our data indicate a role of SIMPLE in exosomes and formation of MVBs. Possible involvement of SIMPLE in microvesicles, however, cannot be excluded. Exosomes and microvesicles are different nanovesicles derived from distinctive cellular compartments. Current models imply that exosomes originate from MVBs, whereas microvesicles derive from shredding of plasma membranes (Huotari and Helenius, 2011;Mittelbrunn and Sanchez-Madrid, 2012). Unfortunately, biochemical characteristics of exosomes and microvesicles are highly similar, in part, due to an integrated circuit for continuous membrane trafficking. Thus it is difficult to identify a specific subset of proteins being secreted by exosomes but not by microvesicles (or vice versa). Given that SIMPLE exhibits punctated intracellular pattern with little staining on the plasma membrane ( Figure  6), we surmise that SIMPLE is mainly affecting exosome formation. The presence of signature binding motifs proposed for endocytic trafficking and their positive and negative roles in exosome secretion of SIMPLE also support this model (Figures 3  and 7). Malformation of MVBs in CMT1C mutation (Figures 9, 12, and 13) further supports a plausible role of SIMPLE in exosomes rather than microvesicles. Hence we propose that SIMPLE mainly plays a role in exosome formation. If our model is correct, CMT1C mutation will mainly affect exosomes, and remaining  Multiple signature motifs for endocytic functions are identified on SIMPLE ( Figure  1). These include the di-Leu (LL) and YKRL motifs for binding of AP complexes, the PTAP motif for binding of ESCRT1 protein Tsg101, the PPxY motifs for binding of Nedd4 type E3 ubiquitin ligases, and a cluster of specific CMT1C point mutation.
Our data indicate that these motifs positively and negatively contribute to the secretion of SIMPLE in exosomes (Figures 3  and 7). We surmise that proper location in the endocytic system, via protein-protein interactions with the LL, YKRL, or PTAP motifs, is critical for subsequent secretion of SIMPLE in exosomes. The PPxY motifs, however, seem to contribute a regulatory function to exosome secretion of SIMPLE. Our findings that SIMPLE encodes PTAP and PPxY regulatory motifs are in parallel with results on the arrestin domain-containing protein 1 (ARRDC1), which is mainly located on the plasma membrane (Nabhan et al., 2012). Nabhan et al. (2012) demonstrate that ARRDC1 recruits Tsg101 and WWP2, a member of the Nedd4 type E3 ubiquitin ligases, to regulate budding of microvesicles from the plasma membrane. These findings may be related to the role of SIMPLE in regulating exosome biogenesis on the endosomal membrane. Thus future investigations to examine the regulatory role of Nedd4 type E3 ubiquitin ligases and Tsg101 in secretion of exosomes/microvesicles are warranted.

SIMPLE in CMT1C
On mutation of endogenous SIMPLE in our CMT1C model, we find reduced secretion of exosomes (Figures 8 and 11). Reduced secretion of exosomes is corroborated by the altered structures in MVBs (Figures 9, 12, and 13). In addition to the reduced concentration of exosomes, scanning electron microscopy analysis reveals that the profile of exosomes secreted was skewed, and smaller exosomes were found upon CMT1C mutation of endogenous SIMPLE (Figures 8 and 11). Quantification of exosomes using nanoparticle tracking analysis, however, only suggests a distorted trend with no obvious enrichment of smaller exosomes upon CMT1C mutation (Figures 8 and 11). Quantification of biological nanovesicles using nanoparticle tracking analysis, unfortunately, is limited to ∼50 nm (Dragovic et al., 2011). Thus quantification of smaller exosomes using nanoparticle tracking analysis, despite being a powerful approach to examine the overall exosome profile, may be underrepresented. Nonetheless, future studies to examine the function and interaction of SIMPLE in late endosome/MVBs, where exosomes are formed, are warranted.
cargoes secreted from CMT1C cells likely will be derived from microvesicles. Future proteomic assays and microarray studies to identify such proteins and RNA species secreted (or not) by CMT1C cells will provide new insights into the biochemical differences between exosomes and microvesicles. Critical biomarkers to differentiate exosomes and microvesicles may be uncovered.
trafficking proteins, such as SIMPLE) in limiting intracellular membranes and on endosomal trafficking warrants careful examination and data interpretation.
Changes in the appearance of endosomal/lysosomal compartments and reduced exosome biogenesis were found upon CMT1C mutation (Simple T115N/+ and Simple T115N/T115N ) and in SIMPLE null (Simple −/− ; Figures 9, 10, 12, and 13 and Supplemental Figure S2). These genetic models therefore demonstrate a role of SIMPLE in late endosomes, in particular in the MVBs, and in exosome production. Morphometric determination of Simple T115N/+ and Simple −/− MEFs, however, reveals distinctive malformations in the ultrastructures (Figure 9). Vacuolated appearance is evident in Simple T115N/+ MEFs, whereas the presence of multilamellar cisternal compartments and accumulation of electron-dense granules are more pronounced in Simple −/− MEFs. In Simple T115N/T115N MEFs (Figure 9), electron microscopy reveals a combined phenotype with a vacuolated appearance, the presence of multilamellar cisternal compartments, and accumulation of electron-dense granules. These observations suggest that CMT1C mutation has an additional effect on the MVBs. Thus loss of expression as in SIMPLE null (Simple −/− ) might Despite the changes in appearance of endosomal/lysosomal compartments upon CMT1C mutation (Figures 9, 10, 12, and 13), we found endogenous wild-type and CMT1C-mutated SIMPLE proteins expressed to similar levels and exhibiting comparable punctated staining in MEFs and B cells (Figures 5, 6, 8, and 10-12). Subcellular fractionation of Simple +/+ and Simple T115N/+ MEFs further demonstrated the exclusive localization of SIMPLE in membrane fractions (Figure 6). Our observation that expression and localization of endogenous SIMPLE is unaffected by CMT1C mutation is in contrast to previous findings of mislocalization and aggregate accumulation in cells overexpressing exogenous CMT1C-mutated SIMPLE (Lee et al., 2011). Indeed, we also observed similar mislocalization and aggregate accumulation of overexpressed CMT1C mutated SIMPLE (data not shown). We surmise that mislocalization and aggregate accumulation of exogenous, CMT1C-mutated SIMPLE is likely an artifact due to overexpression. Similar anomalous observations due to overexpression were found when we expressed exogenous ESCRT protein Tom1 or Hrs in cells (data not shown; Urbe et al., 2000;Katoh et al., 2004;Tumbarello et al., 2012). Hence the peculiarity of overexpression of ESCRT machinery (or related and proposed a gain of toxic function of SIMPLE upon CMT1C mutation. In young CMT1C mice (<12 mo), we found increased hind limb clasping (data not shown). Electrophysiological experiments to determine nerve conduction velocity, however, were indistinguishable between wild-type and CMT1C mice (data not shown), despite the aforementioned phenotype. In addition, in a cohort of ∼50 mice carrying CMT1C mutations (Simple T115N/+ and Simple T115N/T115N mice), we observed that ∼15% of mice exhibited paralysis at old age (>18 mo; data not shown). No incidence of paralysis was found in age-matched Simple +/+ or Simple −/− mice (data not shown), as reported by Somandin et al. (2012). Locomotor defects found in old mice, however, are difficult to interpret due to the complications of age-related factors and deteriorated heath. The low frequency of paralysis in old CMT1C mice also compounds the difficulty in obtaining compelling data with statistical significance. Perhaps, similar to the SIMPLEnull mice (Somandin et al., 2012), CMT1C mice will exhibit neurological phenotypes upon challenge.

Vesicular trafficking and CMT
The role of SIMPLE in exosome production and its defects in CMT1C expand the repertoire of vesicular trafficking proteins whose mutation/deletion causes CMT diseases. Other vesicular trafficking proteins include Rab7, SH3TC2, MTMR2, MTMR13/SBF2, and FIG4, whose mutation/deletion accounts for CMT2B, CMT4C, CMT4B1, CMT4B2, and CMT4J, respectively (Senderek et al., 2003a,b;Verhoeven et al., 2003;Bolino et al., 2004;Meggouh et al., 2006;Chow et al., 2007;Previtali et al., 2007;Tersar et al., 2007;Spinosa et al., 2008;Lupo et al., 2009). Similar to SIMPLE, several of these proteins, such as Rab7, MTMR2, and FIG4, are widely expressed in many different cell types to regulate vesicular trafficking. The molecular rationale for the apparent vulnerability in the peripheral nervous system in CMT diseases, however, is elusive. Future characterization of the endocytic trafficking system in axonal-myelin maintenance and Schwann cell biology is warranted.
On the other hand, although largely uncharacterized, systems other than the peripheral nervous system may be affected in CMT1C patients. In particular, SIMPLE is widely expressed in many different tissues. In addition, SIMPLE is also named LITAF for its role in immune regulation and cytokine expression in sepsis (Tang et al., 2006;Srinivasan et al., 2010;Merrill et al., 2011). It remains elusive, however, whether CMT1C patients exhibit defects in immune regulation and function. It is hard to define pleiotropy within CMT1C patients, as there is significant clinical variability and onset even within families harboring the same mutation not fully recapitulate CMT1C mutation (Simple T115N/+ and Simple T115N/T115N ). Additional gain of function might be elicited upon mutation of SIMPLE. Further biochemical characterizations of the trafficking properties and functions in Simple T115N/+ , Simple −/− , and Simple T115N/T115N cells/mice will provide new mechanistic insights into the autosomal-dominant trait of CMT1C.
The plausible differences in the underlying mechanisms elicited by CMT1C mutation (Simple T115N/+ ) and ablation of SIMPLE (Simple −/− ) were also proposed in a recent study by Somandin et al. (2012), who found no obvious neuropathy in Simple −/− mice even at old age (∼18 mo). They surmised that the loss of SIMPLE expression could not account for neuropathy found in CMT1C patients 5-kb-long homology arm. The FNFL cassette conferred G418 resistance during gene targeting in PTL1 (129B6 hybrid) ES cells. Several targeted ES cells were identified and injected into C57BL/6 blastocysts to generate chimeric mice (chimeras). Male chimeras were bred to ACTB (Flpe/Flpe) females to transmit the floxed SIMPLE/T115N allele. Germline-transmitted SIMPLE/T115N mice (Simple T115N/+ , CMT1C mice) were mated with EIIa-Cre to delete exon 3 and generate Simple −/+ mice. Simple T115N/+ and Simple −/+ mice were backcrossed into C57BL/6 at least six times before use.

Cell culture
Primary mouse embryonic fibroblasts with genotypes of Simple +/+ , Simple T115N/+ , Simple T115N/T115N , and Simple −/− were isolated from E13.5 pups and cultured as described previously (Yang et al., 2006). Primary mouse Schwann cells were isolated from dorsal root ganglia of E13.5 pups and cultured as described previously (Yang et al., 2012). Primary rat Schwann cells and EBV-transformed B cells were cultured as described previously (Shirk et al., 2005;Maurel et al., 2007). Two different clones of control B cells (clones GM and 9) and two different clones of CMT1C B cells (clones 28 and 33) were examined. COS cells and HepG2 cells were cultured in DMEM and MEM, respectively. Transient transfections on COS and HepG2 cells were carried out using Lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer. All media were supplemented with 10% fetal calf serum, 2 mM l-glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml; Invitrogen). Cells were transfected by using Lipofectamine.

Confocal microscopy
Cells were plated on coverslips 24 h before the experiment. Cells were washed three times in cold PBS and fixed in 4% paraformaldehyde for 15 min. For staining of LBPA, cells were fixed and permeabilized in 3.7% (wt/vol) PFA and 0.1% glutaraldehyde in 0.15 mg/ml saponin solution prepared in fixative buffer (5 mM KCl, 137 mM NaCl, 4 mM NaHCO 3 , 0.4 mM KH 2 PO 4 , 1.1 mM Na 2 HPO 4 , 2 mM MgCl 2 , 5 mM 1,4-piperazinediethanesulfonic acid, pH 7.2, 2 mM ethylene glycol tetraacetic acid, and 5.5 mM glucose) for 40 min at 37°C. Paraformaldehyde was quenched by 0.1 M glycine in PBS for 10 min. After incubation in blocking solution (0.5% bovine serum albumin [BSA], 0.1% saponin, and 1% fetal bovine serum in PBS) the cells were incubated with primary antibodies at 4°C overnight, followed by secondary antibodies for 1 h at room temperature. All images were taken with a Leica AOBS SP2 confocal microscope (Leica, Wetzlar, Germany) with a 63× objective lens; primary representative images are shown.

Electron microscopy
Cells were fixed in a mixture of 2.5% glutaraldehyde and 4% paraformaldehyde in PBS for 2.5 h at room temperature (RT). After rinsing six times in PBS, cells were postfixed in 4% uranyl acetate at RT overnight and 1% osmium tetraoxide in PBS for 1 h at RT. Fixed cells were left in 70% ethanol overnight at 4°C and subjected to dehydration with ethanol. Once dehydrated, cells were transitioned twice with propylene oxide and then left in a 1:1 mixture of propylene oxide with Spurr's resin overnight at RT. After embedding and polymerization in Spurr's resin, 70-nm ultrathin sections were made on a Leica Ultracut Ultramicrotome (Leica Microsystems, Buffalo Grove, IL), and sections were collected on 200-mesh copper grids. Sections were poststained with 4% uranyl acetate for 60 min, rinsed, and examined with a FEI Tecnai G2 Twin Spirit Transmission electron microscope (FEI, Hillsboro, OR) operated at 80 kV. Digital images were obtained using an AMT digital camera (AMT, Woburn, MA). For (Bird, 1993;Bennett et al., 2004), which further complicates this issue. Further examination of CMT1C mice and SIMPLE-null mice therefore will provide physiological relevance for the function of SIMPLE.
Reduced production of exosomes by CMT1C-mutated SIMPLE would have intracellular and extracellular pathological consequences. Dysregulated endosomal trafficking and lysosomal stress would affect intracellular homeostasis (Saksena and Emr, 2009;Stuffers et al., 2009). On the other hand, reduced exosome secretion to extracellular milieu would alter intercellular communications (Thery et al., 2009;Mittelbrunn and Sanchez-Madrid, 2012). Of note, either intracellular stress or ineffective extracellular communications would lead to neurological dysfunctions (Jessen and Mirsky, 2008;Taveggia et al., 2010;Garden and La Spada, 2012). For example, it was shown that exosomes might play a prominent role in the propagation of α-synuclein-mediated Parkinson disease and transmission of prion proteins (Vella et al., 2007;Coleman et al., 2009;Emmanouilidou et al., 2010;Alvarez-Erviti et al., 2011;Hasegawa et al., 2011;Danzer et al., 2012). Future studies using Simple T115N/+ , Simple −/− , and Simple T115N/T115N cells/mice will provide new mechanistic insights to address whether intracellular and/or extracellular consequences account for the pathological changes in CMT1C demyelination and other neurodegenerative diseases.

Conclusion
We demonstrated that SIMPLE plays a critical role in exosome formation. Mutation of SIMPLE, which abolishes the proper formation of MVBs and exosome biogenesis, could elicit intracellular and extracellular consequences and place an overwhelming burden on the nervous system, accounting for CMT1C molecular pathogenesis.

Mice
Animal experiments were performed in accordance with the guidelines of the Albert Einstein College of Medicine Institute of Animal Studies. A C>A point mutation was introduced into codon 115 in exon 3 of the SIMPLE gene to mutate the amino acid from Thr to Asn (T115N). A loxP (L83) site and a Frt-Neo-Frt-loxP (FNFL) cassette were then engineered to flank T115N exon 3 of the SIMPLE gene to generate the "floxed/neo" SIMPLE/T115N allele. A gene-targeting vector was constructed by retrieving the 2-kb short homology arm, 1-kb sequence containing exon 3 with T115N mutation, FNFL cassette, and tracking analysis, harvested exosomes were resuspended in PBS, and the concentration of exosomes was determined by using the LM10 nanoparticle characterization system (NanoSight, Amesbury, United Kingdom) equipped with a blue laser (405 nm). Parameters for the settings on NanoSight were as follows: shutter, 500; gain, 256; capture, 60 s; threshold, 13; minimum expected particle size, 30 nm.
immuno-electron microscopy, cells were prepared as described, and sections were collected on nickel grids. Grids were submerged in beakers filled with 95°C sodium citrate buffer, pH 6.0, for 12.5 min. After cooling, grids were blocked in a mixture of 1% BSA with 10% goat serum in PBS at RT for 1 h. Grids were then incubated with primary antibody and secondary antibody conjugated with a 10-nm gold particles (Electron Microscopy Sciences, Hatfield, PA). Grids were rinsed and stained with 4% uranyl acetate for 1 h. Grids for immuno-electron microscopy were examined and imaged as described. For scanning electron microscopy to visualize exosomes, concentrated exosome fractions were resuspended in PBS and spotted onto polylysine-coated coverslips. After adsorption at RT for 40 min, coverslips were fixed with a mixture of 2.5% glutaraldehyde and 4% paraformaldehyde in PBS for 5 min. Coverslips were then rinsed with water and left to air dry overnight. Coverslips were then mounted onto an aluminum specimen stub and coated with gold. Exosomes were examined in an Amray 1910 Field Emission Scanning Electron Microscope operated between 5 and 7 kV (Amray, Bedford, MA). For immuno-electron microscopy labeling of exosomes, exosomes were absorbed into the grid as described. Grids were blocked in a mixture of 1% BSA with 10% goat serum and 0.4% Triton X-100 in PBS for 60 min before incubation with primary antibody at 4°C overnight. After rinses, grids were labeled with secondary antibody conjugated with 2-nm gold particles and silver enhanced using R-Gent SE-EM (Electron Microscopy Sciences). Exosomes were stained for 1 h using 4% uranyl acetate and examined as described. Morphometric determination of the ultrastructures was carried out by counting the appearances of MVBs, vacuoles, multilamellar cisternal compartments, and granules in at least 10 different photographs obtained from distinct sections of electron microscope (EM) grids. Similar morphometric determination was carried out to count the number and measure the size of exosomes in multiple EM photographs. The sum of each phenotype from counting multiple EM photographs is presented.

Exosome analysis
Conditioned media collected from cell culture supernatants were centrifuged at 1000 × g for 10 min and 10,000 × g for 40 min. The supernatant from the final centrifugation was subjected to ultracentrifugation at 100,000 × g for 1 h to isolate exosome pellet. Exosome pellet was resuspended in 0.25 M sucrose solution for ultracentrifugation in 0.25-2.5 M sucrose density gradient as described previously (Trajkovic et al., 2008). For immunoprecipitation assays, exosome pellet was resuspended in either PBS or Triton-lysis buffer (20 mM Tris, pH 7.4, 134 mM NaCl, 2 mM EDTA, 25 mM β-glycerophosphate, 2 mM NaPPi, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 10 μg/ml leupeptin) and subjected to immunoprecipitation using SIMPLE antibodies. For limited trypsin digest, exosome pellet was digested in 0.1% trypsin solution for 8 min at 37°C. For Western blotting, exosome pellet was resuspended in 1× Laemmli buffer and separated by SDS-PAGE for immunoblot analysis. For quantification of exosomes using LactC2-RFP reporter in transfected cells, cells were cotransfected with 0.2 μg of LactC2-RFP and varied amount of SIMPLE constructs (0.2-1 μg). After transfection, culture medium was replaced with phenol red-free DMEM to collect exosomes for 24 h. Collected medium were spun at 1000 × g for 5 min and then 10,000 × g for 40 min to remove cell debris. Isolated supernatant (200 μl) was transferred to a 96-well black-bottom plate. RFP signals in the isolated supernatant were detected by SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA) with excitation and emission at 517 and 610 nm, respectively. For nanoparticle