Cell Motility and the Cytoskeleton (2006)
g-Synuclein Has a Dynamic Intracellular Localization Irina Surgucheva,1,2 Belinda McMahon,1,2 and Andrei Surguchov1,2* 1
Retinal Biology Research Laboratory, Veterans Administration Medical Center, Kansas City, Missouri 2 Department of Neurology, Kansas University Medical Center, Kansas City, Kansas g-Synuclein is a member of the synuclein family consisting of three proteins. Within the last several years increasing attention has focused on these proteins because of their role in human diseases. a-Synuclein relevance to Parkinson’s disease is based on mutations found in familial cases of the disease and its presence in filaments and inclusion bodies in sporadic cases. g-Synuclein is implicated in some forms of cancer and ocular diseases, while b-synuclein may antagonize their pathological functions. In this paper we present data on the localization and properties of g-synuclein in several neuronal and nonneuronal cell cultures. We show that contrary to the current opinion, gsynuclein is not an exclusively cytoplasmic protein, but has a dynamic localization and can associate with subcellular structures. It is present in the perinuclear area and may be associated to centrosomes. On late steps of mitosis g-synuclein is not found in the centrosomes, and redistributes to the midbody in telophase. Under stress conditions a translocation of g-synuclein from the perinuclear area to the nucleus occurs exhibiting nucleocytoplasmic shuttling. g-Synuclein overexpression reduces neurite outgrowth in a greater extent then a-synuclein overexpression. These data support the view that g-synuclein may change its intracellular localization and associate with subcellular structures in response to intracellular signaling or stress. Cell Motil. Cytoskeleton 2006. ' 2006 Wiley-Liss, Inc.
Key words: actin; astrocytes; centrosomes; midbody; nucleus
INTRODUCTION
Synucleins are small highly conserved proteins in vertebrates, especially abundant in neurons and typically enriched in presynaptic terminals [Clayton and George, 1999; Norris et al., 2004; Bennett, 2005]. a-Synuclein forms the major fibrillar components associated with the pathological lesions of neurodegenerative diseases [Spillantini et al., 1997, 1998; Trojanowski et al., 1998; Shults, 2006]. Parkinson’s diseases linked mutations accelerate this aggregation process [Polymeropoulos et al., 1997; Kru¨eger et al., 1998; Zarranz et al., 2004]. Far less is known about two other members of the synuclein family, b-synuclein and g-synuclein. Both b- and g-synuclein are abundant in brain and in spite of high similarity in amino acid sequence with a-synuclein (78 and 60%, respectively) have different properties [Jakes et al., 1994; Buchman et al., 1998a,b; Clayton and George, 1999]. ' 2006 Wiley-Liss, Inc.
g-Synuclein is up-regulated in several types of cancer, stimulates proliferation, invasion, and metastasis and may be used as a tumor marker [Ji et al., 1997; Jia
Contract grant sponsor: NIH, VA Merit Review Grant; Contract grant number: EY 02687; Contract grant sponsor: The Glaucoma Foundation; Contract grant number: QB42308; Contract grant sponsor: Carl Marshall Reeves and Mildred Almen Reeves Foundation; Contract grant sponsor: ADRC; Contract grant number: 99-6403. *Correspondence to: Andrei Surguchov, Retinal Biology Research Laboratory, Veterans Administration Medical Center, Kansas City, MO, 4801 Linwood blvd, Kansas City, MO 66148. E-mail:
[email protected] Received 17 February 2006; Accepted 20 April 2006 Published online in Wiley InterScience (www. interscience.wiley. com). DOI: 10.1002/cm.20135
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et al., 1999; Iwaki et al., 2004; Li et al., 2004]. Recent data suggest that g-synuclein role in cancer may be connected with inhibition of the mitotic checkpoint function and stimulation of chromosomal instability, presumably due to inactivation of checkpoint kinase BubR1 [Gupta et al., 2003; Inaba et al., 2005]. Most probably g-synuclein overrides the mitotic checkpoint control and confers the cellular resistance to antimicrotubule drugcaused apoptosis [Inaba et al., 2005]. In addition, g-synuclein is involved in ocular pathologies [Surguchov et al., 2001a; Surgucheva et al., 2002; Maurage et al., 2003] and some forms of neurodegeneration [Galvin et al., 1999, 2000; Wang et al., 2004]. However, currently little is known about molecular and cellular mechanisms underlying g-synuclein implication in these pathologies. In some cell types g-synuclein has been found in association with the centrosomes; however, most probably it is not a component of centrioles, but is transiently attached to a pericentriolar material (PCM) [Surgucheva et al., 2003]. g-Synuclein inhibits proteasomal activity as the other member of the synuclein family, asynuclein [Snyder et al., 2005]. Interestingly, b-synuclein may antagonize some of the pathological properties of a- and g-synucleins [da Costa et al., 2003; Snyder et al., 2005]. Several recent observations concerning g-synuclein attracted our attention: 1. g-Synuclein can be phosphorylated and as a result of this posttranslational modification its properties are changed [Pronin et al., 2000]. 2. Controversial data exist about synucleins (including g-synuclein) intracellular localization. According to several authors, synucleins are cytoplasmic or membrane-bound proteins [Hashimoto et al., 1997; Buchman et al., 1998a; Clayton and George, 1999; McLean et al., 2000] while others describe their localization basically in the nucleus [Maroteaux et al., 1988], finding their association with histones [Goers et al., 2003]. 3. g-Synuclein affects gene expression. These results were received both for individual genes, for example, metalloproteinases, i.e. MMP-9 and MMP-2 [Surgucheva et al., 2003] and by gene microarray analysis (Surguchov, unpublished). Based on these observations we hypothesize that stress may induce g-synuclein phosphorylation and as a result it translocates to the nucleus, where g-synuclein interferes with transcriptional machinery and thus affects the transcription of the specific genes.
In this paper we present data about the localization and properties of g-synuclein in several neuronal and nonneuronal cell cultures in normal conditions and under stress. We show that indeed, g-synuclein is not an exclusively cytoplasmic protein. It changes its intracellular localization, can translocate to the nucleus, and associates with different subcellular structures. Such dynamic localization and translocation of g-synuclein to subcellular structures may give a clue to its normal function and clarify its role in pathologies. MATERIALS AND METHODS Cell Cultures
Photoreceptor Cell Line. This cell line 661W was a gift from Dr. Al-Ubaidi (University of Illinois College of Medicine, Chicago, IL). An immortalized mouse cell line, 661W cells were originally cloned from retinal tumors of a transgenic mouse line expressing the SV-40 antigen under the control of the interphotoreceptor retinol-binding protein [Krishnamoorthy et al., 1999; Roque et al., 1999]. 661W cells grow in monolayer, exhibit dendritic-like processes, and have a doubling time of about 24 h. When assayed by immunocytochemistry, cultured 661W cells were shown to express the photoreceptor proteins arrestin, IRBP, opsin, rds/peripherin, phosducin, a-rod transducin, and recoverin [Roque et al., 1999]. In addition, coculture studies showed that 661W cells form outer segment-like structures. Thus this cell line provides a valuable tool to study photoreceptor cell biology and related disease processes [Al-Ubaidi et al., 1992]. Retinoblastoma Y79. This human cell line [Reid et al., 1974] was purchased from ATCC and used according to the recommendations of the manufacturer. Cells were grown in RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate, 10 mM HEPES, 1% penicillin/streptomycin. The medium contained 10% of fetal bovine serum. Melanoma Cells. Uveal melanoma cells OM431 were grown in RPMI 1640 medium with 10% fetal calf serum (FCS) diluted with conditioned medium at 1:1 (vol/vol). Cutaneous melanoma cells C8161 were grown in RPMI 1640 medium with 10% FCS, NEAA, 1mM sodium pyruvate, and 2mM glutamine [Ellison et al., 2002]. Human Astrocytoma Cell Line U373 MG. This was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS. Antibodies
Rabbit polyclonal antisera were raised against keyhole limpet hemocyanin conjugates of synthetic peptides
g-Synuclein Localization
derived from the predicted structure of human g-synuclein [Lavedan et al., 1998; Ninkina et al., 1998a] or bovine g-synuclein [Surguchov et al., 1999]. The following peptides were used: Ac-C-DLRPSAPQQEGEASKEKamid (human g-synuclein, amino acids 99-115) and Ac-C-ALKQPVPPQEDEAAKAE-amid (bovine g-synuclein, amino acids 99-115). Ab raised against human gsynuclein was used in all experiments with human cells, while Ab raised against bovine ortholog were applied for the staining of mouse cells. Antisera were screened by ELISA and Western blots using the immunizing peptides and retina extract respectively. Abs were then affinity purified using appropriate peptide immobilized on cyanogen bromide-activated Sepharose 4B. For Western blot analysis a-tubulin Ab was diluted 1:5000, and Ab for bovine g-synuclein (0.76 mg/ml) 1:300. Preadsorbtion of Abs with corresponding antigen abolished the staining completely. Anti-g-tubulin GTU88 (SigmaAldrich, St. Louis, MO) and a-tubulin (DM1-a) were from Sigma (St. Louis, MO). The following Abs were used for centrosome staining: anti-centrin 2 rabbit serum used in a dilution 1:2000 and anti-CTR453 mouse monoclonal used undiluted (gifts from Dr. M. Bornens, Institut Curie, Paris, France). Immunocytochemical Staining
For single and double staining Y79 and 661W, the cells were split on glass coverslips. For Y79 the coverslips were treated with 0.02% poly-D-lysine dissolved in H20 for 30 min directly before splitting. The cells were split at 50–60% confluence and kept at 378C in 5% CO2. Cells were washed briefly with PBS, fixed with 100% methanol for 10 min at 2008C, and then with a methanol: acetone mixture (1:1) for 4 min at 208C. For actin staining, cells were fixed in 3.7% paraformaldehyde for 30 min at RT, then washed and permeabilized with 0.5% triton X-100 in PBS for 15 min at RT. After washing several times with PBS, the samples were blocked by incubation in PBS containing 1% BSA and 0.1% of Triton X-100 for 1 h at RT. For single or double staining, the coverslips were placed down on a drop of primary antibody (Ab) (20–40 ll) diluted in blocking solution and left in humidified chamber at þ48C overnight. We used human g-synuclein Ab at 1:100 dilution, atubulin at 1:10,000, g-tubulin at 1:20,000, centrin2 at 1:2000, CNTR453 without dilution, and bovine g-synuclein at 1:300. After washing with PBS three times for 7 min each, cover slips were incubated with goat-antirabbit Oregon green-conjugated or goat-antimouse Rhodamine Red-conjugated secondary Ab. For double staining by anti-centrin2/anti-g-synuclein, the samples were stained with anti-centrin first, and then with goat antirabbit Rhodamine Red conjugated secondary Ab (Jackson Immunoreseach Lab., West Grove, PA) at 1:400 dilution.
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After intensive washing, samples were incubated with primary g-synuclein Ab, washed with PBS, and then incubated in goat-antirabbit Ab conjugated with Oregon green at 1:200 dilution (Jackson Immunoreseach Lab). To stain nuclei, DAPI (10 mg/ml stock, diluted 1:10,000) was added to the final buffer wash. After the final wash, coverslips were mounted in Vectashield (Vector, Burlingame, CA). Fluorescent images were taken using an Olympus BH-2 fluorescence microscope (Olympus, Japan) equipped with a 568 nm filter for Rhodamine red and a 488 nm filter for Oregon green. Images were recorded by digital photography (Spot, Diagnostic Instruments, Sterling Heights, MI) and stored as computer files. Anti-HSP27 (Victoria, Canada) was used in a dilution 1:200. As controls for the specificity of staining we always included staining without primary Ab. Transfection and Generation of Stable Clones Expressing g-Synuclein
Y79 cells were grown in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. For transfection a pcINeo vector (Promega, Madison WI) containing inserted g-synuclein DNA was used as described previously [Surgucheva et al., 2003]. DNA was purified by centrifugation in a CsCl gradient, and the inserts were sequenced by the dRhodamine terminator method. Cells were plated one day before the transfection on a six well plastic plate (Falcon/BD Biosciences, Bedford, MA). Transfection was carried out when cells reached between 50 and 80% confluency. For each experiment, cells were plated at 1–3 3 105 cells in 2 ml of media. Serum-free medium was added in a small sterile tube as diluent for FuGENE 6 Transfection Reagent (Roche Diagnostic Corporation, Alameda, CA) to make the total volume equal to 100 ll. Then FuGENE 6 reagent was added directly to the medium and mixed by tapping. 1 lg of DNA solution was added to prediluted FuGENE 6. The samples were incubated for 45 min at RT. After incubation the mixture containing DNA and FuGENE was added dropwise to the cells and incubation was continued further for 48 h. Stable clones expressing g-synuclein were isolated by using the limited dilution method in the presence of 400 lg/ml G418. After 48 h the cells were dissociated by trypsin and replated at a density of 1–1.5 3 105 cells/100 mm dish in the presence of 400 lg/ml of G418. After 3 weeks of growth in selective medium G418 resistant colonies were cloned. For the analysis of overexpression, the cells were disrupted by ultrasonic treatment and the extract analyzed by Western blot in 12% polyacrylamide gels. 10–20 lg of total protein was loaded on each well. After transfer the blots were probed with rabbit polyclonal Ab against human g-synuclein (1:500). Antirabbit-HRP conjugated Ab (Amersham Pharmacia Biotech, San Francisco, CA)
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(1:20,000) and the ECL detection system (Amersham Pharmacia Biotech) were used for detection of primary Ab. HT22 stable clones overexpressing a- and g-synuclein were generated in a similar way with slight modifications described previously [Surgucheva et al., 2005a]. Western Blotting
Culture of photoreceptor cells p661W was grown in 35 mm plastic plates until 75% confluency. On the day of experiment a half of the plates were placed in CO2 incubator with temperature 428C, while the incubation of control plates was continued at 378C. Cells were incubated for 30 min, 1 h, and 2 h, harvested, washed by cold PBS, and suspended in a sample buffer with 10 mM b-mercaptoethanol. Protein was measured by BCA method (Pierce, Rockford, IL) and 50 lg of protein was loaded on 12% polyacrylamide gel. Other details of Western blotting are described elsewhere [Surguchov et al., 2001a; Surgucheva et al., 2003]. g-Synuclein and a-tubulin were detected using Abs as described in the section ‘‘Antibodies’’ above. Antirabbit-HRP (Amersham) was diluted 1:50,000 for g-synuclein and antimouse HRP-conjugated was used at 1:40,000 dilution for a-tubulin detection. a-Tubulin was used as an internal control to confirm that equal amount of the sample was present in each well. Measurement of Neurite Outgrowth
Neurite outgrowth assay manufactured by Chemicon (Temecula, CA) was used. HT22 clones overexpressing a- or g-synuclein generated as described earlier [Surgucheva et al., 2005a] were grown for 4 days until 60–70% confluence. On the day of experiments 500 ll of human laminin (10 lg/ml in PBS) (Sigma Aldrich) were added in each well, and inserts were put in each well. The plates were incubated for 4 h at 378C. The cells were removed from the culture dishes by media without trypsin or chelating agents and resuspended in N2 media (Sigma, St. Louis, MO) at concentration 1 3 106 cells/ml. The inserts covered by laminin were put in a 24 3 well plate with 500 ll N2 media and 100 ll cell suspension were incubated overnight at 378C for neurite extension. Then the inserts were removed from the wells, rinsed in PBS, and fixed in 100% iced-cold methanol for 20 min at RT. After this the inserts were placed for 15 min into wells containing 500 ll of neurite stain solution at RT. Cell bodies were removed from the upper membrane surface by whirling a cotton swab. For quantification 200 ll of neurite stain extraction buffer were placed on a piece of parafilm and underside of membrane was positioned with extraction buffer (5 min, RT). Two hundred microliter of the suspension was used for quantification by reading density at 562 nm.
RESULTS Perinuclear and Nuclear Localization
Sequence similarity between a- and g-synuclein, especially at their phylogenetically conserved N-terminal parts and the data on colocalization of a-synuclein with microtubules [Alim et al., 2002] raised the possibility that g-synuclein subcellular localization may be similar. Therefore when we determined g-synuclein intracellular localization we assayed its possible colocalization with a-tubulin and other elements of the cytoskeleton. Immunocytochemical staining of the growing photoreceptor cell culture 661W with Ab to g-synuclein revealed two predominant sites of its localization: (a) Most of g-synuclein is present in the cytoplasm and perinuclear area in the form of particles (dots) (Figs. 1A and 1C, arrows). Although in some cases we observed these particles in close proximity to microtubules, we did not find their colocalization (Figs. 1A–1D). (b) Within the nucleus where weak staining was observed for the cells grown under normal conditions (Figs. 1A and 1C). More intensive immunoreactivity was observed in the nuclei of cells incubated under stress conditions, e.g. at 428C for 12 h (Figs. 1B and 1D). In some cells we observed a patchy staining in a nuclear area (Fig. 1B), while in others the staining was more homogeneous (Fig. 1D). g-Synuclein nuclear localization was also found in other cell types (see below Figs. 3C and 3E and Figs. 4B, 4D, and 4E). Anti-tubulin Ab showed staining of an intracellular network typical for microtubules (Figs. 1A–1C and 1E); however, no association of g-synuclein with microtubules was revealed. In some cells we also observed gsynuclein positive filament-like structures. These structures are relatively short and not abundant (Figs. 1E and 1F, arrows). They are not colocalized with microtubules (E, red) and heat shock protein HSP27 (F, red). Western blot analysis revealed that the same stress conditions (428C) did not induce a significant change in the level of g-synuclein expression in these cells (Fig. 2A) and the expression of a-tubulin used as control (Fig. 2B). However, the expression of the heat shock protein hsp60 increased several fold under these same conditions (not shown).
Effect of Synuclein Overexpression on Neurite Outgrowth
Previously the effect of g-synuclein on cell migration and adhesion was demonstrated [Jia et al., 1999]. Thus we decided to determine the influence of g-synuclein overexpression on neurite outgrowth and compare it with the effect of a-synuclein. As shown on Fig. 2C, after 24 h of growth neurite outgrowth is considerably reduced in clones overexpressing g-synuclein (66% reduction compared to control cells, bar 3, P < 0.03),
Fig. 1. Immunolocalization of g-synuclein in photoreceptor cells 661W. Cells were fixed by methanol and processed for immunofluorescent staining as described in Materials and Methods. (A–D) Localization of g-synuclein (red) and a-tubulin (green) was examined. (A, C) Control cells; (B, D) cells incubated at 428C. g-Synuclein localizes
in the cytoplasm and perinuclear area in the form of dots or particles (arrows) under normal conditions (A, C) and is partially relocated to the nucleus at elevated temperature (B, D). (E, F) Filament-like structures positive for g-synuclein (green, arrows) are not colocalized with microtubules (E, red) and heat shock protein HSP27 (F, red).
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ated with centrosomes on early steps of mitosis, but no centrosome staining by g-synuclein Ab is observed in telophase, Fig. 3A (arrows), suggesting that g-synuclein binding to centrosomes may be cell cycle dependent. (b) Y79 retinoblastoma and astrocytes are not the only types of cells in which g-synuclein associates with centrosomes. g-Synuclein is also colocalized with centrosomal markers in other cell types, e.g. ocular melanoma cells OM431 (Fig. 3B) and cutaneous melanoma C8161 (not shown). g-Synuclein in these cell types colocalizes with a-tubulin (Fig. 3B) and other centrosomal markers, e.g. g-tubulin, centrin 2 and CTR453 (not shown). Midbody Staining
Immunocytochemical staining of astrocytoma cell line U373 (Fig. 3, C1–C3) and two melanoma cell lines OM431 (Fig. 3D) and C8161 (Fig. 3E) demonstrates that in these cells g-synuclein translocates by cytokinesis to the midbody (Figs. 3C–3E)––the cytoplasmic bridge linking the two daughter cells. Comparison of the g-synuclein and a-tubulin staining reveals that g-synuclein codistributes with a-tubulin in the central part of the midbody region. Characteristics of Stable Clones Expressing g-Synuclein
Fig. 2. Western blotting of photoreceptor cell extracts (A and B) and neurite outgrowth in cells overexpressing synucleins (C). A and B: Extracts from cells incubated at 378C (A and B, lanes 1) and at 428C for 30 min (A and B, lanes 2), 1 h (A and B, lanes 3), and 2 h (A and B, lanes 4) were subjected to electrophoresis, blotted, and probed with Abs to g-synuclein (A) and a-tubulin as control (B). C Neurite outgrowth in HT22 clones overexpressing a-synuclein (bar 2) and gsynuclein (bar 3). (1) Control cells transfected by an empty vector. Asterisks indicate that the experimental data significantly differ from controls.
while a-synuclein caused 30% reduction in neurite outgrowth (bar 2, P < 0.05). After 48 h of growth the effect persisted, but became less significant (41% for g-synuclein and 23% reduction for a-synuclein, respectively).
g-Synuclein association with centrosomes in Y79 retinoblastoma cells was demonstrated by us earlier [Surguchov et al., 2001b] and also can be seen on Fig. 3A as well as in other cell types (Fig. 3B). In order to study the effect of g-synuclein overexpression on centrosome association we generated a stable clone by transfecting Y79 cells with an integration vector containing g-synuclein cDNA. We generated 20 stable clones that exhibited different levels of g-synuclein expression, and selected two that showed the strongest signal by Western blot analysis as described earlier [Surguchov et al., 2001b]. Immunofluorescence analysis revealed that the level of g-synuclein associated with centrosomes in these stable clones was significantly increased (Figs. 4A and 4B – g-synuclein immunoreactivity, E, F – merged staining with centrosomal marker g-tubulin), while no effect of g-synuclein overexpression on the staining intensity for g-tubulin was observed (Figs. 4C and 4D).
Centrosome Staining
DISCUSSION
Previously we demonstrated that g-synuclein is a centrosome-associated protein in retinoblastoma Y79 cells and astrocytes [Surguchov et al., 2001b; Surgucheva et al., 2002]. In this paper we extent this observation, demonstrating that (a) g-Synuclein is associ-
The data presented here shows that g-synuclein may have different intracellular localization depending on cell cycle and conditions of growth. In a culture of photoreceptor cells, g-synuclein is present in the perinuclear area where it can form dot-like structures or can be
Fig. 3. Association of g-synuclein with subcellular structures. Immunohistochemical staining by g-synuclein Ab (green) and a-tubulin Ab (red). (A) In Y79 retinoblastoma cells g-synuclein is associated with centrosomes in the majority of cells except cells undergoing late stages of mitosis (arrows); (B and D) melanoma cells OM431, (E) melanoma C8161 cells. (B) centrosome staining in melanoma cells is shown by the arrow. (C–E) Association of g-synuclein with midbody (arrows) of cytokinesis. (C1–C3) The human astrocytoma cell line U373 MG. C1, g-synuclein; C2, a-tubulin; C3, merged. Comparison of the g-synuclein and a-tubulin staining reveals that g-synuclein codistributes with atubulin in the central part of the midbody region.
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Fig. 4. Increased association of g-synuclein with centrosomes in overexpressing clones. Immunohistochemical staining of nontransfected cells (A, C, E) and stable clones overexpressing g-synuclein (B, D, F) by anti-g-synuclein (A, B) and anti-g-tubulin (C, D). (E, F) Double staining by anti-g-synuclein and anti-g-tubulin. Note that higher amount of g-synuclein (green; yellow in merged image) is associated with centrosomes in clones overexpressing g-synuclein (B, F) compared to controls cells (A, E).
localized in the nucleus (Fig. 1). No colocalization of these dots (or particles) with the cytoskeletal proteins, including actin, tubulin, and neurofilament proteins was revealed (not shown). These filament-like structures positive for g-synuclein (green, arrows) are not colocalized with microtubules (Fig. 1E, red) and heat shock protein HSP27 (F, red).
In some cells we also observed g-synuclein positive filamentous structures (Figs. 1E and 1F, arrows). After stress induced by incubation of cells at 428C, some immunoreactivity is translocated from the cytoplasm to the nucleus where either a patchy staining (Fig. 1B) or a more homogenous staining of the nucleus was observed (Fig. 1D). In addition to the photoreceptor cells, localiza-
g-Synuclein Localization
tion of g-synuclein to the nucleus of melanoma cells was also observed (Fig. 3B). The localization of synuclein in the nucleus of neuronal cell soma was first described by [Maroteaux et al., 1988]. However, in later publications a-synuclein was found predominantly in the cytoplasm and/or as a membrane-bound protein [Hashimoto et al., 1997; Clayton and George, 1999; McLean et al., 2000], while g-synuclein was shown to be predominantly cytosolic [Buchman et al., 1998a]. Thus, members of the synuclein family have different subcellular localizations, and they might migrate between different cellular compartments depending on different intracellular and extracellular stimuli. Stress is apparently one of the factors inducing synuclein shuttling between nucleus and cytoplasm. In this paper we present data that g-synuclein is present both in the cytoplasm and/or in the nucleus. A similar subcellular localization and stressinduced translocation from the cytoplasm into the nucleus was previously demonstrated for several chaperones [Rhee et al., 2000; Rodriguez et al., 2000]. Recently chaperonelike activity was demonstrated for a, b-, and g-synuclein [Souza et al., 2000b; Surgucheva et al., 2005b]. A possible mechanism for the regulation of g-synuclein migration between the cytoplasm and the nucleus is protein phosphorylation. g-Synuclein can be phosphorylated by G proteincoupled receptor kinases [Pronin et al., 2000]. Phosphorylation-dephosphorylation was shown to be an important factor affecting the translocation of chaperones between the cytoplasm and the nucleus [Rodriguez et al., 2000]. Earlier it was shown that a-synuclein reduces neurite extension and causes weaker adhesion compared to controls or b-synuclein [Takenouchi et al., 2001]. The authors concluded that reduced neuritic activity in the a-synucleinexpressing cells might be due to alterations in cell adhesion capacity. Later, a possible role of ERK signaling and caveolae was proposed for mediating this response to a-synuclein [Hashimoto et al., 2003]. In this paper we determined the effect of a- and g-synuclein expression on neurite outgrowth using neuronal culture HT22 and found that both synucleins inhibit neurite outgrowth. The effect of g-synuclein on neurite outgrowth points to its possible role in cell– cell and cell substrate interactions. This finding is important because of g-synuclein role in tumorgenesis [Ji et al., 1997; Jia et al., 1999; Bruening et al., 2000; Fung et al., 2003]. g-Synuclein is able to form relatively short intracellular filaments (Figs. 1E and 1F) and is present in processes after actin cytoskeleton is disassembled by latrunculin A treatment (not shown). Previously, different types of filaments were described that contained only one member of the synuclein family, a-synuclein. Interestingly, this protein can form filaments or fibrils both in vitro [Crowther et al., 1998; El-Agnaf et al., 1998; Giasson et al., 1999; Souza et al., 2000a] and in vivo [Spillantini et al., 1998; Goedert et al., 1999]. a-Synuclein
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aggregation into filamentous inclusions plays a role in the development of intraneuronal inclusions. Earlier we described g-synuclein association with the centrosome [Surguchov et al., 2001b] and further extended this conclusion in the current paper. Interestingly, that g-synuclein association with the centrosomes is apparently cycle-cell dependent, since g-synuclein staining disappears in cells on the step of late mitosis (anaphase and telophase, Fig. 3A). Staining of stable cell lines expressing protein with anti-g-synuclein revealed an increase in immunoreactivity in the centrosome (Fig. 4). These data suggest that centrosomes are not saturated with g-synuclein in nontransfected cells and that overexpression of protein in stable clones leads to additional binding of this protein to the centrosome. Although these results open a number of intriguing possibilities for understanding the role of g-synuclein in cell function, further investigations will be required to firmly establish whether g-synuclein is an authentic protein components of centrosomes and whether it plays any role in centrosome function and cytokinesis. This suggests that the binding of g-synuclein to the centrosome may be mediated by the C-terminal domain which is specific for each member of the synuclein family. Interestingly, some similarity in amino acid sequences exists between C-terminal fragments of g-synuclein and conserved region of human centrosome-associated proteins, e.g. AKAP450 or pericentrin [Gillingham and Munro, 2000]: AKAP450 3876--ALTDYITRLEALQ--3888 : : : : :: : : : : g-syn 89--AVTSGVVRKEDLR--101 A dual role for g-synuclein in neurodegeneration and malignancy which could involve common mechanisms has been suggested [Ji et al., 1997; Jia et al., 1998; Ninkina et al., 1998a,b]. Alteration in the organization of the cell cytoskeleton is one characteristic of both types of disorders. Since the centrosome plays a central role in maintaining the organization of cell cytoplasm, disruption of centrosome function could contribute to these diseases. The centrosome functions as the major microtubule organizing center of the cell and as such it determines the number, polarity, and organization of in-terphase and mitotic microtubules. Our finding that g-synuclein localizes to the centrosome may be providing an important clue regarding the role of this protein in normal cell function and disease states. The results presented in this paper show that g-synuclein is associated with centrosome in early phases of mitosis, but is not found on the centrosome on anaphase and telophase. Centrosomes are present in anaphase and telophase
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[Scholey et al., 2003] and the architecture and composition of the centrosome, especially the PCM, changes during the cell cycle. According to a current hypothesis, g-synuclein role in tumorgenesis may be explained by its binding to mitotic checkpoint kinase BubR1 and inhibition of mitotic checkpoint functions [Gupta et al., 2003; Inaba et al., 2005]. The tendency of endogenous g-synuclein expression coinciding with lower BubR1 protein levels was also observed in several breast cancer cell lines [Gupta et al., 2003]. The reducing effect of g-synuclein on BubR1 protein expression can be prevented by treating g-synuclein-transfected cells with MG-132, a selective 26S proteasome inhibitor, implying that the proteasome machinery may be involved in the g-synuclein-induced reduction of the BubR1 protein. Intriguingly, our recent data showed that synucleins are modulators of the proteasomal activity [Snyder et al., 2005]. Further experiments are necessary to get a deeper understanding how these synuclein functions are coordinated and why their malfunction leads to pathology. Our data presented here and showing g-synuclein relocation to the midbody during cytokinesis points to its possible role during cell division and cytokinesis. Similar cell-cycle dependent translocation to the midbody structures has been demonstrated for other proteins, for example for cytoskeletal protein talin [Bellissent-Waydelich et al., 1999]. Significant upregulation of g-synuclein occurs not only in cancer, its expression may be induced by different stimuli, i.e. toxic substances [Espinoza et al., 2005] or cocaine [Brenz Verca et al., 2003]. In addition, g-synuclein levels in the cerebral cortex are differentially associated with dominant and submissive behavior [Pinhasov et al., 2005]. These data suggest the involvement of g-synuclein in different processes related to human health and environmental safety. In conclusion, we hypothesize that g-synuclein localization is cell-context-dependent as well as temporally and spatially regulated. Our studies indicate a nuclear localization pattern of g-synuclein with dynamic distribution in the cell cycle and transient association with centrosomes and other subcellular structures. These data are in contrast with predominantly or exclusively cytoplasmic localization described previously. g-Synuclein association with centrosomes and spindle may explain its role in increased tumorgenesis and other tumor promoting functions via the effect on checkpoint signaling and induction of aneuploidy. Further studies on the functions of g-synuclein, including the use of knockouts and siRNA, should provide important insight into the role of this protein in diverse physiological processes in health and disease.
ACKNOWLEDGMENTS
Supported by VA Merit Review grant, NIH grant EY 02687, The Glaucoma Foundation Grant QB42308, grant from Carl Marshall Reeves and Mildred Almen Reeves Foundation, and grant from ADRC 99-6403. We would like to thank Dr. M. Bornens (Institut Curie, Paris, France) for anti-centrin 2 and anti-CTR453 antibody. Photoreceptor cell line 661W was a gift from Dr. Al-Ubaidi (University of Oklahoma Health Sciences Center, Oklahoma City). Melanoma cells OM431 and C8161 were a kind gift of Timothy Fleming (Washington University, St. Louis), human astrocytoma was a gift of M. Rosario Hernandez (Washington University, St. Louis). Comments and suggestions of Irina Kaverina (Vanderbilt University Medical Center) are highly appreciated. REFERENCES Alim MA, Hossain MS, Arima K, Takeda K, Izumiyama Y, Nakamura M, Kaji H, Shinoda T, Hisanaga S, Ueda K. 2002. Tubulin seeds a-synuclein fibril formation. J Biol Chem 277(3): 2112–2117. Al-Ubaidi MR, Font RL, Quiambao AB, Keener MJ, Liou GI, Overbeek PA, Baehr W. 1992. Bilateral retinal and brain tumors in transgenic mice expressing simian virus 40 large T antigen under control of the human interphotoreceptor retinoid-binding protein promoter. J Cell Biol 119:1681–1687. Bellissent-Waydelich A, Vanier MT, Albiges-Rizo C, Simon-Assmann P. 1999. Talin concentrates to the midbody region during mammalian cell cytokinesis. J Histochem Cytochem 47(11): 1357–1368. Bennett MC. 2005. The role of a-synuclein in neurodegenerative diseases. Pharmacol Ther 105(3):311–331. Brenz Verca MS, Bahi A, Boyer F, Wagner GC, Dreyer JL. 2003. Distribution of a- and g-synucleins in the adult rat brain and their modification by high-dose cocaine treatment. Eur J Neurosci 18(7):1923–1938. Bruening W, Giasson BI, Klein-Szanto AJ, Lee VM, Trojanowski JQ, Godwin AK. 2000. Synucleins are expressed in the majority of breast and ovarian carcinomas and in preneoplastic lesions of the ovary. Cancer 88(9):2154–2163. Buchman VL, Hunter HJ, Pinon LG, Thompson J, Privalova EM, Ninkina NN, Davies AM. 1998a. Persyn, a member of the synuclein family, has a distinct pattern of expression in the developing nervous system. J Neurosci 18(22):9335–9341. Buchman VL, Adu J, Pinon LGP, Ninkina NN, Davies AM. 1998b. Persyn, a member of the synuclein family, influences neurofilament network integrity. Nat Neurosci 1:101–103. Clayton DF, George JM. 1999. Synucleins in synaptic plasticity and neurodegenerative disorders. J Neurosci Res 58(1):120– 129. Crowther RA, Jakes R, Spillantini MG, Goedert M. 1998. Synthetic filaments assembled from C-terminally truncated a-synuclein. FEBS Lett 436(3):309–312. da Costa CA, Masliah E, Checler F. 2003. b-Synuclein displays an antiapoptotic p53-dependent phenotype and protects neurons from 6-hydroxydopamine-induced caspase 3 activation: Crosstalk with a-synuclein and implication for Parkinson’s disease. J Biol Chem 278(39):37330–37335.
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