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• W2166236894 abstract "Voltage-gated sodium channels initiate electrical signaling in excitable cells such as muscle and neurons. They also are expressed in non-excitable cells such as macrophages and neoplastic cells. Previously, in macrophages, we demonstrated expression of SCN8A, the gene that encodes the channel NaV1.6, and intracellular localization of NaV1.6 to regions near F-actin bundles, particularly at areas of cell attachment. Here we show that a splice variant of NaV1.6 regulates cellular invasion through its effects on podosome and invadopodia formation in macrophages and melanoma cells. cDNA sequence analysis of SCN8A from THP-1 cells, a human monocyte-macrophage cell line, confirmed the expression of a full-length splice variant that lacks exon 18. Immunoelectron microscopy demonstrated NaV1.6-positive staining within the electron dense podosome rosette structure. Pharmacologic antagonism with tetrodotoxin (TTX) in differentiated THP-1 cells or absence of functional NaV1.6 through a naturally occurring mutation (med) in mouse peritoneal macrophages inhibited podosome formation. Agonist-mediated activation of the channel with veratridine caused release of sodium from cationic vesicular compartments, uptake by mitochondria, and mitochondrial calcium release through the Na/Ca exchanger. Invasion by differentiated THP-1 and HTB-66 cells, an invasive melanoma cell line, through extracellular matrix was inhibited by TTX. THP-1 invasion also was inhibited by small hairpin RNA knockdown of SCN8A. These results demonstrate that a variant of NaV1.6 participates in the control of podosome and invadopodia formation and suggest that intracellular sodium release mediated by NaV1.6 may regulate cellular invasion of macrophages and melanoma cells. Voltage-gated sodium channels initiate electrical signaling in excitable cells such as muscle and neurons. They also are expressed in non-excitable cells such as macrophages and neoplastic cells. Previously, in macrophages, we demonstrated expression of SCN8A, the gene that encodes the channel NaV1.6, and intracellular localization of NaV1.6 to regions near F-actin bundles, particularly at areas of cell attachment. Here we show that a splice variant of NaV1.6 regulates cellular invasion through its effects on podosome and invadopodia formation in macrophages and melanoma cells. cDNA sequence analysis of SCN8A from THP-1 cells, a human monocyte-macrophage cell line, confirmed the expression of a full-length splice variant that lacks exon 18. Immunoelectron microscopy demonstrated NaV1.6-positive staining within the electron dense podosome rosette structure. Pharmacologic antagonism with tetrodotoxin (TTX) in differentiated THP-1 cells or absence of functional NaV1.6 through a naturally occurring mutation (med) in mouse peritoneal macrophages inhibited podosome formation. Agonist-mediated activation of the channel with veratridine caused release of sodium from cationic vesicular compartments, uptake by mitochondria, and mitochondrial calcium release through the Na/Ca exchanger. Invasion by differentiated THP-1 and HTB-66 cells, an invasive melanoma cell line, through extracellular matrix was inhibited by TTX. THP-1 invasion also was inhibited by small hairpin RNA knockdown of SCN8A. These results demonstrate that a variant of NaV1.6 participates in the control of podosome and invadopodia formation and suggest that intracellular sodium release mediated by NaV1.6 may regulate cellular invasion of macrophages and melanoma cells. In excitable tissues such as muscle and nerve, activation of voltage-gated sodium channels initiates electrical signaling through sodium influx coupled to membrane depolarization (1Catterall W. Goldin A. Waxman S. Pharmacol. Rev. 2005; 57: 397-409Crossref PubMed Scopus (1063) Google Scholar). During muscle contraction or synaptic vesicle release, depolarization is coupled to increases in cytosolic calcium from extracellular and intracellular stores. Increased cytosolic sodium due to entry of sodium through plasma membrane channels may also be sufficient to mobilize calcium from intracellular stores (2Lowe D. Richardson B. Taylor P. Donatsch P. Nature. 1976; 260: 337-338Crossref PubMed Scopus (108) Google Scholar, 3Leblanc N. Hume J. Science. 1990; 248: 372-376Crossref PubMed Scopus (454) Google Scholar, 4Blumenstein Y. Maximyuk O. Lozovaya N. Yatsenko N. Kanevsky N. Krishtal O. Dascal N. J. Physiol. 2004; 556: 121-134Crossref PubMed Scopus (26) Google Scholar, 5Nikolaeva M. Mukherjee B. Stys P. J. Neurosci. 2005; 25: 9960-9967Crossref PubMed Scopus (83) Google Scholar). Non-excitable cells such as bone marrow-derived macrophages also express voltage-gated sodium channels (6Carrithers M. Dib-Hajj S. Carrithers L. Tokmoulina G. Pypaert M. Jonas E. Waxman S. J. Immunol. 2007; 178: 7822-7832Crossref PubMed Scopus (99) Google Scholar, 7Craner M. Damarjian T. Liu S. Hains B. Lo A. Black J. Newcombe J. Cuzner M. Waxman S. Glia. 2005; 49: 220-229Crossref PubMed Scopus (216) Google Scholar, 8Black J. Liu S. Carrithers M. Carrithers L. Waxman S. Ann. Neurol. 2007; 62: 21-33Crossref PubMed Scopus (96) Google Scholar). In human macrophages, variants of the neuronal channel, NaV1.6, and the cardiac channel, NaV1.5, are expressed intracellularly but not at the plasma membrane (6Carrithers M. Dib-Hajj S. Carrithers L. Tokmoulina G. Pypaert M. Jonas E. Waxman S. J. Immunol. 2007; 178: 7822-7832Crossref PubMed Scopus (99) Google Scholar). NaV1.5 localizes to the macrophage late endosome in interferon-γ or lipolysaccharide-activated cells where it mediates endosomal acidification. NaV1.6 localizes to vesicles that are distributed throughout the cytoplasm and some of which are associated with the actin cytoskeleton. NaV1.6 has a less clear functional role in macrophages. Unlike NaV1.5, NaV1.6 is also expressed in unprimed human macrophages and in unprimed and primed mouse macrophages and microglia (7Craner M. Damarjian T. Liu S. Hains B. Lo A. Black J. Newcombe J. Cuzner M. Waxman S. Glia. 2005; 49: 220-229Crossref PubMed Scopus (216) Google Scholar, 8Black J. Liu S. Carrithers M. Carrithers L. Waxman S. Ann. Neurol. 2007; 62: 21-33Crossref PubMed Scopus (96) Google Scholar). Many invasive neoplastic cell lines also express voltage-gated sodium channels, which may regulate their ability to metastasize (9Fraser S. Salvador V. Manning E. Mizal J. Altun S. Raza M. Berridge R. Djamgoz M. J. Cell. Physiol. 2003; 195: 479-487Crossref PubMed Scopus (138) Google Scholar). Adhesion and movement of monocytes, macrophages, and invasive neoplasms are regulated in part by podosome assembly. Podosomes are specialized regions of the F-actin cytoskeleton that mediate local adhesion, invasion, and migration in specific cell types such as macrophages, bone osteoclasts, and other immune-derived cells (10Miyauchi A. Hruska K. Greenfield E. Duncan R. Alvarez J. Barattolo R. Colucci S. Zambonin-Zallone A. Teitelbaum S. Teti A. J. Cell Biol. 1990; 111: 2543-2552Crossref PubMed Scopus (199) Google Scholar, 11Evans J. Matsudaira P. Eur. J. Cell Biol. 2006; 85: 145-149Crossref PubMed Scopus (32) Google Scholar, 12Calle Y. Burns S. Thrasher A. Jones G. Eur. J. Cell Biol. 2006; 85: 151-157Crossref PubMed Scopus (116) Google Scholar, 13Tsuboi S. J. Immunol. 2007; 178: 2987-2995Crossref PubMed Scopus (47) Google Scholar, 14Carman C. Sage P. Sciuto T. de la Fuente M. Geha R. Ochs H. Dvorak H. Dvorak A. Springer T. Immunity. 2007; 26: 784-797Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). They are very dynamic structures that degrade and re-polymerize within seconds to minutes (15Evans J. Correia I. Krasavina O. Watson N. Matsudaira P. J. Cell Biol. 2003; 161: 697-705Crossref PubMed Scopus (135) Google Scholar). Their formation is calcium-dependent (10Miyauchi A. Hruska K. Greenfield E. Duncan R. Alvarez J. Barattolo R. Colucci S. Zambonin-Zallone A. Teitelbaum S. Teti A. J. Cell Biol. 1990; 111: 2543-2552Crossref PubMed Scopus (199) Google Scholar, 16Chellaiah M. Kizer N. Silva M. Alvarez U. Kwiatkowski D. Hruska K. J. Cell Biol. 2000; 148: 665-678Crossref PubMed Scopus (213) Google Scholar). In invasive neoplastic cells, these structures are called invadopodia and may be important determinants for invasiveness and metastasis (17Tarone G. Cirillo D. Giancotti F. Comoglio P. Marchisio P. Exp. Cell Res. 1985; 159: 141-157Crossref PubMed Scopus (342) Google Scholar, 18Chen W. J. Exp. Zool. 1989; 251: 167-185Crossref PubMed Scopus (270) Google Scholar). Our goal here was to characterize the role of intracellular NaV1.6 in the regulation of the actin cytoskeleton. A splice variant of NaV1.6 that lacks exon 18 is expressed at a low level in fetal neurons and many non-neural tissues (19Plummer N.W. McBurney M.W. Meisler M.H. J. Biol. Chem. 1997; 272: 24008-24015Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). We demonstrate the expression of this splice variant of SCN8A, the gene that encodes NaV1.6 in macrophages, and show that pharmacologic antagonism with tetrodotoxin (TTX), 3The abbreviations used are: TTX, tetrodotoxin; m-CSF, macrophage colony-stimulating factor; MCP-1, monocyte chemotractant protein; NMDG, N-methyl-d-glucamine; SBFI, sodium binding benzofuran isopthalate; shRNA, small hairpin RNA; FBS, fetal bovine serum; PBS, phosphate-buffered saline; HBSS, Hanks' balanced salt solution; RFU, relative fluorescent unit. absence of a functional NaV1.6 due to a genetic mutation, or knockdown of NaV1.6 expression with shRNA impairs podosome formation in macrophages. Blockade of the channel also prevents cellular invasion of macrophages and melanoma cells through extracellular matrix. Activation of the channel by the voltage-gated sodium channel agonist veratridine leads to vesicular intracellular sodium release, uptake of sodium by the adjacent mitochondrial compartment, and release of mitochondrial calcium. These findings suggest that a variant of NaV1.6 contributes to the control of macrophage and melanoma cellular invasion through a signaling pathway that may link intracellular sodium channel activation to actin cytoskeleton dynamics. Cells-THP-1 cells, a human premyelomonocytic leukemic cell line, were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), sodium pyruvate, and non-essential amino acids. Differentiation to a macrophage phenotype was induced by treatment with 12-O-tetradecanoylphorbol-13-acetate (10 ng/ml, 72 h). HTB-66 cells, an invasive human melanoma cell line, were grown in Dulbecco's modified Eagle's media supplemented with 10% FBS, sodium pyruvate, and non-essential amino acids. Mice-Resident peritoneal macrophages were obtained from C57BL6/J, NaV1.6-deficient C3HeB/FeJ-Scn8amed/J mice, and wild-type littermates (Jackson Laboratories) by peritoneal lavage with serum supplemented RPMI. C3HeB/FeJ-Scn8amed/J mice and their control littermates were ∼14-21 days of age and were bred in our facility from heterozygous carriers of the med recessive gene. C57BL6/J mice were purchased from Jackson Laboratories and used at 6-8 weeks of age. Macrophages were isolated by centrifugation followed by isolation of mononuclear cells on a lymphocyte separation medium step gradient and adhesion to glass coverslips. Cells were plated at a density of 1 × 106/ml and maintained for 18-24 h in the same media as THP-1 cells. All animal studies were reviewed and approved by the Yale University School of Medicine Institutional Animal Care and Review Committee (IACUC). SCN8A cDNA Sequence Analysis-A PCR-based approach was used to sequence SCN8A cDNA generated from THP-1. mRNA was isolated, reverse transcribed, and analyzed for expression of SCN8A by quantitative PCR as described previously (6Carrithers M. Dib-Hajj S. Carrithers L. Tokmoulina G. Pypaert M. Jonas E. Waxman S. J. Immunol. 2007; 178: 7822-7832Crossref PubMed Scopus (99) Google Scholar). Three-way (“stitched”) PCR was used to generate a full-length coding PCR fragment for sequencing (exon 2-27). First, two separate PCR fragments were synthesized: one (2.52 kb) with the 2F and 15R primers and the other (3.36 kb) with 15F and 27R primers (see Table S1 for primer sequences). These two fragments were gel eluted and purified. Equal amounts of these fragments were used as a template for the three-way PCR using the 2F and 27R primers to amplify a 5.8-kb fragment. Cycle conditions were as follows: denaturation at 95 °C for 5 min × 1 cycle, denaturation at 95 °C for 30 s, annealing at 62 °C for 30 s, and extension at 68 °C for 7 min (×34 cycles), and further extension at 68 °C for 10 min × 1 cycle. Sequencing of the 5.8-kb PCR product was performed at the W.M. Keck facility at Yale University School of Medicine. The primers used for sequencing (“primer walking”) of this product are shown in Table S1. The products were sequenced in both the forward and reverse directions for confirmation. DNAS-TAR (Lasergene) was used for analysis of the sequence data. Immunohistochemistry-Cells grown on multichambered glass coverslips were washed with PBS, fixed with 4% paraformaldehyde in PBS (10 min), and blocked in PBS containing 5% serum (goat or donkey, dependent on secondary antibody), 0.1% Triton X-100, 1% bovine serum albumin, and 0.04 mg/ml normal human IgG. Primary and secondary antibodies were diluted in blocking solution. Rabbit anti-NaV1.6 was obtained from Alomone Laboratories. Goat anti-gelsolin and mouse anti-dynamin II were from Santa Cruz Biotechnology and BD Biosciences, respectively. Secondary donkey antibodies conjugated to Alexa fluorophores, MitoTracker dye, and phalloidin-Alexa 350 and 488 were obtained from Invitrogen. 4′,6′-Diamidino-2-phenylindole was from Vector Laboratories. Images were obtained on a Zeiss Axiovert 200 fluorescent microscope with a ×63 objective (Zeiss Plan Apochromat, 1.4 oil). Electron Microscopy-For fixation for regular epon embedding to show membrane preservation, samples were fixed in 2.5% gluteraldehyde in 0.1 m sodium cacodylate buffer, pH 7.4, with 3% sucrose for 1 h at room temperature. The samples were rinsed 3 times in sodium cacodylate rinse buffer and then pelleted in 2% agar. These were post-fixed in 1% osmium tetroxide for 1 h, en bloc stained in 2% uranyl acetate in maleate buffer, pH 5.2, for a further hour then rinsed, dehydrated, and infiltrated with epon resin and baked overnight at 60 °C. Hardened blocks were cut using a Leica UltraCut UCT. 60-nm sections were collected and stained using 2% uranyl acetate and lead citrate. For sample fixation for immunoelectron microscopy, samples were fixed in 4% paraformaldehyde in 0.25 m Hepes for 1 h. Samples were rinsed in PBS and re-suspended in 10% gelatin, chilled, and trimmed to smaller blocks and placed in cryoprotectant of 2.3 m sucrose overnight on a rotor at 4 °C. They were transferred to aluminum pins and frozen rapidly in liquid nitrogen. The frozen block was trimmed on a Leica Cryo-EMUC6UltraCut and 75-nm thick sections were collected. The frozen sections were collected on a drop of sucrose, thawed, and placed on a nickel formvar/carbon-coated grid and floated in a dish of PBS ready for immunolabeling. For immunolabeling of sections, grids were placed section side down on drops of 0.1 m ammonium chloride for 10 min to quench untreated aldehyde groups, then blocked for nonspecific binding in 1% fish skin gelatin in PBS for 20 min. Single labeled grids were incubated in either a primary antibody rabbit anti-Nav1.6, 1:50 (Alomone), or mouse anti-β-actin (Sigma), 1:200 dilutions, for 30 min. Controls were also done using rabbit IgG (Jackson) and mouse IgG (Jackson) at the same dilution. Secondary antibodies were either 12-nm anti-rabbit or anti-mouse colloidal gold (Jackson) for 30 min. Double labeling was performed as above but by combining the two primaries, rinsing, and then combining secondary direct gold anti-mouse 6 nm (actin) and anti-rabbit 12 nm (Nav1.6). All grids were rinsed in PBS, fixed using 1% gluteraldehyde for 5 min, and rinsed, transferring grids to a UA/methylcellulose drop for 10 min. Samples were all viewed on a FEI Tencai Biotwin TEM at 80 Kv. Images were taken using Morada CCD and iTEM (Olympus) software. Podosome Quantitation-Differentiated THP-1 cells were treated with serum-free media for 4 h in the presence and absence of 300 nm TTX, a concentration that blocks TTX-sensitive sodium channels such as NaV1.6 but not TTX-resistant channels such as NaV1.5. They were then permeabilized in PBS containing 0.1% Triton X-100 for 20 min, incubated with phalloidin-Alexa 488 in 1% bovine serum albumin for an additional 20 min, and washed with PBS. Cells were counterstained with 4′,6′-diamidino-2-phenylindole and analyzed by fluorescent microscopy. Podosomes were identified manually with an event marker (Zeiss Axiovision 4.6.3 software) from images taken with a ×40 objective (LD Plan-Neofluar, 0.6). Live Cell Imaging-For live cell imaging of sodium flux, 12-O-tetradecanoylphorbol-13-acetate-treated THP-1 cells plated on a Delta T4 cell culture plate (Bioptechs) were labeled with 4 μm Sodium Green and 1 μm Corona Red for 40 min at 37 °C in Hanks' buffered salt solution (HBSS). Cells were washed three times with HBSS and then monitored for fluorescence on a heated microscopy stage (37 °C) with a heated ×63 oil immersion objective on a Zeiss Axiovert 200 fluorescent microscope. Two color fluorescent images were taken in multiple Z-planes prior to and following a 1-min stimulation with veratridine (80 μm). Images were deconvoluted using Axiovision Three-dimensional Deconvolution software (Zeiss) and analyzed quantitatively with Axiovision 4.6.3 Automeasurement. Fluorometry-For time-resolved fluorescence analysis of THP-1 cells or freshly isolated mouse peritoneal macrophages, cells were isolated by centrifugation, resuspended in serum-free HBSS at room temperature, and labeled with indicator dyes. For sodium flux measurements, cells were labeled with the ratiometric sodium indicator SBFI-AM (4 μm) for 40 min at room temperature in HBSS and then washed with assay buffer by three successive centrifugations. Assay buffer was either 135 mm NaCl, 4.5 mm KCl, 4 mm EGTA, 11 mm glucose, and 20 mm Hepes, pH 7.4, or the same buffer with NaCl replaced by 145 mm N-methyl-d-glucamine (NMDG). NMDG-containing solutions were titrated to pH 7.4 with HCl. Prior to and following veratridine stimulation (80 μm), the ratio of fluorescence intensities excited at 340/380 nm was monitored at an emission wavelength of 505 nm in a LS-50b spectrophotometer (PerkinElmer Life Sciences). For calibration of intracellular sodium, THP-1 cells in NMDG buffer were mixed with increasing concentrations of extracellular sodium (5, 15, 25, and 50 mm NaCl, balanced in a molar fashion with NMDG) following permeabilization of cells with 0.1% Triton X-100. At least four separate measurements were taken at the indicated concentration to generate a standard curve. By this method, the estimated intracellular sodium concentration was 11.15 ± 0.92 mm (n = 4) in the presence of NMDG buffer. For Sodium Green fluorometric experiments, cells were labeled with 4 μm dye for 40 min and washed as described above. Samples were excited at 507 nm, and emission was monitored at 532 nm. For calcium flux experiments, THP-1 cells were labeled with 1 μm Fluo-4 for 30 min at room temperature. Cells were washed by centrifugation three times with HBSS and then analyzed by fluorometry. In some experiments, cells were pre-treated with 4 mm EGTA or a combination of EGTA and 0.02 mm CGP-36517. Excitation was at 494 nm and emission at 516 nm. For Fura-2 measurements in mouse peritoneal macrophages, cells were allowed to adhere to glass coverslips overnight, washed with HBSS, and labeled with Fura-2 (1 μm) for 30 min at 37 °C. Cells were washed and then incubated in fresh HBSS for an additional 30 min at 37 °C. Excitation was at 340 and 380 nm with ratiometric emission recorded at 510 nm. Calibration was performed using Ca-EGTA buffers in permeabilized cells. Invasion and Migration Assays-Cellular invasion through a reconstituted basement membrane matrix (ECMatrix, Chemicon) was measured in a 96-well plate format. In the presence and absence of TTX (300 nm), cells were serum starved in 0.1% FBS for 24 h prior to plating on the matrix. 100,000 cells were added to each well in 0.1 ml of media with 0.1% FBS in the presence and absence of freshly added TTX and incubated at 37 °C for 24 h. For THP-1 cells, the lower chamber in the plate assay contained varying concentrations of human macrophage colony-stimulating factor (m-CSF) (25, 100, or 400 ng/ml) or no chemoattractant (control condition). THP-1 invasion also was examined in shRNA-treated cells at an m-CSF concentration of 100 ng/ml. For HTB-66 cells, the attractant was either 10% FBS in Dulbecco's modified Eagle's medium (experimental condition) or 0.1% FBS in Dulbecco's modified Eagle's medium (control condition). Cells that invaded through the matrix were dissociated, lysed, and detected by CyQuant GR dye (Invitrogen), using a fluorescent plate reader. Invasion was expressed in relative fluorescent units (RFU) and was determined by subtracting background invasion (control conditions) from that observed in the experimental conditions. Migration was measured in a transwell assay (Chemicon) using a5-μm pore size. THP-1 cells were serum starved for 4 h in the presence and absence of TTX (300 nm) and then analyzed in the migration assay in the presence and absence of freshly added TTX. Human monocyte chemotractant protein (MCP-1) was used as the stimulus (7.5 ng/ml-250 ng/ml for 2 h). Quantitative analysis was performed as described above for invasion assays. Small Hairpin RNA-THP-1 cells were transduced with shRNA lentiviral clones as previously described (6Carrithers M. Dib-Hajj S. Carrithers L. Tokmoulina G. Pypaert M. Jonas E. Waxman S. J. Immunol. 2007; 178: 7822-7832Crossref PubMed Scopus (99) Google Scholar). The following five clones, specific for human SCN8A (encodes NaV1.6), were obtained from Sigma: SHVRSC-TRCN0000044488-92. Percent knockdown was determined by real time PCR (Cepheid) using TaqMan FAM-labeled primers (Applied Biosystems) (Table 1). Clones 89 and 92 were selected for functional assays because of knockdown >75% or absence of significant knockdown, respectively. Knockdown of NaV1.6 in THP clone 89 also was confirmed by Western blot using the anti-NaV1.6 subtype-specific antibody.TABLE 1Real time PCR analysis of SCN8A expressionCell lineSCN8A Ct (threshold cycle)GAPDH CtnTHP-1 differentiated36.84 ± 0.3820.03THP-1 undifferentiated35.98 ± 0.9320.07HTB-6635.94 ± 0.4920.07THP-1 Clone 89 (differentiated)>39.74 (2 runs > 50 cycles)20.03THP-1 Clone 92 (differentiated)36.37 ± 0.2420.03Real time PCR analysis was performed as described under “Experimental Procedures.” Ct values (threshold cycle) were normalized using GAPDH expression as a control (Ct = 20.0). Expression of SCN8A, which encodes NaV1.6, was detected in only one out of three experiments for THP-1 Clone 89 (>50 cycles in two conditions). Knockdown was estimated to be >75%. THP-1 cells were not primed with either interferon-γ or lipopolysaccharide. Under these conditions, expression of SCN5A, which encodes NaV1.5, was not detected. Cycling conditions were the same for each cell line listed. Stage 1 was an initial denaturation at 95 °C for 120 s followed by Stage 2 with denaturation at 95 °C for 15 s and annealing/extension at 70 °C for 60 s for 50 cycles. All runs were performed on a Cepheid SmartCycler, and the conditions were selected per manufacturer's suggestions. Open table in a new tab Real time PCR analysis was performed as described under “Experimental Procedures.” Ct values (threshold cycle) were normalized using GAPDH expression as a control (Ct = 20.0). Expression of SCN8A, which encodes NaV1.6, was detected in only one out of three experiments for THP-1 Clone 89 (>50 cycles in two conditions). Knockdown was estimated to be >75%. THP-1 cells were not primed with either interferon-γ or lipopolysaccharide. Under these conditions, expression of SCN5A, which encodes NaV1.5, was not detected. Cycling conditions were the same for each cell line listed. Stage 1 was an initial denaturation at 95 °C for 120 s followed by Stage 2 with denaturation at 95 °C for 15 s and annealing/extension at 70 °C for 60 s for 50 cycles. All runs were performed on a Cepheid SmartCycler, and the conditions were selected per manufacturer's suggestions. Statistics-Comparisons between groups were made using a Student's t test (unpaired, unequal variance) with a p < 0.05 considered statistically significant (Kaleidagraph 4.03, Synergy Software). Data are expressed as mean ± S.E. Image Processing-Microscopic images were obtained using Axiovision software as described above, exported as TIFF files, compiled in composite figures using Adobe Illustrator, and exported as TIFF files. cDNA Cloning of an SCN8A Transcript from THP-1 Cells Reveals a Splice Variant-cDNA sequence analysis of a full-length coding sequence for SCN8A from THP-1 cells demonstrated expression of the splice variant that lacks exon 18 (Figs. S1 and S2). This exon encodes a 41-amino acid portion of the IIIS3-IIIS4 transmembrane region of NaV1.6. This finding is consistent with prior studies of SCN8A because exon 18N, which was hypothesized to be a non-neuronal exon variant of SCN8A, encodes a stop codon within its sequence and, if expressed, would prevent the translation of a full-length NaV1.6 (19Plummer N.W. McBurney M.W. Meisler M.H. J. Biol. Chem. 1997; 272: 24008-24015Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). A similar splice variant that would encode a truncated, but not full-length, variant of NaV1.6 has been described in another non-excitable cell type, astrocytes (20Oh Y. Waxman S.G. NeuroReport. 1998; 9: 1261-1266Crossref PubMed Scopus (45) Google Scholar). Some NaV1.6 Positive Vesicles Are Localized Adjacent to Melanoma Invadopodia and Macrophage Podosomes-Previously we demonstrated by immunofluorescence and immuno-EM that macrophage NaV1.6 appeared to localize to F-actin fiber bundles (6Carrithers M. Dib-Hajj S. Carrithers L. Tokmoulina G. Pypaert M. Jonas E. Waxman S. J. Immunol. 2007; 178: 7822-7832Crossref PubMed Scopus (99) Google Scholar). Here we further analyzed the relationship between NaV1.6-positive membrane structures and the F-actin cytoskeleton in cells known to form invadopodia and podosomes. In the invasive human melanoma cell line, HTB-66, primary mouse peritoneal macrophages, and the human monocytic cell line, THP-1, differentiated with phorbol ester to a macrophage phenotype, NaV1.6-positive vesicles were observed throughout the cell but in some cellular regions they appeared to cluster around melanoma invadopodia and the core of macrophage podosome structures (Figs. 1A and 2, A and C). Unlike the previous study where NaV1.6/F-actin co-localization was demonstrated in a more longitudinal view in a migrating cell (6Carrithers M. Dib-Hajj S. Carrithers L. Tokmoulina G. Pypaert M. Jonas E. Waxman S. J. Immunol. 2007; 178: 7822-7832Crossref PubMed Scopus (99) Google Scholar), these views are cross-sectional and, in a more detailed fashion, show the NaV1.6-positive regions surrounding the F-actin core rather than localizing to the core itself. In mouse macrophages and differentiated THP-1 cells, this vesicular localization was most intense around centrally located podosomes (Fig. 2, A and C). In addition, in differentiated THP-1 cells, there were clusters of mitochondria concentrated in podosome-dense regions (Fig. 2C). This latter image (Fig. 2C) is a deconvoluted image through only Z-planes that were podosome positive at the sites of cellular attachment.FIGURE 2In macrophages, NaV1.6-positive vesicles localize to cellular regions rich in podosomes. A, phalloidin staining of F-actin (left panel, green) in a mouse peritoneal macrophage shows numerous podosomes (circular structures). Staining for NaV1.6 (middle panel, red) demonstrates positive staining of vesicular-type structures, some of which co-localize to the periphery of the F-actin-rich core of the podosome structure (merged image on the right). B, in a mouse peritoneal macrophage, NaV1.6-containing vesicles (red) also co-localize with the calcium-dependent, actin-regulatory protein gelsolin (green), particularly at the leading edge of the cell as compared with the trailing tail. C, a similar staining pattern was observed in differentiated THP-1 cells, a human monocytic cell line, as compared with mouse primary mouse macrophages (A). Centrally located podosomes (far left panel, green) were concentrated in regions rich in NaV1.6-positive vesicles (middle left panel, red) and mitochondria (stained with Mito-Tracker, middle right panel). Scale bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also investigated the relationship of other actin regulatory proteins with NaV1.6. At the leading edge of a HTB-66 cell, NaV1.6-positive vesicles appeared to co-localize with or were adjacent to regions rich in F-actin and the GTPase Dynamin II, a known regulator of invadopodia and podosomes (21McNiven M. Baldassarre M. Buccione R. Front. Biosci. 2004; 9: 1944-1953Crossref PubMed Scopus (77) Google Scholar, 22Bruzzaniti A. Neff L. Sanjay A. Horne W. De Camilli P. Baron R. Mol. Biol. Cell. 2005; 16: 3301-3313Crossref PubMed Scopus (110) Google Scholar) (Fig. 1B, top). However, in a more detailed view of the invadopodia leading edge from the same cell using a deconvoluted image, there is some degree of co-localization between Dynamin II and NaV1.6 (arrow), but the three prot" @default.
• W2166236894 created "2016-06-24" @default.
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• W2166236894 date "2009-03-01" @default.
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• W2166236894 title "Regulation of Podosome Formation in Macrophages by a Splice Variant of the Sodium Channel SCN8A" @default.
• W2166236894 cites W1798861522 @default.
• W2166236894 cites W1908396212 @default.
• W2166236894 cites W1964616832 @default.
• W2166236894 cites W1975917180 @default.
• W2166236894 cites W1977230497 @default.
• W2166236894 cites W2002352511 @default.
• W2166236894 cites W2004403903 @default.
• W2166236894 cites W2024886848 @default.
• W2166236894 cites W2026357254 @default.
• W2166236894 cites W2037489902 @default.
• W2166236894 cites W2039380715 @default.
• W2166236894 cites W2044296671 @default.
• W2166236894 cites W2051984678 @default.
• W2166236894 cites W2057440436 @default.
• W2166236894 cites W2057570836 @default.
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• W2166236894 cites W2106516212 @default.
• W2166236894 cites W2112749067 @default.
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• W2166236894 cites W2144235003 @default.
• W2166236894 cites W2154803239 @default.
• W2166236894 cites W2155290928 @default.
• W2166236894 cites W2159332152 @default.
• W2166236894 cites W2160045132 @default.
• W2166236894 cites W2162347886 @default.
• W2166236894 cites W2170154403 @default.
• W2166236894 cites W2333603867 @default.
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