Extracellular α-synuclein impairs sphingosine 1-phosphate receptor type 3 (S1PR3)-regulated lysosomal delivery of cathepsin D in HeLa cells.

α-Synuclein (α-Syn)-positive intracellular fibrillar protein deposits, known as Lewy bodies, are thought to be involved in the pathogenesis of Parkinson's disease (PD). Although recent lines of evidence suggested that extracellular α-Syn secreted from pathogenic neurons contributes to the propagation of PD pathology, the precise mechanism of action remains unclear. We have reported that extracellular α-Syn caused sphingosine 1-phosphate (S1P) receptor type 1 (S1PR1) uncoupled from Gi and inhibited downstream G-protein signaling in SH-SY5Y cells, although its patho/physiological role remains to be clarified. Here we show that extracellular α-Syn caused S1P receptor type 3 (S1PR3) uncoupled from G protein in HeLa cells. Further studies indicated that α-Syn treatment reduced cathepsin D activity while enhancing the secretion of immature pro-cathepsin D into cell culture medium, suggesting that lysosomal delivery of cathepsin D was disturbed. Actually, extracellular α-Syn attenuated the retrograde trafficking of insulin-like growth factor-II/mannose 6-phosphate (IGF-II/M6P) receptor, which is under the regulation of S1PR3. These findings shed light on the understanding of dissemination of the PD pathology, that is, the mechanism underlying how extracellular α-Syn secreted from pathogenic cells causes lysosomal dysfunction of the neighboring healthy cells, leading to propagation of the disease.

Features and mechanisms of propofol-induced protein kinase C (PKC) translocation and activation in living cells.

Background and purpose: In this study, we aimed to elucidate the action mechanisms of propofol, particularly those underlying propofol-induced protein kinase C (PKC) translocation. Experimental approach: Various PKCs fused with green fluorescent protein (PKC-GFP) or other GFP-fused proteins were expressed in HeLa cells, and their propofol-induced dynamics were observed using confocal laser scanning microscopy. Propofol-induced PKC activation in cells was estimated using the C kinase activity receptor (CKAR), an indicator of intracellular PKC activation. We also examined PKC translocation using isomers and derivatives of propofol to identify the crucial structural motifs involved in this process. Key results: Propofol persistently translocated PKCα conventional PKCs and PKCδ from novel PKCs (nPKCs) to the plasma membrane (PM). Propofol translocated PKCδ and PKCη of nPKCs to the Golgi apparatus and endoplasmic reticulum, respectively. Propofol also induced the nuclear translocation of PKCζ of atypical PKCs or proteins other than PKCs, such that the protein concentration inside and outside the nucleus became uniform. CKAR analysis revealed that propofol activated PKC in the PM and Golgi apparatus. Moreover, tests using isomers and derivatives of propofol predicted that the structural motifs important for the induction of PKC and nuclear translocation are different. Conclusion and implications: Propofol induced the subtype-specific intracellular translocation of PKCs and activated PKCs. Additionally, propofol induced the nuclear translocation of PKCs and other proteins, probably by altering the permeability of the nuclear envelope. Interestingly, propofol-induced PKC and nuclear translocation may occur via different mechanisms. Our findings provide insights into the action mechanisms of propofol.

Identification of protein kinase C domains involved in its translocation induced by propofol.

Propofol is widely used for general anesthesia and sedation; however, the mechanisms of its anesthetic and adverse effects are not fully understood. We have previously shown that propofol activates protein kinase C (PKC) and induces its translocation in a subtype-specific manner. The purpose of this study was to identify the PKC domains involved in propofol-induced PKC translocation. The regulatory domains of PKC consist of C1 and C2 domains, and the C1 domain is subdivided into the C1A and C1B subdomains. Mutant PKCα and PKCδ with each domain deleted were fused with green fluorescent protein (GFP) and expressed in HeLa cells. Propofol-induced PKC translocation was observed by time-lapse imaging using a fluorescence microscope. The results showed that persistent propofol-induced PKC translocation to the plasma membrane was abolished by the deletion of both C1 and C2 domains in PKCα and by the deletion of the C1B domain in PKCδ. Therefore, propofol-induced PKC translocation involves the C1 and C2 domains of PKCα and the C1B domain of PKCδ. We also found that treatment with calphostin C, a C1 domain inhibitor, abolished propofol-induced PKCδ translocation. In addition, calphostin C inhibited the propofol-induced phosphorylation of endothelial nitric oxide synthase (eNOS). These results suggest that it may be possible to modulate the exertion of propofol effects by regulating the PKC domains involved in propofol-induced PKC translocation.

Constitutive activation of S1P receptors at the trans-Golgi network is required for surface transport carrier formation.

The importance of the G-protein ßγ subunits in the regulation of cargo transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is well accepted; however, the molecular mechanism underlying the G-protein activation at the TGN remains unclear. We show here that sphingosine 1-phosphate (S1P) receptors at the PM were trafficked to the TGN in response to a surface transport cargo, temperature-sensitive vesicular stomatitis virus glycoprotein tagged with green fluorescent protein accumulation in the Golgi. The receptor internalization occurred in an S1P-independent manner but required phosphorylation by G-protein receptor kinase 2 and ß-arrestin association before internalization. Continuously activated S1P receptors in a manner dependent on S1P at the TGN kept transmitting G-protein signals including the ßγ subunits supply necessary for transport carrier formation at the TGN destined for the PM.

Involvement of Receptor-Mediated S1P Signaling in EGF-Induced Macropinocytosis in COS7 Cells.

Macropinocytosis is a highly conserved cellular process of endocytosis by which extracellular fluid and nutrients are taken up into cells through large, heterogeneous vesicles known as macropinosomes. Growth factors such as epidermal growth factor (EGF) can induce macropinocytosis in many types of cells, although precise mechanism underlying EGF-induced macropinocytosis remains unclear. In the present studies we have shown the involvement of S1P signaling in EGF-induced macropinocytosis in COS7 cells. First, EGF-induced macropinocytosis was strongly impaired in sphingosine kinase isozymes, SphK1 or SphK2-depleted cells, which was completely rescued by the expression of the corresponding wild-type isozyme but not the catalytically inactive one, suggesting the involvement of sphingosine 1-phosphate (S1P) in this phenomenon. Next, we observed that EGF-induced macropinocytosis was strongly inhibited in S1P type 1 receptor (S1P1R)-knockdown cells, implying involvement of S1P1R in this event. Furthermore, we could successfully demonstrate EGF-induced trans-activation of S1P1R using one-molecular fluorescence resonance energy transfer (FRET) technique. Moreover, for EGF-induced Rac1 activation, a step essential to F-actin formation and subsequent macropinocytosis, S1P signaling is required for its full activation, as judged by FRET analysis. These findings indicate that growth factors such as EGF utilize receptor-mediated S1P signaling for the regulation of macropinocytosis to fulfil vital cell activity.

Apical-basal polarity inhibits epithelial-mesenchymal transition and tumour metastasis by PAR-complex-mediated SNAI1 degradation.

Loss of apical-basal polarity and activation of epithelial-mesenchymal transition (EMT) both contribute to carcinoma progression and metastasis. Here, we report that apical-basal polarity inhibits EMT to suppress metastatic dissemination. Using mouse and human epithelial three-dimensional organoid cultures, we show that the PAR-atypical protein kinase C (aPKC) polarity complex inhibits EMT and invasion by promoting degradation of the SNAIL family protein SNAI1. Under intact apical-basal polarity, aPKC kinases phosphorylate S249 of SNAI1, which leads to protein degradation. Loss of apical-basal polarity prevents aPKC-mediated SNAI1 phosphorylation and stabilizes the SNAI1 protein to promote EMT and invasion. In human breast tumour xenografts, inhibition of the PAR-complex-mediated SNAI1 degradation mechanism promotes tumour invasion and metastasis. Analyses of human breast tissue samples reveal negative correlations between PAR3 and SNAI1 protein levels. Our results demonstrate that apical-basal polarity functions as a critical checkpoint of EMT to precisely control epithelial-mesenchymal plasticity during tumour metastasis.

Activation of atypical protein kinase C by sphingosine 1-phosphate revealed by an aPKC-specific activity reporter.

Atypical protein kinase C (aPKC) isozymes are unique in the PKC superfamily in that they are not regulated by the lipid second messenger diacylglycerol, which has led to speculation about whether a different second messenger acutely controls their function. Here, using a genetically encoded reporter that we designed, aPKC-specific C kinase activity reporter (aCKAR), we found that the lipid mediator sphingosine 1-phosphate (S1P) promoted the cellular activity of aPKC. Intracellular S1P directly bound to the purified kinase domain of aPKC and relieved autoinhibitory constraints, thereby activating the kinase. In silico studies identified potential binding sites on the kinase domain, one of which was validated biochemically. In HeLa cells, S1P-dependent activation of aPKC suppressed apoptosis. Together, our findings identify a previously undescribed molecular mechanism of aPKC regulation, a molecular target for S1P in cell survival regulation, and a tool to further explore the biochemical and biological functions of aPKC.

Essential Role of Sphingosine Kinase 2 in the Regulation of Cargo Contents in the Exosomes from K562 Cells.

Sphingosine 1-phosphate (S1P) is a bioactive phosphorylated product of sphingosine catalyzed by sphingosine kinase (SphK) and implicated in diverse cellular functions including vesicular trafficking. In the present study we have shown the importance of one of the subtypes of SphK, SphK2, in the regulation of cargo content in exosomes released from human myeloid leukemia K562 cells. First, SphK2 has been shown to localize with N-Rh-PE-positive late endosomes in the cells. Next, siRNA-mediated knockdown of Sphk2 but not SphK1 resulted in a reduction of cargo content in purified exosomes. The involvement of SphK2 in this phenomenon was further investigated by pharmacological approaches. When cells were treated with N,N-dimethylsphingosine (DMS), one of the most frequently used inhibitors for SphK, cargo contents in purified exosomes were enhanced unexpectedly. Finally, it has been shown that DMS has a potency to stimulate SphK2 activity depending on the substrate sphingosine- and the inhibitor-doses as estimated by in vitro assay systems using a purified SphK2. These findings suggest that SphK2/S1P signaling plays an important role in the regulation of cargo content in exosomes in K562 cells.

Extracellular α-synuclein drives sphingosine 1-phosphate receptor subtype 1 out of lipid rafts, leading to impaired inhibitory G-protein signaling.

α-Synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, are thought to be involved in the pathogenesis of Lewy body diseases, such as Parkinson's disease (PD). Although growing evidence suggests that cell-to-cell transmission of α-Syn is associated with the progression of PD and that extracellular α-Syn promotes formation of inclusion bodies, its precise mechanism of action in the extracellular space remains unclear. Here, as indicated by both conventional fractionation techniques and FRET-based protein-protein interaction analysis, we demonstrate that extracellular α-Syn causes expulsion of sphingosine 1-phosphate receptor subtype 1 (S1P1R) from the lipid raft fractions. S1P1R regulates vesicular trafficking, and its expulsion involved α-Syn binding to membrane-surface gangliosides. Consequently, the S1P1R became refractory to S1P stimulation required for activating inhibitory G-protein (Gi) in the plasma membranes. Moreover, the extracellular α-Syn also induced uncoupling of the S1P1R on internal vesicles, resulting in the reduced amount of CD63 molecule (CD63) in the lumen of multivesicular endosomes, together with a decrease in CD63 in the released exosomes from α-Syn-treated cells. Furthermore, cholesterol-depleting agent-induced S1P1R expulsion from the rafts also resulted in S1P1R uncoupling. Taken together, these results suggest that extracellular α-Syn-induced expulsion of S1P1R from lipid rafts promotes the uncoupling of S1P1R from Gi, thereby blocking subsequent Gi signals, such as inhibition of cargo sorting into exosomal vesicles in multivesicular endosomes. These findings help shed additional light on PD pathogenesis.

Erratum: Impairment of PDGF-induced chemotaxis by extracellular α-synuclein through selective inhibition of Rac1 activation.

This corrects the article DOI: 10.1038/srep37810.

Erratum: Extracellular α-synuclein induces sphingosine 1-phosphate receptor subtype 1 uncoupled from inhibitory G-protein leaving ß-arrestin signal intact.

This corrects the article DOI: 10.1038/srep44248.

Quantitative Analysis of Cargo Density in Single-extracellular Vesicles by Imaging.

Function of extracellular vesicles such as exosomes and microvesicles is determined by their wide ranges of cargoes inside them. Even in the pure exosomes or microvesicles the cargo contents are very heterogeneous. To understand this heterogeneous nature of extracellular vesicles, we need information of the vesicles, which will give us some parameters including vesicle size, number and cargo content of each vesicle. Here, we describe a new method to quantify cargo density in single-extracellular vesicles. Staining of extracellular vesicles in a membrane lipid content-proportionate manner and immobilization of extracellular vesicles onto glass substrate allow us to obtain cargo density information of single-extracellular vesicles. This protocol will be useful to analyze the effects of various drugs or genetic manipulation on vesicle generation and maturation including cargo sorting into heterogeneous extracellular vesicles.

Involvement of Gßγ subunits of Gi protein coupled with S1P receptor on multivesicular endosomes in F-actin formation and cargo sorting into exosomes.

Exosomes play a critical role in cell-to-cell communication by delivering cargo molecules to recipient cells. However, the mechanism underlying the generation of the exosomal multivesicular endosome (MVE) is one of the mysteries in the field of endosome research. Although sphingolipid metabolites such as ceramide and sphingosine 1-phosphate (S1P) are known to play important roles in MVE formation and maturation, the detailed molecular mechanisms are still unclear. Here, we show that Rho family GTPases, including Cdc42 and Rac1, are constitutively activated on exosomal MVEs and are regulated by S1P signaling as measured by fluorescence resonance energy transfer (FRET)-based conformational changes. Moreover, we detected S1P signaling-induced filamentous actin (F-actin) formation. A selective inhibitor of Gßγ subunits, M119, strongly inhibited both F-actin formation on MVEs and cargo sorting into exosomal intralumenal vesicles of MVEs, both of which were fully rescued by the simultaneous expression of constitutively active Cdc42 and Rac1. Our results shed light on the mechanism underlying exosomal MVE maturation and inform the understanding of the physiological relevance of continuous activation of the S1P receptor and subsequent downstream G protein signaling to Gßγ subunits/Rho family GTPases-regulated F-actin formation on MVEs for cargo sorting into exosomal intralumenal vesicles.

DHHC5-mediated palmitoylation of S1P receptor subtype 1 determines G-protein coupling.

Sphingosine 1-phosphate (S1P) is a pleiotropic lipid mediator involved in the regulation of immune cell trafficking and vascular permeability acting mainly through G-protein-coupled S1P receptors (S1PRs). However, mechanism underlying how S1PRs are coupled with G-proteins remains unknown. Here we have uncovered that palmitoylation of a prototypical subtype S1P1R is prerequisite for subsequent inhibitory G-protein (Gi) coupling. We have identified DHHC5 as an enzyme for palmitoylation of S1P1R. Under basal conditions, S1P1R was functionally associated with DHHC5 in the plasma membranes (PM) and was fully palmitoylated, enabling Gi coupling. Upon stimulation, the receptor underwent internalisation leaving DHHC5 in PM, resulting in depalmitoylation of S1P1R. We also revealed that while physiological agonist S1P-induced endocytosed S1P1R readily recycled back to PM, pharmacological FTY720-P-induced endocytosed S1P1R-positive vesicles became associated with DHHC5 in the later phase, persistently transmitting Gi signals there. This indicates that FTY720-P switches off the S1P signal in PM, while switching on its signal continuously inside the cells. We propose that DHHC5-mediated palmitoylation of S1P1R determines Gi coupling and its signalling in a spatio/temporal manner.

Phospholipase D is Dispensable for Epidermal Growth Factor-Induced Chemotaxis.

α-Synuclein (α-Syn) is implicated in several neurodegenerative disorders, including Parkinson's disease, known collectively as the synucleinopathies. α-Syn is known to be secreted from the cells and may contribute to the progression of the disease. Although extracellular α-Syn is shown to impair platelet-derived growth factor-induced chemotaxis, molecular mechanism of α-Syn-induced motility failure remains elusive. Here we have aimed at phospholipase D (PLD) as a potential target for α-Syn and examined the involvement of this enzyme in α-Syn action. Indeed, extracellular α-Syn caused inhibition of agonist-induced PLD activation. However, inhibition of hydrolytic activity of PLD by 1-butanol treatment showed little or no effect on agonist-induced chemotaxis. These results suggest that some signaling pathways other than PLD may be involved in α-Syn-induced inhibition of chemotaxis.

Extracellular α-synuclein induces sphingosine 1-phosphate receptor subtype 1 uncoupled from inhibitory G-protein leaving ß-arrestin signal intact.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. The presence of α-synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, is the cytopathological hallmark of PD. Increasing bodies of evidence suggest that cell-to-cell transmission of α-Syn plays a role in the progression of PD. Although extracellular α-Syn is known to cause abnormal cell motility, the precise mechanism remains elusive. Here we show that impairment of platelet-derived growth factor-induced cell motility caused by extracellular α-Syn is mainly attributed to selective inhibition of sphingosine 1-phosphate (S1P) signalling. Treatment of human neuroblastoma cells with recombinant α-Syn caused S1P type 1 (S1P1) receptor-selective uncoupling from inhibitory G-protein (Gi) as determined by both functional and fluorescence resonance energy transfer (FRET)-based structural analyses. By contrast, α-Syn caused little or no effect on S1P2 receptor-mediated signalling. Both wild-type and α-Syn(A53T), a mutant found in familiar PD, caused uncoupling of S1P1 receptor, although α-Syn(A53T) showed stronger potency in uncoupling. Moreover, S1P1 receptor-mediated ß-arrestin signal was unaltered by α-Syn(A53T). These results suggest that exogenous α-Syn modulates S1P1 receptor-mediated signalling from both Gi and ß-arrestin signals into ß-arrestin-biased signal. These findings uncovered a novel function of exogenous α-Syn in the cells.

Impairment of PDGF-induced chemotaxis by extracellular α-synuclein through selective inhibition of Rac1 activation.

Parkinson's disease (PD) is characterized by α-synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies. Although it is known that extracellular α-Syn is detected in the plasma and cerebrospinal fluid, its physiological significance remains unclear. Here, we show that extracellular α-Syn suppresses platelet-derived growth factor (PDGF)-induced chemotaxis in human neuroblastoma SH-SY5Y cells. The inhibitory effect was stronger in the mutant α-Syn(A53T), found in hereditary PD, and the degree of inhibition was time-dependent, presumably because of the oligomerization of α-Syn. PDGF-induced activation of Akt or Erk was not influenced by α-Syn(A53T). Further studies revealed that α-Syn(A53T) inhibited PDGF-induced Rac1 activation, whereas Cdc42 activation remained unaffected, resulting in unbalanced actin filament remodeling. These results shed light on the understanding of pathological as well as physiological functions of extracellular α-Syn in neuronal cells.

Ongoing activation of sphingosine 1-phosphate receptors mediates maturation of exosomal multivesicular endosomes.

During late endosome maturation, cargo molecules are sorted into intralumenal vesicles (ILVs) of multivesicular endosomes (MVEs), and are either delivered to lysosomes for degradation or fused with the plasma membranes for exosome release. The mechanism underlying formation of exosomal ILVs and cargo sorting into ILVs destined for exosome release is still unclear. Here we show that inhibitory G protein (Gi)-coupled sphingosine 1-phosphate (S1P) receptors regulate exosomal MVE maturation. Gi-coupled S1P receptors on MVEs are constitutively activated through a constant supply of S1P via autocrine activation within organelles. We also found that the continuous activation of Gi-coupled S1P receptors on MVEs is essential for cargo sorting into ILVs destined for exosome release. Our results reveal a mechanism underlying ESCRT-independent maturation of exosomal MVEs.

Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells.

In mammals and birds, thermoregulation to conserve body temperature is vital to life. Multiple mechanisms of thermogeneration have been proposed, localized in different subcellular organelles. However, visualizing thermogenesis directly in intact organelles has been challenging. Here we have developed genetically encoded, GFP-based thermosensors (tsGFPs) that enable visualization of thermogenesis in discrete organelles in living cells. In tsGFPs, a tandem formation of coiled-coil structures of the Salmonella thermosensing protein TlpA transmits conformational changes to GFP to convert temperature changes into visible and quantifiable fluorescence changes. Specific targeting of tsGFPs enables visualization of thermogenesis in the mitochondria of brown adipocytes and the endoplasmic reticulum of myotubes. In HeLa cells, tsGFP targeted to mitochondria reveals heterogeneity in thermogenesis that correlates with the electrochemical gradient. Thus, tsGFPs are powerful tools to noninvasively assess thermogenesis in living cells.