Journal: Journal of Bacteriology

Article Title: Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

doi: 10.1128/JB.01795-14

Figure Lengend Snippet: MtrB interactions with FtsI and Wag31. (A) BACTH analysis of MtrB interactions with FtsI and Wag31. E. coli BTH101 cotransformants with plasmids expressing Wag31/Wag31, Wag31/MtrB, Wag31/MtrBH305Y, MtrB/FtsI, and MtrBH305Y/FtsI were spotted on indicator agar plates as previously described (18), wherein blue and white spots indicate positive and negative interactions, respectively. Cotransformants with GCN4/GCN4 and empty vector/MtrB, representing positive and negative controls, respectively, were also spotted. (B) Recombinant colonies expressing the indicated combinations of proteins were propagated in LB broth, and β-galactosidase activity was measured as described in the text. Data shown are means ± standard deviations from three independent experiments. (C) Pulldown assays. Wag31 interacts with MtrB and not with FtsZ. E. coli lysates with MBP-Wag31 were incubated with His10-MtrBsol (i) or His10-FtsZ (iii) bound to Ni-NTA resin with rocking at 4°C. The Ni-NTA resin was washed five times, and the bound proteins were eluted with buffer containing imidazole (see below and Materials and Methods) (17). MBP-Wag31 was incubated with Ni-NTA (ii) and processed as described for panels i and iii. Various fractions were separated in SDS-polyacrylamide gels and transferred to PVDF membranes, and the proteins were visualized by immunoblotting using anti-MtrB, anti-Wag31, anti-FtsZ, or anti-MBP antibody (see Fig. S2 in the supplemental material). L, load; W, wash; E, elution fractions; W4 and W5, washes 4 and 5, respectively; E1, E2, and E3 are elutions with buffers containing 100 mM, 300 mM, and 1 M imidazole, respectively. For co-IP assays (iv) 1 ml of M. smegmatis lysate mixed with 60 μg of purified MBP-MtrB was incubated overnight with anti-Wag31 antibodies coupled to magnetic beads (BioMag Plus amine particles). The beads were washed five times with IP wash buffer, and the MtrB immunoprecipitates were eluted with 100 mM citrate (pH 3.1). The eluted proteins were neutralized with 1 N NaOH, separated by SDS-PAGE, and visualized following immunoblotting with anti-Wag31 or anti-MtrB antibody. P, pure protein markers for MBP-MtrB and His-Wag31; L, load; W, wash 5; E1 and E2, elutions 1 and 2; EC, elution from buffer control where beads were incubated with buffer instead of lysate. White arrowhead, MBP-MtrB, MtrB, or Wag31; black arrowhead, IgG band. M, molecular mass marker. α, anti.

Article Snippet: In vitro phosphorylation experiments revealed that MtrB phosphorylates MtrA ( 4 ) and that phosphorylated MtrA (MtrA∼P) binds to the origin of replication ( oriC ) and the promoters of fbpB , ripA , and dnaA ( 5 , – 7 ).

Techniques: Expressing, Plasmid Preparation, Recombinant, Activity Assay, Incubation, Western Blot, Co-Immunoprecipitation Assay, Purification, Magnetic Beads, SDS Page, Control, Marker

Journal: Journal of Bacteriology

Article Title: Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

doi: 10.1128/JB.01795-14

Figure Lengend Snippet: Loss of MtrB or depletion of FtsI increases phosphorylation of Wag31. (A) M. smegmatis WT, the ΔmtrB strain, FtsI-depleted cultures (FtsI depletion for 12 h), and the ΔmtrB Pami::mtrB, ΔmtrB Pami::mtrAY102C (where mtrAY102C is the Y-to-C change at position 102 encoded by mtrA) and ΔmtrB Pami::mtrBH305Y strains were grown as described in the legends to Fig. 3 and ​and4.4. The complemented ΔmtrB strains were grown with 0.2% acetamide. Wag31∼P/Wag31 ratios were determined by immunoblotting with anti-phospho-Ser/Thr and anti-Wag31 antibodies. Whole-cell lysates (5 μg protein) from the above strains were resolved by SDS-PAGE in 12% gels, and immunoblotting was performed with anti-phospho-Ser/Thr antibodies. The blots were then stripped and reprobed with anti-Wag31 antibodies. Wag31∼P/Wag31 (anti-phospho-Ser/Thr and anti-Wag31) ratios for various strains were normalized to those in the WT and plotted (B). The data shown are represented as the averages ± standard errors from three independent experiments. *, P < 0.05. (C) The Wag31 levels in the indicated strains were measured by immunoblotting, as previously described (36), and normalized to SigA levels. The data are represented as the means ± standard errors from three independent experiments.

Article Snippet: In vitro phosphorylation experiments revealed that MtrB phosphorylates MtrA ( 4 ) and that phosphorylated MtrA (MtrA∼P) binds to the origin of replication ( oriC ) and the promoters of fbpB , ripA , and dnaA ( 5 , – 7 ).

Techniques: Phospho-proteomics, Western Blot, SDS Page

Journal: Journal of Bacteriology

Article Title: Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

doi: 10.1128/JB.01795-14

Figure Lengend Snippet: MtrB activity and localization are compromised in the absence of FtsI. (A and B) qRT-PCR analysis of MtrA targets. Total RNA was extracted from ΔftsI::Pami-ftsI (A) and Δwag31::Pami-wag31 (B) strains grown without and with 0.2% acetamide (see details in text) and reverse transcribed, and qRT-PCR was performed as described in Materials and Methods. The expression levels of select genes, that is, dnaA, ripA, fbpB, wag31, mtrA, mtrB, and ftsI, relative to the housekeeping gene sigA were compared, and the values are presented as fold difference. In panel A fold expression levels upon FtsI depletion (growth in the absence of acetamide for 12 h) were normalized to those in the presence of acetamide (FtsI+). In panel B fold expression levels upon Wag31 depletion (growth in the absence of acetamide for 15 h) were normalized to those in the presence of acetamide (Wag31+). Wag31 depletion beyond 15 h led to extreme cell distortion and eventually cell lysis; hence, expression studies were not carried out beyond 15 h. (C and D) MtrB-GFP localization. M. smegmatis ΔftsI::Pami-ftsI (C) and Δwag31::Pami-wag31 (D) strains expressing Ptet::mtrB-gfp were grown without and with 0.2% acetamide, as for panels A and B. For the visualization of MtrB-GFP, anhydrotetracycline was added at 10 ng/ml for 1 h. Bright-field (i and iii) and fluorescence (ii and iv) microscopy and imaging were carried out as described in the text. The ΔftsI::Pami-ftsI or Δwag31::Pami-wag31 strain was grown with (i and ii) or without (iii and iv) acetamide. For panel C, percent septal MtrB-GFP localizations from two independent experiments were scored, and averages ± standard errors are given in the respective fluorescent panels. Each experiment included >100 cells from each condition. White arrows, MtrB-GFP septal localization; black arrows, distorted cell shape upon Wag31 depletion.

Article Snippet: In vitro phosphorylation experiments revealed that MtrB phosphorylates MtrA ( 4 ) and that phosphorylated MtrA (MtrA∼P) binds to the origin of replication ( oriC ) and the promoters of fbpB , ripA , and dnaA ( 5 , – 7 ).

Techniques: Activity Assay, Quantitative RT-PCR, Reverse Transcription, Expressing, Lysis, Fluorescence, Microscopy, Imaging

Journal: Journal of Bacteriology

Article Title: Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

doi: 10.1128/JB.01795-14

Figure Lengend Snippet: FtsI localization and activity are altered in the absence of MtrB. (A) GFP-FtsI localization was examined in M. smegmatis WT (i and ii), the ΔmtrB strain (iii and iv), and merodiploid strains overproducing (↑) MtrB (v and vi) or MtrBH305Y (vii and viii). In all of the strains, the GFP-FtsI fusion protein was produced from Pami::gfp-ftsI following induction with 0.2% acetamide for 3 h, visualized by bright-field (left panels) and fluorescent (right panels) microscopy, and imaged as described in the text. (B) The exponential cultures of the M. smegmatis WT strain (i and ii) and the ΔmtrB strain (iii and iv) were grown in the presence of Van-FL for 2 h and were imaged by bright-field (i and iii) and fluorescence (ii and iv) microscopy. (C) Loss of MtrB increases sensitivity to vancomycin. M. smegmatis WT and ΔmtrB strains were grown for 6 h, and 1 × 105 cells were spread on 7H10 agar plates. Etest antibiotic strips (ampicillin or vancomycin) were placed on the agar plates, plates were incubated for 4 days at 37°C, and MICs were measured as per the supplier's protocol.

Article Snippet: In vitro phosphorylation experiments revealed that MtrB phosphorylates MtrA ( 4 ) and that phosphorylated MtrA (MtrA∼P) binds to the origin of replication ( oriC ) and the promoters of fbpB , ripA , and dnaA ( 5 , – 7 ).

Techniques: Activity Assay, Produced, Microscopy, Fluorescence, Incubation

Journal: Journal of Bacteriology

Article Title: Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

doi: 10.1128/JB.01795-14

Figure Lengend Snippet: Wag31 localization is altered in the absence of MtrB. (A) Pami::wag31-mCherry expressing M. smegmatis WT (i and ii), the ΔmtrB strain (iii and iv), and M. smegmatis overproducing MtrB (v and vi) or MtrBH305Y (vii and viii) were grown with 0.2% acetamide for 3 h and visualized as described in the legend to Fig. 3A. Top panels are bright-field images; bottom panels are fluorescence images. Arrow, septal localization; arrowhead, polar localization. Percent septal Wag31-mCherry localizations from two independent experiments were scored, and averages ± standard errors are given in the respective fluorescent panels. Each experiment included >132 cells from each strain. Black arrows, septa in multiseptate ΔmtrB strain; white arrows and arrowheads, septal and polar localizations, respectively. (B) FtsZ-GFP localization in WT (i and ii) and the ΔmtrB strain (iii and iv). Arrow, FtsZ-GFP rings.

Article Snippet: In vitro phosphorylation experiments revealed that MtrB phosphorylates MtrA ( 4 ) and that phosphorylated MtrA (MtrA∼P) binds to the origin of replication ( oriC ) and the promoters of fbpB , ripA , and dnaA ( 5 , – 7 ).

Techniques: Expressing, Fluorescence

Journal: Journal of Bacteriology

Article Title: Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

doi: 10.1128/JB.01795-14

Figure Lengend Snippet: MtrB interacts with PknA and PknB. Interactions of MtrB and MtrA with PknA or PknB were examined using BACTH assays, as previously described (17). MtrA, MtrB, FtsI, PknA, and PknB fusions to the T18 and T25 fragments of adenylate cyclase in BACTH vectors (see Table S1 in the supplemental material) were used to transform E. coli BTH101, and the recombinants were plated on LB agar supplemented with X-Gal and isopropyl-β-d-thiogalactopyranoside. Green-blue colonies, indicative of positive interactions, were subsequently propagated in LB broth, and β-galactosidase activity was measured as described in the text. GCN4/GCN4 and MtrB/MtrA are shown as positive controls, and MtrB/empty vector (vector control) is shown as the negative control. The data shown are the means ± standard deviations from three independent experiments.

Article Snippet: In vitro phosphorylation experiments revealed that MtrB phosphorylates MtrA ( 4 ) and that phosphorylated MtrA (MtrA∼P) binds to the origin of replication ( oriC ) and the promoters of fbpB , ripA , and dnaA ( 5 , – 7 ).

Techniques: Activity Assay, Plasmid Preparation, Control, Negative Control

Journal: Nucleic Acids Research

Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

doi: 10.1093/nar/gkv275

Figure Lengend Snippet: RHC-Glo splicing reporter minigene assay. Upper panel: schematic representation of the RHC-Glo splicing reporter minigene and the sub-cloned sequences. Lower panel: Agarose gel electrophoresis of RT-PCR minigene splicing products expressed in HEK293 cells. Inclusion or exclusion of exon 2 is indicated on the right. The gel pictures are representative results from two experiments.

Article Snippet: Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA).

Techniques: Mini Gene Assay, Clone Assay, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

doi: 10.1093/nar/gkv275

Figure Lengend Snippet: The balanced interplay between an hnRNP A1 binding ESS and two SRSF1 binding ESEs dictate MTRR pseudoexon activation. ( A ) Schematic representation of the sequences from MTRR -minigenes used and the nucleotide changes introduced. A pictogram of hnRNP A1 position weight matrix is shown above the putative ESS and the invariant position 2 of this motif is underscored. A pictogram of the SRSF1 position weight matrix is shown above the previously described ESE1 and the ACADM like ESE. Indicated positions 361, 362 and 365 are relative to the ACADM coding sequence. Nucleotide positions that were changed are underscored. ( B ) Splicing minigene assay. MTRR -minigenes were transiently transfected into HEK293. After RNA isolation the splicing products were analyzed by RT-PCR. The upper panel shows the average of pseudoexon inclusion from two measurements of each duplicate. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument. The lower panel shows a sample agarose gel electrophoresis displaying pseudoexon inclusion levels in the different cell lines. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inserted between minigene exons. Ψ marks the pseudoexon. ( C ) Binding of hnRNPA1 and SRSF1 proteins. Biotinylated RNA oligonucleotides were used in a pull-down experiment with HeLa nuclear extract followed by SDS PAGE and western blot analysis using antibodies against SRSF1 and hnRNP A1. Blank indicates a control lane from pull down without RNA oligonucleotides. NE is nuclear extract. The displayed blots are representative result from at least three pull-down experiments.

Article Snippet: Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA).

Techniques: Binding Assay, Activation Assay, Sequencing, Mini Gene Assay, Transfection, Isolation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, SDS Page, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

doi: 10.1093/nar/gkv275

Figure Lengend Snippet: SSO treatment in HEK293 cells transfected with wild-type, mutant and TCGGGA MTRR -minigenes. ( A ) Splicing minigene assay. MTRR -minigenes were transiently transfected into HEK293. 24 h after the cells were transfected with SSOs targeting either the 5′ss, 3′ss or both ESEs. After RNA isolation the splicing products were analyzed by RT-PCR. The upper panel shows a sample agarose gel electrophoresis displaying pseudoexon inclusion levels. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inclusion. Ψ marks the pseudoexon. ( B ) Quantification of pseudoexon inclusion. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument.

Article Snippet: Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA).

Techniques: Transfection, Mutagenesis, Mini Gene Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

doi: 10.1093/nar/gkv275

Figure Lengend Snippet: SSO treatment restores MTRR splicing in patient cells. ( A ) Fibroblasts from a patient harboring the c.903+469T>C mutation were transfected with SSOs that target either the 5′ss, the 3′ss, both ESEs (ESE-SSO), or a non-targeting sequence (NT). Control fibroblasts were treated in parallel. RNA was extracted after 48 h. A representative agarose gel electrophoresis of the RT-PCR products is shown. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inclusion between exon 6 and exon 7. Ψ marks the pseudoexon. ( B ) Quantification of pseudoexon inclusion in patient fibroblasts following SSO treatment. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument.

Article Snippet: Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA).

Techniques: Mutagenesis, Transfection, Sequencing, Control, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

doi: 10.1093/nar/gkv275

Figure Lengend Snippet: SSO treatment partially restores enzymatic activity of methionine synthase reductase (MTRR). Fibroblasts from a patient homozygous for the c.903+469T>C mutation were transfected with an SSO that target both ESEs (ESE-SSO) or a non-targeting sequence (Ctr). Fibroblasts were then labeled with [ 57 Co]cyanocabalamine and MTRR activity was determined indirectly by measuring methylcobalamine (MeCbl) synthesis (expressed as% of total labeled cobalamin derivatives). Adenosylcobalamine (AdoCbl) synthesis was measured as a control. The vertical lines represent the range of duplicate determinations in a representative experiment.

Article Snippet: Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA).

Techniques: Activity Assay, Mutagenesis, Transfection, Sequencing, Labeling, Control

Journal: Nucleic Acids Research

Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

doi: 10.1093/nar/gkv275

Figure Lengend Snippet: Splicing model of the MTRR pseudoexon activation induced by the c.903+469T>C mutation. The wild-type sequence harbors a very weak SRSF1 binding ESE1, a flanking ACADM like SRSF1 binding ESE and a hnRNP A1 binding ESS. In the wild-type MTRR gene binding of hnRNP A1 to the TAGGGA high affinity ESS inhibits splicing because this initiates cooperative spreading of hnRNPA1 to weaker sites in a 3′-to-5′ direction thereby directly blocking access to the 5′ splice site and other splicing regulatory elements. Simultaneously binding of hnRNP A1 to the ESS inhibits binding of SRSF1 both to the very weak SRSF1 motif in ESE1 and to the ACADM -like ESE motif. This also affects recognition of the weak 5′ss by U1 snRNP, since this may be dependent on SRSF1 binding to the ESEs. When the c.903+469T>C mutation is present it changes the weak SRSF1 motif in ESE1 to a very strong site, which may act synergistically with the ACADM -like SRSF1 ESE to recruit U1snRNP to the weak 5′ss despite binding of hnRNP A1 to the flanking ESS does not seem to be affected. SRSF1 binding to the ESEs also antagonizes the cooperative spreading of hnRNPA1 binding from the TAGGGA high affinity ESS. Together this leads to pseudoexon activation and the abnormal splicing of the MTRR transcript observed in patients.

Article Snippet: Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA).

Techniques: Activation Assay, Mutagenesis, Sequencing, Binding Assay, Blocking Assay

Journal: The Journal of Neuroscience

Article Title: Mitochondrial Exchanger NCLX Plays a Major Role in the Intracellular Ca 2+ Signaling, Gliotransmission, and Proliferation of Astrocytes

doi: 10.1523/JNEUROSCI.5721-12.2013

Figure Lengend Snippet: NCLX mediates mitochondrial Ca2+ efflux in astrocytes. A–E, Primary astrocytes were cotransfected with mtRP and siNCLX or siControl and were analyzed after 3 d. A, Two-photon microscopy image of astrocytes transfected with mtRP reveals typical mitochondrial pattern of the fluorescent pericam sensor. Scale bar, 10 μm. B–E, Mitochondrial Ca2+ imaging. B, Resting mitochondrial Ca2+ levels of astrocytes transfected with siNCLX- or control siRNA, R/R0 values were determined from fluorescent mtRP imaging before application of the stimulus and normalized to the control condition (n = 5 cultures, n = 134 and 118 regions), C, Mitochondrial Ca2+ signals of control and siNCLX-transfected astrocytes; 100 μm ATP in Ca2+-free HEPES buffer was applied as indicated, averaged curves ± SEM. D–E, Average rates of the mitochondrial Ca2+ efflux (slopes; D) and influx (amplitudes, E) in siControl and siNCLX-transfected astrocytes, comparing n = 10 (n = 38 regions) and n = 11 (n = 64 regions) experiments for each condition (***p < 0.001, *p < 0.05, Wilcoxon, Mann–Whitney U test). F–H, Effect of CGP37157 on mitochondrial Ca2+ efflux. F, Mitochondrial Ca2+ signals from control and CGP37157 (CGP)-treated astrocytes. Cells were superfused with Ca2+-free HEPES buffer ± 20 μm CGP37157and 100 μm ATP was applied as indicated; shown are averaged curves ± SEM. G–H, average rates of mitochondrial efflux (G) and influx (H). Values are given as means with SEM for n = 15 (n = 106 regions) and n = 11 (n = 75 regions) control and CGP37157 experiments, respectively (***p < 0.001, *p < 0.05, Wilcoxon, Mann–Whitney U test).

Article Snippet: To monitor mitochondrial Ca 2+ responses, primary astrocytes were transfected with mtRP ( Nagai et al., 2001 ) Expression of mtRP reached maximal intensity ∼48–72 h after transfection. mtRP expression pattern manifested a typical network-like mitochondrial distribution consistent with the strict mitochondrial localization of this Ca 2+ reporter ( A ).

Techniques: Microscopy, Transfection, Imaging, Control, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: Mitochondrial Exchanger NCLX Plays a Major Role in the Intracellular Ca 2+ Signaling, Gliotransmission, and Proliferation of Astrocytes

doi: 10.1523/JNEUROSCI.5721-12.2013

Figure Lengend Snippet: A, immunoblot analysis of NCLX expression in brain extracts of newborn mice, primary murine astrocytes, and the murine astrocytoma cell line GL261 (top). GAPDH serves as an internal loading control (bottom). B, NCLX is enriched in the mitochondria of astrocytes. NCLX expression in indicated cellular compartments: total, crude ER, cytosol, plasma membrane (PM), ER, and mitochondria (Mito)-enriched fractions (top). The membranes were stripped and reprobed for PM (N-cadherin), ER (Sec-62), and mitochondrial (ANT) markers (bottom). C, Astrocytes are viable after 100% efficient siRNA transfection. Top row shows untreated control astrocytes (no siRNA); bottom row shows astrocytes treated with siGLO and siNCLX (siGLO + siNCLX). Left: Astrocytes accumulated calcein (green), indicating their viability. Nuclei are marked with Hoechst 33342 in blue. Right: Red fluorescence of the same area; untreated astrocytes display dim autofluorecence, whereas siRNA (siGLO + SiNCLX)-treated cells show punctate stain, consistent with intracellular accumulation of the transfection marker siGLO. Scale bar, 20 μm. D, Quantitative RT-PCR analysis after cotransfection with mtRP and siNCLX or siControl, respectively. Untreated and mock-treated (tranfection agent only) cells were used as additional controls. Experiment was performed four times, each in triplicate. Values are given as means with SEM (*p < 0.05, Wilcoxon-Mann–Whitney U test). E, Immunoblot analysis showing NCLX expression in untreated and mock-treated astrocytes, or transfected with siControl or siNCLX (top). GAPDH serves as an internal loading control (bottom).

Article Snippet: To monitor mitochondrial Ca 2+ responses, primary astrocytes were transfected with mtRP ( Nagai et al., 2001 ) Expression of mtRP reached maximal intensity ∼48–72 h after transfection. mtRP expression pattern manifested a typical network-like mitochondrial distribution consistent with the strict mitochondrial localization of this Ca 2+ reporter ( A ).

Techniques: Western Blot, Expressing, Control, Clinical Proteomics, Membrane, Transfection, Fluorescence, Staining, Marker, Quantitative RT-PCR, Cotransfection, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: Mitochondrial Exchanger NCLX Plays a Major Role in the Intracellular Ca 2+ Signaling, Gliotransmission, and Proliferation of Astrocytes

doi: 10.1523/JNEUROSCI.5721-12.2013

Figure Lengend Snippet: NCLX controls SOCE in astrocytes. A–C, Imaging of cytosolic Ca2+ in fura-2 AM-loaded primary murine astrocytes. Astrocytes were cotransfected with siControl or siNCLX and siGLO red indicator. Cells were perfused in Ca2+-free HEPES buffer and 100 μm ATP was applied to empty ER Ca2+ stores; then 5 mm Ca2+ was re-added as indicated by the bar to trigger the activation of SOCE. A, Traces of average fura-2 AM fluorescence (normalized averages) during SOCE. B–C, Amplitudes (B) and rates of SOCE (C) in siControl (n = 9, n = 175) and siNCLX-transfected astrocytes (n = 9, n = 131). Values are given as mean with SEM (***p < 0.001, Wilcoxon, Mann–Whitney U test). D–F, Untransfected astrocytes were perfused in Ca2+-free HEPES buffer without (Control) or with 20 μm CGP37157 or with 1 μm FCCP. After adding 100 μm ATP to empty the ER stores, 5 mm Ca2+ was added as indicated. D, Mean Ca2+ response curve of all experiments (normalized averages). E–F, Statistical analysis of the amplitudes (E) and SOCE rates (F) in astrocytes treated with CGP37157(CGP), FCCP, or without any drug (Control). Values are given as mean with SEM summarized from n = 8 (n = 376 cells), n = 7 (n = 425 cells), and n = 5 (n = 168 cells) experiments for control, CGP37157, and FCCP-treated astrocytes, respectively (***p < 0.001, Kruskal-Wallis rank-sum test followed by Bonferoni's posttest and Wilcoxon test pair comparisons). G–J, SOCE-induced mitochondrial Ca2+ signals in astrocytes cotransfected with mtRP and siControl or siNCLX using the same paradigm described in A. G, Representative curve showing the averaged mitochondrial Ca2+ response. H–J, Rates of the mitochondrial Ca2+ efflux (H), influx (I), and cumulative fluorescence (J) from n = 9 experiments (n = 59 and n = 43 regions of interest for siControl- and siNCLX-transfected astrocytes, respectively; ***p < 0.001, Student's t test for unpaired samples and two-tailed and Mann–Whitney U test).

Article Snippet: To monitor mitochondrial Ca 2+ responses, primary astrocytes were transfected with mtRP ( Nagai et al., 2001 ) Expression of mtRP reached maximal intensity ∼48–72 h after transfection. mtRP expression pattern manifested a typical network-like mitochondrial distribution consistent with the strict mitochondrial localization of this Ca 2+ reporter ( A ).

Techniques: Imaging, Activation Assay, Fluorescence, Transfection, MANN-WHITNEY, Control, Two Tailed Test