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MLC1 trafficking and membrane expression in astrocytes: Role of caveolin-1and phosphorylation

Angela Lanciotti a, Maria Stefania Brignone a, Serena Camerini a, Barbara Serafini a, Gianfranco Macchia a,Carla Raggi b, Paola Molinari c, Marco Crescenzi a, Marco Musumeci c, Massimo Sargiacomo b,Francesca Aloisi a, Tamara Corinna Petrucci a, Elena Ambrosini a,⁎a Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italyb Department of Ematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italyc Department of Pharmacology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy

a b s t r a c ta r t i c l e i n f o

Article history:Received 24 June 2009Revised 23 October 2009Accepted 12 November 2009Available online 26 November 2009

Keywords:LeukoencephalopathyCaveolin-1AstrocytesMLCDystrophin glycoprotein complex (DGC)Glial cellsPKCPKAEndocytosisRafts

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophycaused by mutations in the MLC1 gene that encodes a membrane protein of unknown function. In the brainMLC1 protein is mainly expressed in astrocyte end-feet, localizes in lipid rafts and associates with thedystrophin glycoprotein complex (DGC). Using pull-down and co-fractionation assays in cultured humanand rat astrocytes, we show here that MLC1 intracellular domains pull-down the DGC proteins syntrophin,dystrobrevin, Kir4.1 and caveolin-1, the structural protein of caveolae, thereby supporting a role for DGC andcaveolar structures in MLC1 function. By immunostaining and subcellular fractionation of cultured rat orhuman astrocytes treated with agents modulating caveolin-mediated trafficking, we demonstrate that MLC1is also expressed in intracellular vesicles and endoplasmic reticulum and undergoes caveolae/raft-mediatedendocytosis. Inhibition of endocytosis, cholesterol lowering and protein kinases A- and C-mediated MLC1phosphorylation favour the expression of membrane-associated MLC1. Because pathological mutationsprevent MLC1 membrane expression, the identification of substances regulating MLC1 intracellulartrafficking is potentially relevant for the therapy of MLC.

© 2009 Elsevier Inc. All rights reserved.

Introduction

Megalencephalic leukoencephalopathy with subcortical cysts(MLC) is a rare congenital autosomal recessive leukodistrophycharacterized bymacrocephaly, whitematter swelling and subcorticalcysts associated with myelin vacuolation, blood–brain barrier damageand enlargement of extracellular spaces (Van der Knaap et al., 1995,1996; Singhal et al., 1996; Pascual-Castroviejo et al., 2005). Clinically,MLC patients manifest deterioration of motor functions, ataxia,spasticity, epileptic seizures and slow mental decline (Van der Knaapet al., 1996; Topku et al., 1998).

Almost all MLC patients carry mutations in a recently identifiedgene namedMLC1 (Topku et al. 2000; Leegwater et al., 2001). To date,about 50 different mutations have been described without a cleargenotype-phenotype correlation (Boor et al., 2006). The MLC1 geneencodes a membrane protein with unknown function, named MLC1that contains eight putative trans-membrane domains and shortamino- and carboxylic-cytoplasmic terminals (Leegwater et al., 2001;

Teijido et al., 2004; Boor et al., 2005). In the human brain, MLC1 isexpressed in astrocyte end-foot processes facing blood vessels andmeninges, ependymal cells lining the ventricles and Bergmann glia inthe cerebellum (Schmitt et al., 2003; Teijido et al., 2004; Boor et al.,2005; Ambrosini et al., 2008). In the mouse brain also some neuronalpopulations express MLC1 (Teijido et al., 2007).

MLC1 expression in astrocytes, but not in oligodendrocytes, themyelin forming cells, and brain damages consistent with fluid balancealterations in the brain of MLC patients suggest thatMLC pathogenesismight result from astrocyte dysfunction in the maintenance ofextracellular fluid homeostasis (Simard and Nedergaard, 2004). Dueto similarities in brain damages observed in MLC patients and inpatients affected by congenital muscular dystrophy with braininvolvement (Van Der Knaap et al., 1997; Boor et al. 2007), arelationship between MLC1 and the dystrophin-associated glycopro-tein complex (DGC) has been suggested. This multiprotein complex ismainly expressed in muscle tissue but also in astrocytes and neuronswhere it plays a role in brain development and in the regulation of ionand fluid homeostasis (Amiry-Moghaddam et al., 2004). Indeed, anassociation betweenMLC1 and some DGC proteins has been proposedin recent studies (Boor et al., 2007; Ambrosini et al., 2008). Moreover,we have found that a fraction of MLC1 localizes in specialized

Neurobiology of Disease 37 (2010) 581–595

⁎ Corresponding author. Fax: +39 0649387134.E-mail address: elena.ambrosini@iss.it (E. Ambrosini).Available online on ScienceDirect (www.sciencedirect.com).

0969-9961/$ – see front matter © 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.nbd.2009.11.008

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membrane microenvironments, the caveolar-lipid rafts (Ambrosiniet al., 2008), which are involved in protein trafficking and formation ofsignalling complexes (Head and Insel, 2007; Silva et al., 2007).

Recent findings indicating that pathological MLC1 mutationshamper MLC1 expression in astrocytic membranes (Duarri et al.,2008) point to the importance of elucidating the mechanismsunderlying MLC1 intracellular trafficking and membrane expressionfor therapeutic purposes. Using a combined biochemical, proteomicand immunocytochemical approach inwhole brain tissue andastrocytecultures, in this study we sought to better understand the relationshipbetween MLC1, DGC components and caveolin-1 (cav-1), the mainproteic component of caveolar structures. Furthermore, we investigat-ed the possible role of cav-1, which has been reported to regulate thetraffic of some membrane proteins in several cell types includingastrocytes (Silva et al., 2007), and of MLC1 structural modifications inMLC1 intracellular trafficking andmembrane localization in astrocytes.

Material and methods

Cell cultures and treatments

Astrocyte-enriched cultures (95% purity) were generated from 1-to 2-day-old newborn rats, as previously described (Agresti et al.,1991). Cells weremaintained in culture in DMEMmedium (Euroclone,UK) supplemented with 10% FCS (Gibco, BRL, Gaithersburg, MD, USA)and antibiotics (penicillin/streptomycin, Euroclone) in 5% CO2

atmosphere. For cell treatments, astrocytes plated in polylysinated60-mmdiameter dishes were washed in serum-free (SF) medium andincubated in SF medium plus different concentrations of stimulatoryagents for 1 h at 37 °C. The following agents were used: beta-methyl-cyclodextrin (BMC) (20 mM), cholesterol (5 mM), genistein (100 μg/ml), okadaic acid (100–300 nM), nocodazole (40 μM), phorbol 12-myristate 13-acetate (PMA) (500 pM to 500 μM), and forskolin (1 and10 μM) (all from Sigma Aldrich, St. Louis, MO). After stimulation, cellswerewashed in PBS, collected by scraping, and centrifuged at 2,700×gat 4 °C for 20 min. Cell pellets were solubilized as described below. Forimmunofluorescence staining, cells were treated with the sameconcentrations of stimulatory agents, except for BMC that was usedat a concentration of 10 mM.

Cell transfection

Subconfluent primary astrocytes grown on polylysinated glasscoverslips were transfected with a cav-1–GFP encoding vector previ-ously generated (Molinari et al., 2008) using Lipofectamine reagent(Gibco BRL), following manufacturer instructions. At 48 h post-transfection, cells were fixed with 4% paraformaldehyde and immu-nostained as described below.

Generation of an astrocytoma cell-line overexpressing cav-1

The sequence encoding cav1-GFP-tagged protein derived fromcav-1–GFP encoding vector (Molinari et al., 2008) was subcloned intothe retroviral expression vector pQCXIH (Clontech). The latter vectorwas used to generate retroviral particles by transfecting a HEK 293based packaging cell line and following the manufacturer instruction(Clontech). U251 astrocytoma cell line stably expressing His-MLC1and overexpressing cav1-GFP was obtained by infecting cells with theretroviral lysate as described previously (Molinari et al., 2008) andmaintained in culture in presence of the selection medium containinghygromycin.

Biochemical enrichment of histidine-tagged proteins

An astrocytoma cell line stably overexpressing histidine-taggedMLC1 (U251-HisMLC1)was generated as describedpreviously (Ambro-

sini et al., 2008). Pellets obtained from the astrocytoma cell line (about1×107 cells) were resuspended in buffer A (150 mM NaCl, 10 mMHepes, 1 mM EGTA, 0.1 mM MgCl2 pH 7.4), 0.5% Triton X-100 and aprotease inhibitor cocktail (Sigma), incubated on ice for 20 min andthen centrifuged at 16,000×g for 10 min at 4 °C. The supernatantsobtained were incubated overnight at 4 °C with 100 μl (50% v/vsuspension) of Ni-NTA Agarose (Qiagen, Hilden, Germany) and thenextensively washed with a buffer containing 10 mM imidazole, 20mMTris–HCl pH 7.4, 150 mM NaCl, and 0.2% Triton X-100. Elution wascarried out using imidazole at a concentration ranging from 50 to200 mM, and the eluted proteins were analyzed by SDS–PAGE andWestern blot (WB).

Recombinant protein preparation and pull-down assays

DNA coding for the N-terminal and C-terminal cytoplasmic regionsof human MLC1 (aa 1–56 and 320–377, respectively) were amplifiedby RT-PCR from human brain cDNA and cloned in pGEX-6P-3 vector inframe with the GST protein (Amersham-Pharmacia-Biotech, Piscat-away, NJ) using XhoI/EcorI restriction sites. The constructs weresequenced to confirm the in frame insertion of the GST-MLC1 domainsand used to transform BL21 E. coli cells. To express GST-fusedproteins, cells were incubated for 3 h at 37 °C in the presence of 1 mMIPTG. GST-fusion proteins were purified by affinity chromatographyon GST-Bind™ Resin (Novagen, Madison, WI) following the manufac-turer's instructions. GST and GST fusion proteins were used in in vitroprotein-binding assays, as described previously (Kaelin et al., 1991),with the followingmodifications: primary rat astrocytes were lysed inbuffer containing 1% Triton X-100, 0.5% sodium deoxycholate,150 mM NaCl, 10 mM Hepes (pH 7.4) and protease inhibitor cocktail.Lysates were passed through a 26-gauge needle, incubated in ice for20 min and centrifuged at 15,000×g for 20 min at 4 °C. Thesupernatant was pre-cleared by incubation with the GST-bound toglutathione–agarose overnight at 4 °C and then incubated withagarose-bound GST-MLC1 N-terminal or C-terminal regions withgentle rocking for 2 h at 4 °C. Following exhaustive washes with 0.2%Triton X-100, 0.15 M NaCl, 10 mM Hepes pH 7.4, 1 mM DTT andinhibitor protein cocktail, protein-bound beads were eluted with 0.1 Mglycine pH 3 or 1% SDS. Aliquots (0.5 ml) of eluted proteins wereprecipitated with acetone (1:4 v/v) and analysed by SDS–PAGE andWB. In some experiments, we performed pull-down assays by usinga caveolin-1 enriched protein extract derived from pneumocytesobtained as previously described (Lisanti et al., 1994).

Immunofluorescence and confocal microscopy analysis

Control or stimulated astrocytes (see above for the concentrationof stimulatory agents) were grown on polylysinated coverslips, fixedfor 10 min with 4% paraformaldehyde and washed with PBS. After 1 hof incubation with blocking solution (5% BSA in PBS), cells wereincubated for 1 h at RT with the following primary antibodies (Abs)diluted in PBS: affinity purified anti-MLC1 polyclonal Ab (1:50, AtlasAB, AlbaNova University Center, Stockholm, Sweden), anti-GFAPmonoclonal Ab (mAb) (1:100, BD Transduction Laboratories, Lex-ington, KY), Alexa-fluor (488)-conjugated Phalloidin (1:100, Molec-ular Probe, Inc., Eugene OR), Alexa-fluor (555)-conjugated anti-His(1:100, Qiagen), anti-EEA1mAb (1:50, BD Transduction Laboratories),anti-Clatrin heavy chain mAb (1:40, BD Transduction Laboratories),anti-LAMP-1 mAb (1:40, Developmental Studies Hybridoma Bank,University of Iowa, Iowa City, IA), anti-BiP/GRP78 mAb (1:40, BDTransduction Laboratories), anti-TGN38 mAb (1:40, BD TransductionLaboratories), anti-tubulinmAb (1:40, Molecular Probes, Eugene, OR),anti-cav-1 polyclonal Ab (1:50, Santa Cruz Biotechnology, Inc., SantaCruz, CA) or anti-pentaHis mAb (1:100, Qiagen). Cells were thenincubated for 45 min at RT with biotinilated secondary antibody at aconcentration of 4.3 μg/ml (Biotin-SP-AffiniPure goat anti-rabbit IgG

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H+L; Jackson Immunoresearch Laboratories, West Grove, PA) in PBS+2% normal goat serum, followed by 30-min incubation at RT with 2 μg/ml streptavidin-TRITC (Jackson, UK) in PBS. To reveal mAb binding, aFITC-conjugated donkey anti-mouse (1:100, Jackson, UK) was used.Coverslips were washed, sealed in Vectashield medium (Vector Lab,Burlingame, CA) and analyzed with a laser scanning confocal micro-scope (LSM 5 Pascal, Carl Zeiss).

Brain and astrocyte subcellular fractionation procedures

Brains dissected from Wistar rats were immediately frozen inliquid nitrogen and stored at −80 °C. The brains were homogenizedon ice in buffer A containing 50 mM Tris–HCl (pH 7.4), 1 mM EDTA,1 mM EGTA, 1 mM DTT and protease inhibitor cocktail (4.5 ml ofbuffer for 1.5 g of tissue). The homogenized extract was centrifugedfor 15 min at 3000×g at 4 °C, the cytosolic supernatant #1 wasrecovered and the membrane pellet was resuspended in lysis buffer Bcontaining 1% Triton X-100, 0.5% sodium deoxycholate, 150 mM NaCl,20 mM Hepes (pH 7.4), and protease inhibitor cocktail. Lysates werepassed through a 26-gauge needle, incubated on ice for 20 min andcentrifuged at 15,000×g for 20 min at 4 °C to obtain supernatant #2.The supernatants were analyzed by SDS–gel electrophoresis and WB.In some experiments, cytosolic supernatant #1 was ultracentrifugedat 100,000×g for 2 h at 4 °C before WB analysis. To separate cytosolicand membrane fractions from cultured astrocytes, pellets derivedfrom at least 8×106 cells were extracted with the same protocol using50 μl buffer/1×106 cells. In the case of protein phosphatasetreatment, 30 μl of the cytosolic andmembrane fractions were treatedwith 0.05 U/ml alkaline phosphatase (Roche Diagnostic GmbH,Mannheim, Germany) for 30 min at 37 °C.

Electrophoresis and Western blotting

Protein samples were separated using gradient (4–12%) precastedgels (Invitrogen) and gels were transferred to nitrocellulose (What-man GmbH, Dassel, Germany). Membranes were then incubatedovernight at 4 °C with the following Abs: anti-MLC1 pAb (1:500, inhouse generated), anti-cav-1 pAb (1:1000, Upstate ), anti-Kir4.1 pAb(1:400, Alomone), anti-syntrophin mAb (1:2000, MA-1-745, AffinityBioReagents), anti-β-DG mAb (1:25, NCL-43 DAG, NovocastraLaboratories Ltd., Newcastle Upon Tyne, UK), anti-cav-2 mAb(1:1000, Santa Cruz Biotecnology, INC), anti-cav-3 mAb (1:1000,Santa Cruz Biotecnology), anti-EEA1 mAb (1:5000, BD TransductionLaboratories), anti-GRP78/BiP mAb (1:250, BD Transduction Labora-tories), anti-flotillin mAb (1:1000, BD Transduction Laboratories),anti-dystrobrevin mAb (1:750, BD Transduction Laboratories), anti-Rab5 (FL-215) pAb (1:1000, Santa Cruz Biotecnology), anti-Rab11 (H-87) pAb (1:1000, Santa Cruz Biotecnology), and anti-tubulin mAb(1:1000, Molecular Probes), in PBS + 3% BSA and then incubated for1 h with horseradish peroxidase-conjugated anti-mouse/rabbit Abs(1:500; Pierce, Rockford, IL), for 1 h at RT. Immunoreactive bandswere visualized using an enhanced chemiluminescence reagent(Pierce), and exposed on X-ray films.

In vitro phosphorylation assays

Agarose-bound-, GST-MLC1 N-terminal and GST-MLC C-terminalregions were phosphorylated in vitro by protein kinase A catalyticsubunit (PKA) or protein kinase C (PKC) (both from Promega,Madison, WI). Briefly, 100 μl (50% v/v) suspensions of agarose-bound proteins were equilibrated in protein kinase buffer containing40mMTris–HCl pH 7.4; 20mMMg acetate; 0.2 mMATP/10 μCi γ[32P]ATP (3.000 Ci/mmol) for PKA and 20 mM Hepes pH 7.4; 1.67 mMCaCl2; 1 mM DTT; 10 mM MgCl2; 0.15 mM ATP/10 μCi 32P-γATP(3000 Ci/mmol); 0.6 mg/ml phosphatidyl serine for PKC, respective-ly, and incubated for 1 h at 30 °C with 100 units of PKA, or incubated

for 30 min at 30 °C with 0.4 units of PKC. After extensive washes withPBS, proteins were analyzed by SDS–PAGE. The gel stained withCoomassie blue was dried and radioactive bands were revealed byautoradiography. In phosphorylated protein samples for MALDI-ToFanalysis and nanoflow reversed-phase liquid chromatography tan-dem mass spectrometry (RP-LC-MS/MS) (see Supplementary mate-rial for experimental procedures), γ[32P]ATP was omitted in theincubation mixture.

Results

MLC1 protein derived from human astrocytoma cells interacts with DGCcomponents and caveolin-1

To elucidate the relationship between MLC1 and DGC proteins, weperformed a co-purification assay in which we evaluated the presenceof some DGC components and of the associated protein cav-1 insamples enriched in MLC1 protein. Because neither our in-housedeveloped nor the commercially available anti-MLC1 pAbwere able toimmunoprecipitate MLC1, we used an astrocytoma cell line (U251)over-expressing histidine (His)-tagged MLC1 (Ambrosini et al., 2008)and the NiNTA agarose resin that specifically binds His taggedproteins to enrich His-MLC1 and its interactors from cell extracts.WB of the samples obtained from His-purification indicated that boththe previously described higher (60 kDa) and lower (36 kDa)molecular weight (MW) MLC1 main components (Teijido et al.,2004; Ambrosini et al. 2008) were strongly enriched in samplesderived from His-MLC1-U251 cells but not from the parental cell lineused as control (Fig. 1A). Using specific antibodies against some of theDGC proteins, we found that dystrobrevin, syntrophin and Kir4.1, butnot β-DG, co-purified with His-MLC1 (Fig. 1B). These data indicatethat MLC1 likely associates with some of the cytoplasmic componentsof the DGC and not with the transmembrane protein β-DG. In linewith our previous finding that in astrocytes MLC1 localizes indetergent resistant caveolar lipid-raft domains together with DGCproteins and cav-1 (Ambrosini et al., 2008), we found that also cav-1co-purified with His-tagged MLC1 (Fig. 1B).

Association of MLC1 intracellular N- and C-terminal domains with DGCcomponents and caveolin oligomers

The finding that MLC1 interacts with some of the cytosoliccomponents of the DGC and with cav-1 prompted us to identify thespecific MLC1 domains involved in these associations. To this purpose,the N-terminal (aa 1–55) and the C-terminal (aa 322–377) of thehuman MLC1, both of which are localized intracellularly (Boor et al.,2005), were expressed as GST-fused proteins and used to pull-downproteins from cultured rat astrocyte extracts. WB analysis of the pull-down assay-derived samples indicated that, similarly to what wasobserved in astrocytoma cells, also in normal astrocytes dystrobrevin,Kir4.1 and syntrophin, but not β-DG, interact with MLC1. GST-fusedNH-terminal, but not control GST–protein alone, pulled-downdystrobrevin, syntrophin and Kir4.1 (Fig. 2A). Among the differentKir4.1 forms recognized by the anti Kir4.1 mAb, only the Kir4.1 dimerwas detected in the pulled-down sample (Fig. 2A, arrow). Syntrophinand Kir4.1 were also detected using GST-MLC1 C-terminal peptide(Fig. 2A). These data indicate that the above-mentioned DGCcomponents may bind to both MLC1 intracellular domains. Alterna-tively or in addition, it is possible that the result of this experimentmight be due to the formation of dimeric or oligomeric structuresbetween recombinant MLC1 C- and/or N-terminal domains andnative MLC1 present in astrocytic extracts which could leave the N- or-C-terminals of the latter available to interact with DGC proteins. Ifthis were the case, we cannot exclude that other regions of the MLC1protein could be involved in the binding of DGC components. Asshown in Fig. 2A, we also observed the interaction of the N- and C-

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terminal MLC1 domains with α-tubulin, the main component ofmicrotubules that is involved in protein distribution in caveolar-lipidraft compartments and in the trafficking of caveolar structures(Mundy et al., 2002; Head and Insel, 2007). In the same experiments,we found that cav-1 binds both MLC1 intracellular domains, whilecav-2, another caveolin isoform, which in most cells forms oligomericcomplexes with cav-1 (Zschocke et al., 2005; Silva et al., 2007), wasdetected only among proteins interacting with the MLC1 C-domain(Fig. 2B). Conversely, MLC1 intracellular domains did not pull-downcav-3, the muscle specific cav isoform that is also expressed inastrocytes (Ikezu et al., 1998; Silva et al., 2005, 2007). When using 1%of SDS instead of glycine (0.1 M, pH 3) to elute GST-MLC1-boundproteins we were able to solubilize and reveal both cav-1 isoforms αand β (Fig. 2B, arrows). With this protocol, we also obtainedadditional cav-1 immunoreactive bands of higher molecular weight(about 40 kDa) (Fig. 2B) which likely represent cav-1 homo and/orhetero-oligomers (Lisanti et al., 1993; Sargiacomo et al., 1995).

To understand whether MLC1 and cav-1 interacted directly weperformed a pull-down assay using recombinant His-tagged MLC1and GST–cav-1 but failed to detect an interaction between the tworecombinant proteins (data not shown). Due to the possibility thatMLC1–cav-1 interaction requires that cav-1 is in the native oligomericand/or post-translationally modified form, we used GST-MLC1 N- andC-terminal proteins to pull-down cav-1 from an enriched preparationof caveolins obtained frommouse pneumocytes, cells that abundantlyexpress cav-1 (Lisanti et al., 1994). We found that both GST-MLC1 N-and C-terminals, but not GST alone, interact preferentially withcaveolar hetero-oligomeric structures that are highly enriched in thepulled-down samples and, to a lesser extent, with the cav-1 monomer

(Fig. 2C). Although it cannot be excluded that some additionalproteins or lipids in the pneumocyte enriched cav-1 fraction mightmediate MLC1–cav-1 interaction, this result strongly suggests thatMLC1 interacts with cav-1 through its intracellular domains.

MLC1 co-immunolocalizes with cav-1 in intracellular vesicles inastrocytes

To understand the relationship between MLC1 and cav-1 inastrocytes we next performed immunolocalization experiments.Because the available anti-cav-1 mAb failed to detect cav-1 in normalastrocytes and an anti-MLC1 mAb is not available yet, we used ratastrocytes transiently transfected with a plasmid expressing cav-1tagged with GFP at its COOH terminal. C-terminally GFP-tagged cav-1was previously shown to be a reliable marker for endogenous cav-1(Pelkmans et al., 2001; Mundy et al., 2002). Immunofluorescencestaining with an anti-MLC1 pAb raised against the intracellular MLC1-NH domain revealed that MLC1 immunoreactivity was present inintracellular vesicles, mainly distributed in the perinuclear area, andcolocalized with cav-1–GFP (Fig. 3A). The predominant perinucleardistribution of cav-1 in astroglial cells has been described previously(Cameron et al., 1997; Ikezu et al., 1998). To exclude possible artefactsdue to GFP–cav-1 overexpression, we also verified the MLC1–cav-1colocalization in the stably transfected human astrocytoma cell lineexpressing His-tagged MLC1 by using an anti-cav-1 pAb coupled withanti-HismAb to reveal His-taggedMLC1. In agreementwith the resultsobtained with transiently transfected primary astrocytes, MLC1 andcav-1were found to colocalizemainly in intracellular vesicles and veryrarely at the plasma membrane level (Fig. 3B and arrow).

Fig. 1. Histidine(His)-tagged MLC1 protein co-fractionates with DGC components and caveolin-1 (cav-1). (A) WB analysis of protein samples derived from U251 astrocytoma cellsand from U251 expressing His-tagged MLC1 after Ni-NTA agarose resin purification used to enrich His-MLC1 protein. The 60-kDa and 36-kDa MLC1 components are enriched inU251-His-MLC1-derived samples but not in those from the parental cell line U251 used as control for non-specific protein binding to the Ni-NTA resin. One representativeexperiment out of two is shown. (B)WB of the His-MLC1-enriched samples eluted from Ni-NTA agarose with 50 and 200mM Imidazole using specific Abs against DGC components.Dystrobrevin (DB), syntrophin (Synt), Kir4.1 and cav-1, but not β-dystroglycan (β-DG), co-elute with MLC1. The Ab against Kir4.1 stains several bands: the monomer, dimerand tetramer are indicated by arrowheads. The asterisk in panel A indicates unspecific bands recognized by anti-MLC1 Ab. The asterisks in panel B indicate unspecific bandsrecognized by anti-Kir4.1Ab. One representative experiment out of three is shown.

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Immunocytochemical analysis of the intracellular distribution of MLC1in rat astrocytes

The cav-1–GFP distribution observed in the above experimentswas comparable with the caveolar endocytic vesicular structurespresent in the cytosol, also termed caveosomes, that act as anintermediate station in caveolar endocytosis and have been describedin stably cav-1–GFP transfected CHO cells (Pelkmans et al., 2001,Mundy et al., 2002). This caveolar vesicular endocytic system isreported to be connected to smooth endoplasmic reticulum (ER) ortrans-Golgi membranes via a microtubule system sensitive tonocodazole treatment (Mundy et al., 2002; Pelkmans et al., 2001;Pelkmans and Helenius, 2002). To verify the possible association ofMLC1 intracellular vesicles and cytoskeletal structures in astrocyteswe performed immunostainings with anti-MLC1 pAb in combinationwith anti-tubulin mAb to stain microtubules, anti-GFAP mAb to stainastrocytic intermediate filaments, or Alexa-Fluor-conjugated phalloi-din to stain actin filaments. We observed that MLC1 positive vesiclesco-localize with tubulin and GFAP in perinuclear areas (Figs. 4A andB). Conversely, in the majority of astrocytes MLC1 does not colocalizewith actin, although a partial colocalizationwas occasionally observedin astrocyte perinuclear area and end-feet (Fig. 4C).

To verify whether MLC1 intracellular expression is associated withthe ER and/or Golgi compartment we performed double immunoflu-orescence stainings with anti-MLC1 pAb and anti-BiP/GRP78 or anti-TGN38 mAbs, that respectively stain ER and Golgi organelles. Weobserved that perinuclear MLC1 immunoreactivity colocalizes withthe ER marker BiP/GRP78 but not with the trans-Golgi marker TGN38

(Figs. 5A and B) and that MLC1 localization in these area is completelyprevented by treatment of astrocytes with the microtubule disruptingagent nocodazole (Figs. 5C and D).

Because MLC1 positive vesicles outnumbered the cav-1–MLC1double positive ones and in order to better clarify the intracellularcompartmentalization of MLC1, we also performed double immuno-fluorescence stainings with MLC1 pAb and mAbs recognizing intra-cellular small vesicular organelles, such as early endosomes (EEA1),clathrin vesicles (clathrin heavy chain) and lysosomes (Lamp-1). Weobserved that MLC1 protein is present mainly in early endosomes andrarely in lysosomes, but not in clathrin vesicles (Figs. 5E–G). Tosummarize, these results indicate that intracellular MLC1 localizespredominantly in vesicular structures and ER compartment and thatthis distribution is sensitive to microtubule system disorganization.Based on these observations, it is proposed thatMLC1 and cav-1mightinteract in caveosome structures that are connected to the ERcompartments through microtubules. In addition the presence ofMLC1 in early endosomes and lysosomes suggests that one or bothMLC1 components expressed in astrocytes (Ambrosini et al., 2008) canfollow the route of the endo-lysosomal pathways.

In order to evaluate whether mutations in MLC1 might alter itsintracellular localization we transfected primary rat astrocytes withvectors encoding MLC1 carrying two different pathological mutations(S280L and S246R) that were previously overexpressed in astrocytesand heterologous cells and were shown to behave as a more severemutant retained in the ER and a milder one partially able to reach theplasma membrane, respectively (Duarri et al., 2008). In agreementwith these data (Duarri et al., 2008), immunostainings with anti-

Fig. 2. Pull-down of rat astrocyte proteins by N-terminal (aa 1–55) and C-terminal (aa 322–377) domains of human MLC1. (A) WB analysis of astrocyte extracts (Input) and pulled-down samples: dystrobrevin (DB), Kir4.1 dimer (arrow), syntrophin (Synt) and α-tubulin (α-tub) interact with the GST-MLC1 N-terminal but not with control GST alone. Synt,Kir4.1 dimer andα-tub are also pulled down by the GST-MLC1 C-terminal, while β-DG is not detected among the pulled-down proteins. (B)WB of pulled-down proteins eluted withglycine (0.1 M, pH 3): cav-1 binds to both MLC1 N- and C-terminal domains, while cav-2 only binds the MLC1 C-terminal. Cav-3 is never detected among the pulled-down proteins.An additional band of about 40-kDa MW, consistent with cav-1 homo and/or hetero-dimers (arrowhead), is observed after 1% of SDS elution of GST-bound proteins. In the samesample, α and β cav-1 isoforms are also found (arrows). (C) GST-MLC1-N- and C-terminals pull-down cav-1 from an enriched preparation of caveolins obtained from a mousepneumocyte extract. MLC1 N- and C-terminals, but not GST alone, interact preferentially with caveolar hetero-oligomeric structures (upper arrowhead) and onlymarginally with thecav-1 monomer (lower arrowhead). One representative experiment out of two is shown.

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MLC1 pAb and confocal analysis of transfected astrocytes showed thatboth mutated proteins accumulated in the perinuclear area (mostlikely in ER structures), while only the S246R mutant protein wasoccasionally found in the astrocytic membranes and end-feet(Supplementary Fig. S1, panel A). Co-immunostainings for mutatedMLC1 and EEA1 (early endosome marker) or cav-1 proteins indicatedthat MLC1 mutants only rarely co-localized with caveosomes or earlyendosomes (Supplementary Fig. S1, panels B and C).

Biochemical analysis of the association of different MLC1 componentswith intracellular structures in rat brain tissue

We previously reported that different MLC1 components can bedetected in whole brain and cultured astrocyte extracts: the 60-kDaMLC1 component that associates with raft membrane compartmentsand can be extracted from cell and tissue samples with strongdetergents; the 36-kDa MLC1 protein that is extracted in milderconditions; and a minor component of 30 kDa often found in thedetergent solubilized fractions (Ambrosini et al., 2008). To elucidatethe association of these MLC1 components with specific intracellularorganelles, we developed a further fractionation procedure thatallowed us to separate from rat brain tissue two proteic fractionsselectively enriched in the differentMLC1 components (as described inMaterial and methods). For these experiments, rat brain tissue, ratherthan cultured astrocytes, was used to obtain higher protein amounts.After mechanical and detergent-mediated solubilisation followed bysequential centrifugation and ultracentrifugation steps we found thatboth the 60- and 30-kDa MLC1 proteins are associated with a proteinfraction which encompasses the plasma membrane (cav-1, cav-2,flotillin-positivemembranes) ER structures (Bip/GPR78-positive) and

part of the recycling endosome organelles (Rab5/11-positive). Con-versely, the 36- kDa MLC1 component was exclusively detected in thecytosolic fraction obtained by mechanical disruption of the tissue inthe absence of detergents and in the supernatant obtained afterultracentrifugation of this cytosolic fraction (Fig. 6). In the latterfraction, which encompasses cytosolic proteins and proteins associat-ed with small vesicle and organelle membranes, the 36-kDa MLC1protein co-separates together with the highly enriched EEA1 protein,the marker of early endosomes. In the same fraction, but into a lesserextent, MLC1 also co-fractionates, with Rab5 and Rab11, small GTPaseproteinsmarkers of early and recycling endosomes that are involved invesicle trafficking between intracellular compartments (Fig. 6). In lightof these findings and of immunolocalization data, we propose thatwhile the 60-kDa MLC1 component is associated with intracellularcaveolar positive structures the monomeric 36-kDa MLC1 mighttraffic to early endosomes and be sorted to the recycling endosomepathway.

Effects of raft caveolin-mediated endocytosis activators and inhibitors onMLC1 intracellular localization

The intracellular vesicular coimmunolocalization observedbetweenMLC1 and cav-1 and the results obtained so far suggested a possibleinvolvement ofMLC1 in caveolar/raft-mediated endocytosis pathways.To investigate this possibility, we evaluated MLC1 expression anddistribution in protein extracts derived from rat astrocytes aftertreatment with drugs used to inhibit or stimulate caveolar-dependentendocytosis. It is well established that cholesterol is essential for theformation and structural integrity of lipid rafts domains and thatcholesterol depleting agents, like beta-methyl cyclodextrin (BMC), or

Fig. 3. Co-immunolocalization of MLC1 and cav-1 in astrocytes. (A) Double immunofluorescence staining of cultured rat astrocytes transfected with GFP–cav-1 vector shows thatMLC1 (red) and cav-1 (green) colocalize in perinuclear vesicular structures. (B) Immunostaining of stably transfected U251 cells expressing His-tag MLC1 with anti-His mAb toreveal MLC1 (red) and anti-cav-1 pAb (green) unravels a similar vesicular colocalization of MLC1 and cav-1. The arrow in B points to one cell where MLC1–cav-1 colocalization ispresent on the membrane. Bars=20 μM.

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tyrosine kinase inhibitors, like genistein, can inhibit caveolar raft-mediated endocytosis (Parton and Richards, 2003; Lajoie and Nabi,2007). Conversely, cholesterol itself and the phosphatase inhibitorokadaic acid have been used to stimulate caveolar-raft mediatedendocytic pathway (Parton et al., 1994; Thomsenet al., 2002; Sharmaetal., 2004). To follow the fate of MLC1 components after treatment withthe abovementioned agents, rat astrocyteswere solubilized to separatethe cytosolic (36 kDa) from the membrane-associated (60 kDa) MLC1fraction as described above. Due to the low level of expression, the 30-kDa MLC1 protein was not analyzed in this set of experiments. WBanalysis revealed that BMCand genistein induced an increase in the 60-kDa MLC1 component and a decrease in the 36-kDa fraction (Figs. 7Aand B). Accordingly, immunofluorescence stainings of BMC- andgenistein-treated astrocytes revealed that MLC1 immunoreactivitywas distributed more diffusely in the cell body and along astrocyticextensions relative to control cells, where most of the staining wasconfined around the perinuclear area (Figs. 7C–E). In BMC-treated cellsMLC1 was organized in discrete clusters, probably associated with theinternal side of the plasma membrane (Fig. 7D), while after genisteintreatment MLC1 immunoreactivity showed a punctate distribution onastrocytic membranes (Fig. 7E, arrows) and a cytoplasmic localizationoverlappingwithGFAP. The latter result suggests thatgenistein-inducedMLC1 trafficking in astrocytes could be mediated by the interaction ofMLC-containing vesicles with GFAP+ filaments, similarly to whatobserved for cell surface trafficking of some glutamate transporters(Hughes et al., 2004). In the same set of experiments we also found thatastrocyte treatmentwith cholesterol induced amarked downregulationof the 60-kDa MLC1 component normally associated with the plasmamembrane and a minimal increase in the cytoplasmic 36-kDa MCL1component (Fig. 7F). Consistent with cholesterol-mediated stimulation

of MLC1 caveolar endocytosis, immunostaining of cholesterol-treatedastrocytes revealed an increase in intracellular MLC1-positive vesiclesscattered through the cytoplasm, apparently translocating from themembrane to the internal perinuclear area (Fig. 7G, arrow).

Astrocyte treatment with 100 nM of the phosphatase inhibitorokadaic acid, another agent known to promote lipid raft/caveolaeendocytosis, led to an increase in the cytoplasmic 36-kDa componentand, contrary towhatwas expected, to a similar increase in the 60-kDamembrane-associated fraction (Fig. 7H). Being okadaic acid aphosphatase inhibitor, we hypothesized that the MLC1 membranecomponent could be totally or in part phosphorylated and that itsmembrane expression might be influenced by its phosphorylationstate and thus by phosphatase inhibition. Indeed, when astrocyteswere treated with a higher concentration of okadaic acid (300 nM)which is reported to inhibit most of the cellular phosphatases weobserved that, respect to control cells, the 60-kDa MLC1 proteinshifted to a higher MW, an effect that is indicative of a possiblephosphorylation event (Fig. 7I). Immunofluorescence staining ofastrocytes treated with 100 nM of okadaic acid showed an increaseinMLC1-positive intracellular vesicles (Fig. 7L, arrow) a result that canbe related to the increased levels of the 36-kDa component observedin the WB. Overall these results indicate that MLC1 undergoes raft-mediated endocytic pathways and suggest that the MLC1 60-kDamembrane component may be subjected to phosphorylation.

Effect of cav-1 overexpression on MLC1 intracellular distribution inastrocytes

Several studies have shown that cav-1 overexpression negativelyregulates cav-mediated endocytosis (Le et al., 2002; Lajoie and Nabi,

Fig. 4. Relationship between MLC1-positive vesicles and astrocytic cytoskeletal structures. (A) Double immunostainings with anti-MLC1 pAb (red) and anti-tubulin mAb (green)reveal a partial colocalization of MLC1 vesicles and microtubules in the perinuclear area (arrow). (B) MLC1-positive vesicles (red) colocalize in the perinuclear area with GFAP(green) (arrow). (C) Double immunostaining with anti-MLC1 pAb (red) and Alexa Fluor (488)-conjugated phalloidin (green) to stain actin filaments reveals overlap of MLC1 andphalloidin immunoreactivities in the end-feet and perinuclear area (arrows) of one astrocyte out of five shown. Bars=20 μM.

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Fig. 5.MLC1 localization in intracellular organelles and effect of nocodazole treatment in rat astrocytes. (A) Double immunofluorescence staining with anti-MLC1 pAb (red) and anti-GPR78 mAb (green) shows colocalization between MLC1 and the ER marker (arrow). (B) Double immunofluorescence with anti-MLC1 pAb (red) and anti-TGN38 mAb (green)indicates that MLC1 does not localize in the trans-Golgi. (C, D) Different distribution of MLC1 (red) in control and nocodazole(40 μM)-treated astrocytes stained with GFAP mAb(green). In control cells MLC1 is mainly localized in the perinuclear area (arrow) a distribution completely prevented by nocodazole treatment. (E) MLC1 (red) and early endosomeantigen (EEA1) (green) partially colocalize. (F) Double immunostaining for MLC1 (red) and Lamp-1 (green) shows presence of MLC1in a small fraction of lysosomes (arrows).(F) No colocalization is observed between MLC1 (red) and clathrin vesicle heavy chain protein (green). Bars=20 μM.

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2007; Yuan et al., 2007; González et al., 2007; Hagiwara et al., 2009).The same studies however also highlighted that caveolar-mediatedendocytosis is a complex and highly regulated trafficking pathwayresulting from a balance of different cellular factors such as cell lipidcomposition, cholesterol content, caveolin expression levels andcaveolae formation (Sharma et al., 2004; Echarri et al., 2007). Tounderstand whether MLC1 intracellular expression and trafficking inastrocytes could be regulated by cav-1 overexpression we stablyinfected the U251 cell line overexpressing His-MLC1 with a retroviralvector producing the cav-1–GFP fusion protein. Similarly to whatobserved in astrocytes treated with agents inhibiting cav-mediatedendocytosis (Figs. 7A and B), WB analysis of cav-1–GFP transfectedastrocytoma cells cultured for 10 days revealed a significant decreaseof the 36-kDa MLC1 cytosolic fraction and a very small increase inthe 60-kDa membrane component with respect to control cellsstably infected with the GFP-expressing vector alone (Fig. 8A).Differently from transiently transfected astrocytes analysed 48 h post-transfection and U251 His-MLC1 astrocytoma cells (Fig. 3), anti-MLC1immunostaining of cav-1–GFP transfected astrocytoma cells culturedfor 10 days showed the lack of cav-1–MLC1 double positive intracellularvesicles inmost of the cav-1 overexpressing cells (Fig. 8B). These resultsfurther support the involvement of cav-mediated endocytosis pro-cesses in MLC1 intracellular trafficking (or positioning) and revealthat in astrocytes MLC1 endocytosis is negatively regulated by cav-1overexpression.

MLC1 is phosphorylated in vitro by PKC and PKA on NH and COOHterminals

Data obtained with okadaic acid treatment prompted us to verifywhether MLC1 is a phosphorylated protein. Because MLC1 sequence

analysis in protein data base (Swiss prot data base, Motifs scan)suggests potential consensus sequence for protein kinase C (PKC)phosphorylation in the cytoplasmic MLC1 domains we testedwhether MLC1 could be phosphorylated by PKC. Interestingly,studies in astrocytes provided evidence of a cytosolic associatedcav-1 complex containing PKC and tubulin (Ito et al., 2002, 2006).We also investigated the possibility that MLC1 might be phosphor-ylated by PKA, an enzyme localized in membrane caveolar lipidrafts, directly interacting with cav-1 and often involved in theregulation of membrane protein localization in these compartments(Razani and Lisanti, 2001; Cohen et al., 2004). We performed an invitro kinase assay to verify that MLC1 could be a PKC and PKAsubstrate using recombinant GST-MLC1 N- and C-terminals, and PKCor PKA catalytic subunits. The autoradiography of the samplesphosphorylated in vitro by PKC and PKA in the presence of γ[32P]ATP showed that PKC phosphorylated both GST MLC1 N- andC-terminal domains whereas PKA phosphorylated only the GST-MLC1 N-terminal (Fig. 9A).

In vitro mapping of MLC1 residues phosphorylated by PKC and PKA

To map residues phosphorylated by PKA and PKC, in vitrophosphorylated recombinant GST-MLC1 N- and -MLC1 C-terminalswere analyzed by SDS–PAGE; the electrophoretic bands weredigested in gel by trypsin and the peptide mixtures analyzed bymass spectrometry. The constructs were identified by MALDI ToF.MS/MS spectra of peptides derived from the PKA-phosphorylatedN-terminal region of MLC1 (Supplementary Fig. S2A) allowed theidentification of the phoshorylated residue corresponding to serine27 (S27) of the human MLC1 (Fig 9B). MS/MS spectra of peptidesderived from PKC-phosphorylated GST-MLC1 C- and N-terminalregions (Supplementary Fig. S2B) indicated that Serine S27 in theMLC1 N-terminal region and S339 in the MLC1 C-terminal werephosphorylated (Fig. 9B).

Analysis of in vivo MLC1 phosphorylation and effects of PKC- andPKA-induced phosphorylation on MLC1 intracellular trafficking

To confirm that MLC1 could be phosphorylated in vivo, weperformed a dephosphorylation assay on astrocyte membrane andcytosolic protein extracts. We observed that the 60-kDa MLC1membrane associated fraction, but not the 36-kDa protein,underwent a shift to a lower molecular weight, suggestive ofdephosphorylation (Fig. 9C). To analyse whether PKA- and PKC-induced MLC1 phosphorylation could affect MLC1 intracellulartrafficking we treated primary cultured astrocytes with increasingconcentrations of phorbol ester, a general stimulator of PKC enzyme(PMA, from 500 pM up to 300 μM), and of forskolin, a PKA stimulatingagent (FSK, 1 and 10 μM). WB analysis of total protein extracts fromstimulated cells revealed an increase in the membrane MLC1components, as compared to control cells, at the concentration of500 nM PMA and 10 μM FSK (Supplementary Fig. S3).

To better evaluate the effects of the phosphorylation inducingagents on the different MLC1 components, after stimulation with theoptimal PMA (500 nM) and FSK (10 μM) concentrations astrocyteswere collected, fractionated in cytosolic and membrane fractions asdescribed above, and analysed by WB and immunofluorescence.Panel A of Fig. 10 indicates that PMA stimulation induced an increasein both MLC1 membrane components and a decrease of cytosolic 36-kDa MLC1 as compared to control cells. Similarly to PMA, FSK(10 μM) treatment induced a downregulation of the 36-kDa MLC1component and a lower increase in the 60-kDa band compared toPMA (Fig. 10B). Immunofluorescence stainings revealed an increasein the intensity and distribution of MLC1 immunoreactivity along theastrocytic processes after treatments with PMA and FSK as comparedto control cultures (Figs. 10C–E). As observed for genistein, an

Fig. 6. Subcellular fractionation of MLC1 components from rat brain samples. WBanalysis performed on rat brain protein samples after subcellular fractionation showsthat both the 60- and 30-kDa MLC1 components are present in the detergent solublefraction (lane M) that includes cav-1, cav-2 and flotillin positive membranes, BiP/GRP78 positive ER structures and Rab5/11 positive endosomal organelles. The 36-kDacomponent is found in the low-ionic strength cytosolic fraction (lane C) that stillcontains cav-1, cav-2, flotillin and Bip/GRP78. After purification of the latter fraction byultracentrifugation at 100,000×g for 2 h at 4 °C, the 36-kDa MLC1 and Rab 5/Rab11proteins, but not other membrane proteins, are still detected along with a highlyenriched EAA1 positive fraction (lane C⁎). One representative experiment out of three isshown.

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increase in the colocalization of vesicular MLC1 and GFAP immunor-eactivities was also observed after PMA and FSK stimulation,suggesting the possible involvement of intermediate filaments in

the PMA- and FSK-induced MLC1 surface trafficking. Of note, a similarmechanism has been described for glutamate transporters in primaryastrocytes (Hughes et al., 2004).

Fig. 7. Effects of inhibitors and activators of cav-mediated endocytosis onMLC1 protein expression and distribution in astrocytes. Cultured rat astrocyteswere incubated for 1 h at 37 °CwithBMC(10mM),genistein (100μg/ml), cholesterol (5mM)orokadaic acid (OA,100and300nM)andthenanalysedbyWBafter subcellular fractionationof the cytosolic (C) andmembrane(M)fractions (A, B, F, H, I) or subjected to double immunofluorescence stainingswith anti-MLC1pAb (red) andanti-GFAPmAb (green) (C, D, E, G, L). (A, B) BMCandgenistein treatments cause anincrease in membraneMLC1 (60 kDa) and a decrease of the cytosol associated MLC1 (36 kDa) component. (C, D) Differently from control astrocytes (C), where MLC1 immunoreactivity ismainly present around the perinuclear area, in BMC-treated astrocytes (D) MLC1 is localized in discrete clusters distributed throughout the cell body and processes. (E) In genistein-treatedastrocytes,MLC1 immunoreactivity ispresentalong astrocyticprocesses in apunctate distributionon themembrane(arrows) and in intracellular vesicles colocalizingwithGFAP intermediatefilaments. (F) Cholesterol treatment induces amarkeddownregulationof theMLC160-kDa proteinandaminimal increase in the36-kDa protein. (G) In cholesterol-treatedastrocytes,MLC1-containing vesicles arepresent in theperinuclear area and on the plasmamembrane (arrow). (H)OA treatment (100 nM) induces an increase of the 36- and60-kDa MLC1 components.(I) Attheconcentrationof 300nM,OAalso induces a shift to ahigherMWof the60-kDa MLC1component. (L) InOA(100nM)-treatedastrocytesMLC1positive intracellular vesicles are sparse inthecytoplasm (arrow). In all WB experiments actin staining is shown as a control of the equal amount of protein loaded into the gel; one representative experiment out of three is shown.Bars=20 μM.

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Discussion

By focusing our study on the molecular mechanisms andregulatory factors involved in the intracellular distribution andmembrane trafficking of MLC1 in astrocytes we have shown thatMLC1 interacts with defined components of the DGC and with cav-1

and can be subjected to cav-mediated endocytosis and endo-lysosomal pathways. Moreover, we have found that MLC1 intracel-lular trafficking in astrocytes is influenced by PKC- and PKA-mediatedMLC1 phosphorylation. These findings contribute to shed light on themechanisms regulating MLC1 compartmentalization in the brain andare potentially relevant for understanding how MLC1 mutations that

Fig. 9. In vitro phosphorylation of MLC1 N- and C-terminal regions by protein kinase A (PKA) and protein kinase C (PKC). (A) Comassie blue staining (CB) and autoradiography (32P)of recombinant GST-MLC1 N- and C-terminal regions after SDS–PAGE and in vitro phosphorylation by PKC or PKA catalytic subunits. Both GST-MLC1 N- and C-terminal regions arephosphorylated by PKC, while only the GST-MLC1 N-terminal domain is phosphorylated by PKA. (B) Sequence of MLC1 N- and C-terminal regions expressed as GST-fused proteins.The residues phosphorylated by PKA and PKC identified byMS/MS are indicated (arrows). In the MLC1 N-terminal, the serine27 (S27) is phosphorylated by both kinases. In theMLC1C-terminal, S339 (S339) is phosphorylated by PKC. (C) The 60-kDa MLC1 component is phosphorylated in vivo as indicated by the shift to a lowerMWwhen astrocyte membrane (M)and cytosolic (C) protein extracts were incubated for 30 min at 37 °C with 0.05 U/ml alkaline phosphatase. One representative experiment out of three is shown.

Fig. 8. Effect of cav-1 overexpression on MLC1 expression and distribution in human astrocytes. (A) Subcellular fractionation and WB of His-MLC1 U251 cells stably infected with aretroviral vector expressing cav-1–GFP indicates a decrease in the expression of the 36-kDa MLC1 component relative to control cells stably infected with the same vector carryingthe GFP protein alone. Anti-cav-1 pAb detects the recombinant cav-1–GFP fusion protein (arrow), but not endogenous cav-1, in cav-1–GFP infected cells. Actin staining is shown as acontrol of the equal amount of protein loaded into the gel. (B) Double immunofluorescence of infected cells with anti-cav-1 pAb and HismAb to stain His-taggedMLC1 reveals lack ofintracellular colocalization of cav-1 and MLC1 in most of the infected cells. Only occasionally, MLC1–cav-1 colocalization is observed (arrow in B). Bar=20 μM.

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hamper the correct localization of this protein in astrocytic mem-branes lead to MLC disease (Duarri et al., 2008).

Interaction of MLC1with DGC components and cav-1

By using pull-down assay and fractionation techniques we showthat in astrocytes the MLC1 intracellular domains interact with thecytosolic components of the DGC, syntrophin and dystrobrevin, andwith Kir4.1. Because dystrobrevin binds syntrophin, which in turn canbind Kir 4.1 also in glial cells (Connors et al., 2004 ), it is conceivablethat due to their interaction with one or more of the DGC componentsMLC1 cytoplasmic domains can pull-downmost of the DGC associatedproteins. The present data extend our previous observations (Ambro-sini et al., 2008) by showing that MLC1 association with the DGCoccurs through its interaction with cytoplasmic DGC components andnot with themembrane protein β-DG. Althoughwe still do not knownif MLC1 can directly bind some of these proteins these results stronglyreinforce the hypothesis that DGC proteins play a role in MLC1membrane expression and function. We also found that MLC1interacts with cav-1, the structural protein of caveolae. Cav-1associates with DGC (Halayko and Stelmack, 2005) and is involvedin the compartmentalization of signal transduction components, lipidmetabolism, endocytosis and intracellular trafficking of severalmembrane proteins in several cell types (Wyse et al., 2003; Headand Insel, 2007, Parton and Simons, 2007), including astrocytes (Silvaet al., 2007). We observed that cav-1 extracted from astrocytes andpneumocytes, which express high levels of this proteins (Williamsand Lisanti, 2004), interacts with the MLC1 NH- and COOH-terminals.It is possible that cav-1 interaction with both domains might be

needed to stabilize MLC1 in caveolar structures (caveolae and/orcaveosomes). However, since we failed to reveal a direct interactionbetweenMLC1 and cav-1 recombinant proteins, it cannot be excludedthat a protein or lipid present in cell extracts might mediate MLC1–cav-1 interaction or that cav-1 must be structurally and/or post-translationally modified to bind MLC1 (Nomura and Fujimoto, 1999;Echarri et al., 2007, Aoki et al., 2007). The latter possibility issupported by the finding that oligomeric organized cav-1 structurespreferentially interact with MLC1.

MLC1 is expressed in intracellular vesicles and ER and undergoes cav-1mediated endocytosis

The presence and function of cav-1 in regions outside the caveolarplasma membrane, like intracellular vesicles, Golgi-ER structures andrecycling endosomes has been already documented in different celltypes (Pelkmans et al., 2004, Head and Insel, 2007, Aoki et al., 2007),including astrocytes (Megias et al., 2000, Ito et al., 2002, 2006). Thecav-1 vesicular trafficking has been mainly attributed to lipid-raftcaveolar structures emerging from the trans-Golgi to reach plasmamembrane (Tagawa et al., 2005) and/or to the caveolar endocyticvesicles (caveosomes) involved in the endocytosis of plasmamembrane proteins transported to trans-Golgi, endoplasmic reticu-lum (ER) or recycled to the plasma membrane (Pelkmans et al., 2001,2004; Mundy et al., 2002). The intracellular MLC1–cav-1 vesicularcolocalization and the lack of colocalization between MLC1 and thetrans-Golgi and clathrin vesicle markers, together with the microtu-bule dependent MLC1 distribution in ER structures in culturedastrocytes, indicate a possible role of cav-1 in regulating MLC1

Fig. 10. Effects of PKC and PKA phosphorylation on MLC1 intracellular localization. Primary cultured rat astrocytes were treated for 1 h at 37 °C with 500 nM of PMA or with 10 μMFSK to stimulate PKC and PKA, respectively. (A) WB of the cytosolic and membrane fractions of control and treated astrocytes indicates that PMA stimulation induces an increase inthe 60-kDa MLC1 and a reduction of the cytosolic 36-kDa protein. (B) Similar data are obtained with FSK. Cav-1 is shown as a control of the equal amount of proteins loaded into thegel. One representative WB experiment out of three is shown. (C) Immunofluorescence stainings of PMA- or FSK-stimulated astrocytes stained with GFAP mAb (green) and MLC1pAb (red) reveals a wider distribution of MLC1 along the cellular processes. Bars=20 μM.

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trafficking from the plasmamembrane to intracellular compartments,particularly to the ER. Importantly, astrocyte treatment with factorsaffecting cav-1-mediated endocytosis such as cholesterol, BMCD andgenistein, and cav-1 overexpression alter MLC1 localization confirm-ing the cav-1 dependent endocytic MLC1 intracellular trafficking. Asimilar caveolin-mediated intracellular transport to ER structures hasbeen previously described for the simian virus 40 (SV40) antigenretrograde trafficking (Pelkmans et al., 2001; 2002) and for somecellular proteins (Benlimame et al., 1998; Le and Nabi, 2003; Khan etal., 2006; Zito et al., 2007). It has been previously reported thatmutated MLC1 proteins are subjected to the ER associated qualitycontrol (ERAD), which ensures the correct protein folding and leadsmisfolded mutated proteins to proteasome degradation (Duarri et al.,2008). In addition to being subjected to this conformationalcheckpoint, the native MLC1 protein present in large amounts inER-associated structures could play a role in there and/or represent alarge intracellular pool of MLC1 that allows the protein to regulate itscell surface expression by cycling on and off the plasma membrane.Some unassembled subunits of complex hetero-oligomeric mem-brane proteins have been reported to localize in the ER andredistribute to the cell surface and endosomal compartmentsfollowing heterodimer formation (Schwappach, 2008; Casbon et al.,2009). Moreover, it cannot be ruled out thatMLC1 acts as a chaperone,as it has been recently hypothesized (Duarri et al., 2008), which isinvolved in the cycling of cargo proteins between ER and plasmamembranes. The findings that only 36-kDa MLC1 co-fractionates withEAA1 and Rab5-11 and that MLC1 colocalizes with EEA1 marker inastrocytes suggest that this component might be associated with theearly/recycling endosome pathway. Thus, MLC1 components couldfollow different endo-exocytic pathways, as observed for othermembrane proteins (Lajoie and Nabi, 2007). Indeed, raft-dependentendocytosis is a highly complex process in which different cargos canfollow different routes and it is known that caveolae can exchangecontents with other endocytotic pathways (Parton and Simons 2007,Parton and Richards 2003; Mundy et al., 2002). In this regard, acaveosome/early endosome communication system has been recent-ly described in which Rab 5 targets caveolar vesicles to earlyendosomes (Pelkmans et al., 2004). Because we did not observecolocalization of MLC1 and clathrin vesicles it is possible that aftercaveolar internalization of the dimeric membrane-associated 60-kDaMLC1 component the disaggregation of the dimer leads to theformation of the 36-kDa MLC1 monomeric protein that could betotally or in part sorted to early endosomes via Rab 5 to be recycled tothe surface, brought back to the ER or sorted to lysosome fordegradation. At present, it cannot be excluded that cav-1 might alsohave a role in the stabilization of MLC1 at the plasmamembrane level.

Preliminary experiments performed with transfected mutantsindicated that mutations may affect MLC1 intracellular trafficking andsorting in the endo-lysosomal compartment. However, it cannot beruled out that the colocalization of MLC1mutants and EEA1 and cav-1proteins observed in some transfected astrocytes is due to partialrescuing of MLC1 intracellular trafficking caused by the presence ofendogenous wild type MLC1 that could dimerize or oligomerize withthe mutant proteins. Further experiments in MLC1-deficient expe-rimental systemswill be performed to define precisely the intracellularsorting of mutated MLC1.

MLC1 is phosphorylated by PKA and PKC and phosphorylation influencesMLC1 intracellular trafficking

Reversible protein phosphorylation is the most common mecha-nism by which protein function can be dynamically regulated. Here,we show that the MLC1 60-kDa membrane component can bephosphorylated by PKC and PKA. Although we cannot exclude thepresence of additional phosphorylation sites outside the C and Ndomains we speculate that the phosphorylation of these domains can

play a key role in regulating MLC1 trafficking and function. Bothenzymes phosphorylated the same Ser27 localized in the NHintracellular domain. The analysis of the amino acids surroundingthe S27 revealed the presence of an ER retentionmotif (RGR) localizednear the phosphorylation site. The ER retention mediated by RXRmotifs is an important quality control mechanism used by ionchannels/transporters to ensure the proper assembly and traffickingof multimeric complexes. Protein assembly and/or PKA and PKCinduced phosphorylation of sites flanking RXR allow masking ofretention signal favouring protein exit from ER, thus creating a readilyreleasable pool of receptors/channels sensitive to the activation ofintracellular signalling pathways (Scott et al., 2003). Since it has beendemonstrated that MLC1 oligomerizes in ER (Teijido et al., 2004), it ispossible that phosphorylation of the NH MLC1 domain occurs in theER and regulates MLC1 assembly, ER exit and delivery to the plasmamembrane. In preliminary experiments, we found that overexpres-sion of truncated MLC1 lacking the first 30 amino acids including theRXR retention signal increased MLC1 membrane localization in HeLacells that are reported not to express endogenousMLC1 (Lanciotti andAmbrosini, unpublished data). In line with this observation, PMA andFSK treatments, which stimulate PKC and PKA activity, respectively,led to an increase in both the MLC1 membrane component and inMLC1 intracellular trafficking. Alternatively or in combination, it isalso possible that PKC and PKA are involved in regulating MLC1caveolar-mediated endocytosis and recycling pathways. It is knownthat several PKC and PKA isoforms associate with membrane raftswhere they can phosphorylate caveolar-raft resident proteins (Biet al., 2001; Dalskov et al. 2005) and that PKC activation favourscav-mediated endocytosis (Sharma et al., 2004). However, our datashowing a PMA-induced increase in the expression of MLC1membrane component suggest inhibition rather than activation ofcav-mediated endocytosis, as previously observed for some Ca2+

channels (Cha et al., 2008). The finding that PMA treatment does notaffect MLC1 distribution in rafts (data not shown) indicates thatprobably PKC-induced MLC1 phosphorylation does not alter cav-1–MLC1 interaction. Previous results indicate that PKA-mediatedphosphorylation of aquaporin-4 is not required for channel internal-ization but is involved in retaining the channel in a vesicle-recyclingcompartments, allowing it to escape the degradation pathway(Carmosino et al., 2007). Such a mechanism could be hypothesizedalso for PKA-mediated effect on MLC1.

Future perspective for MLC disease

Overall, our results indicate that in astrocytes MLC1 is subjected to acomplex intracellular trafficking pathway with several regulatory stepsthat can influence MLC1 membrane expression. Given the hypothesisthat MLC pathogenesis results from the lack of MLC1 membraneexpression (Duarri et al., 2008), our data provide clues for theidentification of potentially useful therapeutic agents. Although phar-macological strategies that induce mutated MLC1 protein to overcomethe ERAD control have been suggested (Teijido et al., 2004, Duarri et al.,2008), specific treatments for MLC patients are not available yet. Wepropose that, due to their ability to increaseMLC1membraneexpression,agents affecting cav-mediated endocytosis, intracellular cholesterollevels and protein phosphorylation could be explored for theirtherapeutic potential in patients carrying mutations in the MLC1 gene.

Acknowledgments

We thank Dr. Antonella Bernardo for providing astrocyte cellcultures. This study is supported by grants of the Italian Ministry ofHealth, ELA Foundation (France) and Myelinet Cost Action to E.A., byISS-NIH Collaborative Programme on Rare diseases (7/DR1) to T.C.Pand by FIRB 2003 (Grant RBNE03FMCJ-002) from the Italian Ministryfor University and Research to M.S.

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Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.nbd.2009.11.008.

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