Journal: Leukemia

Article Title: Mitochondrial fusion is a therapeutic vulnerability of acute myeloid leukemia

doi: 10.1038/s41375-023-01835-x

Figure Lengend Snippet: A–E PDX AML cells or normal CD34 + hematopoietic cells were incubated with vehicle or 10–30 μM of the small compound OPA1 inhibitor MYLS22 in methylcellulose. A , B Quantification of mitochondrial length using MTDR/DAPI staining and confocal imaging (63x objective. Scale bars = 2 μm) in PDX cells. C L-CFU assays on PDX AML cells after 7–10 days ( n = 3). D Colony formation from normal human CD34 + hematopoietic progenitor cells after 10 days ( n = 4). Left panel: BFU-E, right panel: CFU-GM. E Representative contour plots (left panel) and cell-cycle phase quantification (right panel) using Ki67/DAPI staining ( n = 4). F – I Mice were treated with vehicle or 30 mg/kg MYLS22 by daily intraperitoneal injection during 7 days ( n = 12 mice per arm). G Representative contour plots of hCD45 versus mCD45. H Quantification of hCD45 + human AML cells. I Quantification of mCD45 + murine hematopoietic cells. J MFN2 and OPA1 promote mitochondrial fusion, driving mitochondrial oxidative phosphorylation (OxPhos) and ROS production, which favor leukemic cells proliferation (left panel). After depletion of MFN2 or OPA1, or inhibition of OPA1 by the small compound MYLS22, inhibition of mitochondrial fusion results in ROS depletion and transition from G 1 to G 0 phase of cell cycle (right panel). Vertical bars indicate standard deviations. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mitotempo and MYLS22 were from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Incubation, Staining, Imaging, Injection, Phospho-proteomics, Inhibition

Journal: Cellular and Molecular Life Sciences

Article Title: Cdk5 regulates IP3R1-mediated Ca 2+ dynamics and Ca 2+ -mediated cell proliferation

doi: 10.1007/s00018-022-04515-8

Figure Lengend Snippet: Cdk5 −/− MEFs exhibit increased Nrf2 level, and scavenging ROS with mito-tempo or GSH prevents the increase in ROS and Nrf2 level in these cells. A Cdk5 −/− MEFs show upregulated expression of Nrf2 and its downstream targets, Prx1 and Prx2. Lysates of wt and Cdk5 −/− MEFs were analyzed by SDS-PAGE and immunoblotting for Cdk5, Nrf2, Prx1 and Prx2. Actin blot was used to assess protein loading. B Wt and Cdk5 −/− MEFs treated with an ROS scavenger, mito-tempo (10 µM) or GSH (10 µM), and then stained with 5 µM DCFDA for 30 min were examined for cytoplasmic ROS level by live-cell imaging using an Olympus I ×71 fluorescence microscope at 160 × magnification. Scale bar = 100 µm. C MEFs treated with mito-tempo or GSH were also analyzed by SDS-PAGE and immunoblotting for Nrf2. The graph (lower panel) shows the ratios of levels of Nrf2 vs actin calculated following densitometric analysis of blots using NIH Image J 1.61

Article Snippet: Mito-tempo and antibodies against Cdk5 (C-8), tubulin (D-10), IP3R1 (E-8), p21 CIP1 (L-17), Prx1 (N-19) and actin (I-19) were from Santa Cruz Biotech (Manassas, VA, USA).

Techniques: Expressing, SDS Page, Western Blot, Staining, Live Cell Imaging, Fluorescence, Microscopy

Journal: Frontiers in Plant Science

Article Title: Mitochondrial pleomorphy in plant cells is driven by contiguous ER dynamics

doi: 10.3389/fpls.2015.00783

Figure Lengend Snippet: Effects of light, dark and sugar on mitochondria length . (A–E) Pie diagram showing the relative percentage of small (less than 0.85 μm) vs. elongated (longer than 0.85 μm) mitochondria in a cellular population for under different conditions. Data based on Arabidopsis mito-GFP transgenics grown for 7 days in light (70 μmol m −2 s −1 ) and dark on MS medium with no sucrose (A1,A2,B,C) and with 3% sucrose (A3,A4,D,E) . Mitochondria in seedlings grown in the dark were predominantly elongated (B,D) as compared to the punctate mitochondria in plants grown in light (C,E) . (F,G) Dark grown plants grown without sucrose were transferred to light (70 μmol m −2 s −1 ) for 1 h (F) and 12 h (G) without sucrose. Putative fission sites on tubular mitochondria are indicated (arrows).

Article Snippet: The 3-dimensional (3D) iso-surface volume rendering of images of mito-GFP RER double transgenics was carried out using IMARIS (v. 6.4.0; Bitplane AG).

Techniques:

Journal: Frontiers in Plant Science

Article Title: Mitochondrial pleomorphy in plant cells is driven by contiguous ER dynamics

doi: 10.3389/fpls.2015.00783

Figure Lengend Snippet: Average size of ER polygons and mitochondria correlates under light and dark growth conditions . (A,B) Representative images from 8 day old plants of RER Arabidopsis transgenics grown in the light (164 μmol m −2 s −1 ) (A) and complete darkness (B) show the visible difference in the size of ER polygons. (C) A comparison of mitochondria and ER size from plants grown in the light and dark. Mitochondria from mito-GFP plants ( n = 200 mitochondria per treatment) were significantly longer when plants were grown in the dark than in the light (1.07 ± 0.34 and 0.78 ± 0.15 μm, dark and light, respectively; p < 0.01). The average ER polygon area was also significantly larger in dark grown plants than those grown in the dark (55.07 ± 39.44 and 3.27 ± 2.05 μm, dark and light respectively; n = 120 ER polygons per treatment; p < 0.01). Standard error bars are shown. (D) “Corrals,” regions with small ER polygons formed between large ER polygons (boxed in area) were observed in dark grown plants. Mitochondria enmeshed in these regions were small while mitochondria elsewhere in the cell appeared elongated. The number of small ER polygons increased upon exposure to light and coincided with the increase in population of small mitochondria. Scale bars in (A,B,D) = 10 μm.

Article Snippet: The 3-dimensional (3D) iso-surface volume rendering of images of mito-GFP RER double transgenics was carried out using IMARIS (v. 6.4.0; Bitplane AG).

Techniques: Comparison

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: A pipeline for multidimensional confocal analysis of mitochondrial morphology, function, and dynamics in pancreatic β-cells.

doi: 10.1152/ajpendo.00457.2019

Figure Lengend Snippet: Fig. 1. General workflow and comparison of mitochondrial identification using global vs. adaptive thresholding methods. A: schematic of the general workflow required for mitochondrial analysis by confocal microscopy. Shaded boxes represent the steps that are addressed and detailed in this paper. B: 2 representative examples of object identification using global thresholding (“default” method) vs. adaptive thresholding (radius 1.25 m, C 11) on images of MIN6-cell mitochondria labeled with mitochondria-targeted yellow fluorescent protein (mito-YFP). The number of identified objects (mitochondria) and their total area are indicated below the images. Scale bar, 1 m. C: part of the mitochondrial network in a MIN6 cell co-transfected with mito-dsRed and mitochondria-targeted photoactivatable green fluorescent protein (mito-PAGFP). Top: all mitochondria imaged in the mito-dsRed channel. Bottom left: a single mitochondrion (green) was labeled by laser-based mito-PAGFP activation at the point indicated by the arrow. Bottom right: object identification using global vs. adaptive threshold algorithms applied to the dsRed channel; in each image, the object that is identified as contiguous with the PAGFP-labeled mitochondrion is shown in green. Comparison with the original image shows that the adaptive method more accurately distinguished the photo-labeled mitochondrion, whereas global thresholding artificially merged it with adjacent mitochondria. Scale bar, 1 m. D: quantitative comparison of the degree to which global and adaptive thresholding under- or overestimated the PAGFP-labeled mitochondrion in 5 test images. The corresponding images and details of the estimation algorithm are shown in Supplemental Fig. S3. 2D, 2-dimensional; 3D, 3-dimensional; 4D, 4-dimensional.

Article Snippet: The mitochondria-targeted YFP (mito-YFP) and mitochondria-targeted photoactivatable GFP (mito-PAGFP) plasmids were gifts from Dr. Mark Cookson (42) and Dr. Richard Youle (Addgene; no. 23348) (23), respectively.

Techniques: Comparison, Confocal Microscopy, Labeling, Transfection, Activation Assay

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: A pipeline for multidimensional confocal analysis of mitochondrial morphology, function, and dynamics in pancreatic β-cells.

doi: 10.1152/ajpendo.00457.2019

Figure Lengend Snippet: Fig. 4. Limitations of 2-dimensional (2D) morphometric analysis and the importance of deconvolution for the quality and accuracy of 3-dimensional (3D) mitochondrial analysis. A: schematic illustrating the effect of object orientation in 3D space on the image capture in a horizontal 2D slice. The apparent 2D morphology of the same tubular object (shown in green) will depend on its orientation relative to the confocal plane. If a curved object (shown in blue) intersects the confocal plane at several locations, it will erroneously be identified as separate objects. B: MIN6 cells were co-transfected with mito-dsRed and mitochondria-targeted photoactivatable green fluorescent protein (mito-PAGFP) and photoactivation induced at the point indicated by an arrowhead. Scale bars, 3 m. Top: 2D image of Mito-dsRed and mito-PAGFP channels after photoactivation. Bottom: objects identified after preprocessing and thresholding of the 2D cross-section. C: full 3D imaging and reconstruction (rendered using Huygens Professional software) of the same mitochondrial population shown in B. Note that the photo-labeled mitochondrion in 2D appears as a series of separate mitochondria, whereas 3D visualization correctly identifies it as 1 contiguous organelle. D: a full z-stack was acquired from a mitochondria-targeted yellow fluorescent protein (mito-YFP)-expressing MIN6 cell that was 11 m in height. Top: maximum projection views of the z-stack before and after deconvolution. The confocal image stack was deconvolved using either ImageJ DeconvolutionLab (Richardson-Lucy algorithm) or Huygens Professional (Classical Maximum Likelihood Estimation) software for 40 iterations. Dotted line indicates the position of the axial section shown below. Bottom: axial sections (xz-plane) of the raw and deconvolved image stacks. The reduction in axial stretching of objects can be seen in the deconvolved stacks, with the best improvement achieved using the Huygens algorithm (see additional details in Supplemental Fig. S6 and Supplemental Table S1). E: 3D renderings of the z-stack before and after deconvolution with ImageJ or Huygens Professional. All 3D visualizations were generated using the Huygens 3D object renderer, with a unique color assigned to separate objects.

Article Snippet: The mitochondria-targeted YFP (mito-YFP) and mitochondria-targeted photoactivatable GFP (mito-PAGFP) plasmids were gifts from Dr. Mark Cookson (42) and Dr. Richard Youle (Addgene; no. 23348) (23), respectively.

Techniques: Transfection, Imaging, Software, Labeling, Expressing, Generated

Journal: Cell Reports

Article Title: Thyroid hormone regulates glutamine metabolism and anaplerotic fluxes by inducing mitochondrial glutamate aminotransferase GPT2

doi: 10.1016/j.celrep.2022.110409

Figure Lengend Snippet:

Article Snippet: Anti-Thyroid hormone receptor antibody (C3)-Chip Grade , ABCAM , ab-2743; RRID: AB_2043044.

Techniques: Recombinant, Modification, Lysis, Western Blot, SYBR Green Assay, Activity Assay, Reporter Assay, Generated, Software, Imaging