Journal: Frontiers in Physiology

Article Title: Loss of Drosophila Clueless differentially affects the mitochondrial proteome compared to loss of Sod2 and Pink1

doi: 10.3389/fphys.2022.1004099

Figure Lengend Snippet: Mitochondrial respiratory chain proteins are greatly decreased in clu mutants. (A) A pie chart showing clu specific less abundant proteins in different categories as percentage of the whole [total = 92 ]: Mitochondrial Respiratory Chain (MRC) components, Mitochondrial Ribosomal Proteins (Mitochondrial RP) and Others. (B) Bar graph showing the number of subunits, in percentages, from each entire MRC are either decreased (in green) or remain unaltered (in magenta). (C) Heat maps showing the levels of affected individual MRC proteins of different complexes in clu , Sod2 , and Pink1 mutants. The proteins chosen are decreased in clu mutants. Color coded scale bars (green: less abundant and magenta: unaltered) are representing the log2[Fold Change] of individual protein in each mutant compared to wild type. (D) Western blots showing the levels of representative proteins in clu , Sod2 , and Pink1 . Tom20 was used as a loading control. (E) Proteins from isolated mitochondria were run to determine the levels of native MRC complexes on a BN-PAGE (left panel, stained with colloidal blue, right panel, stained with SilverQuest Silver Staining Kit). (F) In-gel activity assays. Dark purple bands indicate CI activity and brown bands indicate CIV activity. (G,H) The activities from CI (G) and CIV (H) were determined by measuring band intensity of the respective complexes using Fiji software. Band intensities from three gels ( n = 3) for CI and two gels ( n = 2) for CIV were used for the measurements. Error bars: S.E.M. calculated in GraphPad-PRISM software. p values were calculated in GraphPad-PRISM software using an unpaired t -test and each mutant was compared to the wildtype control. Statistical significance = p < 0.0001 (****) and p < 0.003 (**).

Article Snippet: For gene expression analysis, quantitative PCR was performed using TaqMan Gene Expression Master Mix (Thermo Fisher Scientific, Waltham, MA, United States) in a 10 μL reaction with 2 μL diluted cDNA and one of the following TaqMan probes: Dm01806850_g1 (Tom20), Dm02136274_g1 (ND-42), Dm01820354_g1 (ND-19), Dm01804649_g1 (Cyp4ac2), Dm02145551_g1 (Mil), Dm01822473_s1 (Hsp23), Dm01816546_s1 (Pepck), Dm01835343_g1 (ND-30), Dm01794109_g1 (COX4), Dm01830822_g1 (Levy) and Dm02151827_g1 (RPL32) (endogenous control) (Thermo Fisher Scientific, Waltham, MA).

Techniques: Mutagenesis, Western Blot, Control, Isolation, Staining, Silver Staining, Activity Assay, Software

Journal: Cells

Article Title: Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis.

doi: 10.3390/cells9041034

Figure Lengend Snippet: Figure 1. Mitochondria-nucleus contacts. Mesenchymal stem cells (MSCs) were cultured in low-glucose (LG), adipogenic (AD) and osteogenic (OS) media for 3, 7, and 21 days, as indicated. (a) Representative 3D images of mitochondrial morphology, as detected by anti-TOM20 Ab immunofluorescence, and contact sites identified by creating the isosurface of the colocalization channel between nuclear staining (Hoechst) and TOM20. Magnification 40x. Scale bar 10 µm (in zoom panel, scale bar 5 µm). (b) Quantification of mitochondrial total volume, number, and total volume and number of contacts. Data are derived from ≥45 acquired cells/condition from ≥3 independent experiments. Data are shown as the mean ± SD. ANOVA, * p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001 with respect to the LG condition at the same time point.

Article Snippet: The following primary antibodies were used for immunofluorescence images: TOM20 [sc-11415] (1:100) from Santa Cruz Biotechnology, H3 [14269] (1:100), H3K9ac [9649] (1:100), and H3K9me3 [13969] (1:100) from Cell Signaling (Danvers, MA, USA).

Techniques: Cell Culture, Staining, Derivative Assay

Journal: Cells

Article Title: Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis.

doi: 10.3390/cells9041034

Figure Lengend Snippet: Figure 2. Mitochondria in MSC differentiation processes. (a) Representative immunoblot and (b) quantification of HSP60, TOM20, VDAC, and TIM23 protein levels normalized to GAPDH levels. (c) XF phenograms representing the metabolic switching of MSCs during differentiation, as detected using an Extracellular Flux Analyzer (Seahorse Bioscience). (d,e) Oxygen consumption rate (OCR) measurements and relative derived parameters after the addition of oligomycin [1 µM], 1 µM carbonylcyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP) [1 µM], and antimycin A [2 µM] + rotenone [2 µM] in MSCs cultured in low-glucose (LG), adipogenic (AD), and osteogenic (OS) media for (d) 7 and (e) 21 days. Data are derived from ≥45 acquired cells/condition from ≥3 independent experiments and are shown as the mean ± SD. ANOVA, * p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001 with respect to the LG condition at the same time point.

Article Snippet: The following primary antibodies were used for immunofluorescence images: TOM20 [sc-11415] (1:100) from Santa Cruz Biotechnology, H3 [14269] (1:100), H3K9ac [9649] (1:100), and H3K9me3 [13969] (1:100) from Cell Signaling (Danvers, MA, USA).

Techniques: Western Blot, Derivative Assay, Cell Culture

Journal: Cells

Article Title: Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis.

doi: 10.3390/cells9041034

Figure Lengend Snippet: Figure 4. Inhibition of the citrate transport system impaired mitochondrial behavior. MSCs were cultured in low-glucose (LG), adipogenic (AD), and osteogenic (OS) media; where indicated, BTC [5 mM] and iCTP [500 µM] were added to inhibit the transport of citrate produced in mitochondria to the cytosol. (a) Representative images and (b) analysis of mitochondrial total volume and number detected by the anti-TOM20 Ab and the total volume and number of mitochondrion-nucleus contact sites. Magnification 40x. Scale bar 10 µm (in zoom panel, scale bar 5 µm). Data are derived from ≥45 acquired cells/condition from ≥3 independent experiments and are shown as the mean ± SD. ANOVA-test. * p < 0.5, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The following primary antibodies were used for immunofluorescence images: TOM20 [sc-11415] (1:100) from Santa Cruz Biotechnology, H3 [14269] (1:100), H3K9ac [9649] (1:100), and H3K9me3 [13969] (1:100) from Cell Signaling (Danvers, MA, USA).

Techniques: Inhibition, Cell Culture, Produced, Derivative Assay

Journal: Cells

Article Title: Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis.

doi: 10.3390/cells9041034

Figure Lengend Snippet: Figure 5. Citrate from mitochondria increases the level of α-ketoglutarate to promote osteogenesis. (a) Citrate can be converted to acetyl-CoA by citrate lyase (inhibited by BMS) or to α-ketoglutarate (αKG), two metabolites that can mediate mitochondrion-nucleus communication. (b,c) Analysis of relative mRNA levels of osteogenic markers (RUNX2, RANKL, osteocalcin (OC), osteopontin (OPN), osterix (OSX), and alkaline phosphatase (ALP)) in MSCs cultured in low-glucose (LG) and osteogenic media (OS); where indicated, BMS [1 mM], BTC [5 mM], and αKG [1 mM] were added for 21 days. (d) Representative images and analysis of mitochondrial total volume and number detected by the anti-TOM20 Ab and the total volume and number of mitochondrion-nucleus contact sites in MSCs cultured in LG and osteogenic media (OS); where indicated, BTC [5 mM] and αKG [1 mM] were added for 21 days. (e) Representative images and quantification of H3K9me3 in MSCs cultured in LG and OS media; where indicated, BTC [5 mM] and αKG [1 mM] were added for 21 days. Magnification 40x. Scale bar 10 µm (in zoom 5 µm). Data are derived from ≥45 acquired cells/condition from ≥3 independent experiments and are shown as the mean ± SD. ANOVA-test. * p < 0.5, ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies were used for immunofluorescence images: TOM20 [sc-11415] (1:100) from Santa Cruz Biotechnology, H3 [14269] (1:100), H3K9ac [9649] (1:100), and H3K9me3 [13969] (1:100) from Cell Signaling (Danvers, MA, USA).

Techniques: Cell Culture, Derivative Assay

Journal: bioRxiv

Article Title: Phosphoproteomics of CD2 signaling reveals an AMPK-dependent regulation of lytic granule polarization in cytotoxic T cells

doi: 10.1101/795963

Figure Lengend Snippet: (A) Colocalization analysis and (B) immunofluorescence image of LAMP1 and AMPK signals in CTLs. Quantitative measurements were taken for 30 CTLs (n=3 donors). (C) Immunofluorescence images of CTLs electroporated with LAMP1-AIP-mCherry or Tom20-AIP-mCherry constructs and stained with antibodies against perforin (granules). (D) Quantitative analysis of lytic granule polarization in antibody-stimulated mCherry-positive CTLs overexpressing either LAMP1-AIP-mCherry or Tom20-AIP-mCherry (n=3 experiments). (E) Colocalization analysis of immunofluorescence signals in CTLs stained with antibodies against AMPK and perforin (granules) (n=3 experiments). (F) Immunofluorescence image of a CTL stained with antibodies against AMPK, LAMP1 and perforin (granules). (G) CLEM analysis of lytic granules and AMPK-positive compartment in freshly isolated CTLs. N, nucleus; LD, lipid droplet; AMPK, AMPK-positive vesicle; LG, lytic granule. Scale bar, 5 μm. Shown are mean values ± SD; ns, not significant; two-way ANOVA test ****p < 0.0001.

Article Snippet: For overexpression of LAMP1-AIP and Tom20-AIP, CTLs were nucleofected with 2 μg of purified LAMP-mChF-AIP and Tom20-mChF-AIP plasmids (a gift from Takanari Inoue, Addgene plasmids # 61524 and # 61512).

Techniques: Immunofluorescence, Construct, Staining, Isolation

Journal: bioRxiv

Article Title: CLIC4 is a cytokinetic cleavage furrow protein that regulates cortical cytoskeleton stability during cell division

doi: 10.1101/723940

Figure Lengend Snippet: (A) Schematic of Mitotrap used in this experiment. In all cases cells expressing endogenously tagged GFP-CLIC4 were co-transfected with Cry2-GFP-VHH and CIB-Tom20 plasmids and pulsed with a 488nm laser to re-target GFP-CLIC4 at the mitochondria. (B) Interphase cell exposed to 488nm to activate Mitotrap. Mitochondria is labeled in red and endogenous GFP-CLIC4 in green. (C-D) Still images from time-lapse microscopy where Mitotrap was activated. Arrows point to blebs or cytokinesis failure induced by GFP-CLIC4 Mitotrap. (E) Quantification of time required for cells to complete mitotic cell division in control and Mitotrapped cells. Data shown are the means and standard deviations.

Article Snippet: For the Mitotrap experiments, cells expressing endogenous GFP-CLIC4 were transfected with Cry2-VHH Addgene #58370) and Tom20-CIB-stop (Addgene #117243).

Techniques: Expressing, Transfection, Labeling, Time-lapse Microscopy