Journal: eLife

Article Title: Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity

doi: 10.7554/eLife.51845

Figure Lengend Snippet: ( A ) When the fluorogen MG2I (chemical structure shown on left) is bound to the fluorogen-activating protein (FAP) dL5** (right), excitation by far-red light causes generation of singlet oxygen. ( B ) Diagrams of transgene constructs eno2:gal4FF (above) and UAS:COX4-COX8-dL5**-mCer3 (below). Transactivation of the UAS enhancer by Gal4 in the neurons of double transgenic Tg( eno2:gal4ff ); Tg( UAS:COX4-COX8-dL5**-mCer3 ) ‘NeuMitoFAP’ zebrafish results in expression of the dL5**-mCerulean3 fusion protein in the mitochondrial matrix. ( C ) Merged phase contrast and mCerulean3 epifluorescence images, showing live Tg( UAS:COX4-COX8-dL5**-mCer3 ) (above) and Tg( eno2:gal4ff ); Tg( UAS:COX4-COX8-dL5**-mCer3 ) (NeuMitoFAP; below) zebrafish larvae at 5 days post-fertilization. mCerulean3-expressing structures are labeled (Tel, telencephalon; MB, midbrain; HB, hindbrain; SC, spinal cord; R, retina, ENS enteric nervous system). ( D ) Confocal z-plane projection showing mCerulean3 expression in the posterior lateral line ganglion (PLLG) and lateral line nerve (LLN) of a NeuMitoFAP zebrafish. Individual axonal mitochondria are indicated (arrows). ( E ) Brain sections from NeuMitoFAP zebrafish were labeled for nuclei (DAPI; magenta), dL5**-mCerulean3 (cyan) and mitochondria (TOM20; yellow). Single confocal planes of the individual channels are shown in the upper row. The lower row shows: the three channels overlaid; the output of a Boolean (mCerulean3 AND TOM20) map; and representative regions of interest that were analyzed in panels F and G. ( F ) Scatter plot of TOM20 signal (y-axis) versus mCerulean3 signal (x-axis) in each pixel within regions of interest. ( G ) Pearson correlation coefficient of signal intensity for mCerulean3 versus TOM20 (left) compared with mCerulean3 versus DAPI (right). Each data point shows a region of interest corresponding to an individual mCerulean3-expressing cell, bars show mean ± SE; ****p<0.0001, 2-tailed t-test. ( H ) Normalized excitation and emission spectra of the fluorophores and light sources used in this study.

Article Snippet: To generate the responder construct, a 1.74 kb Pme I/ Xho I restriction fragment was released from pcDNA3.1-cox4-cox8-dL5-2xG4S-mCerulean3 ( ; ) (Addgene 73208) and ligated into the Xho I and Klenow-blunted Eco R1 sites of pT2-5UASMCS to yield pTol2-5UAS:cox4-cox8-dL5-2xG4S-mCerulean3 (2xG4S encodes a flexible GGGGSGGGGS linker between dL5** and mCerulean3 to allow correct folding of both protein domains).

Techniques: Construct, Transgenic Assay, Expressing, Labeling

Journal: eLife

Article Title: Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity

doi: 10.7554/eLife.51845

Figure Lengend Snippet: ( A ) Visual motor responses were evaluated in Tg( eno2:gal4FF ); Tg( UAS:COX4-COX8-dL5-mCer3 ) (‘NeuMitoFAP’) zebrafish treated with MG2I. Motor response to cycles of green light (200 Lux; 10 min) and abrupt darkness (10 min) were measured in 96-well plates, using an infrared machine vision/automated tracking system ( ; ). In each graph, the y-axis shows swimming speed (mm/s) and the x-axis time (minutes). The light-colored trace shows the mean instantaneous swimming speed for 12 zebrafish at each frame transition (4 Hz), averaged over 3 cycles of light-dark responses; data for each minute of the response were binned and averaged, allowing calculation of the mean ± SE (round markers and error bars) swimming speed for the 12 zebrafish during each minute of the stimulus cycle. The timing of the light and dark segments of the stimulus cycle are shown above the graphs. Each group of 12 zebrafish was analyzed both before (upper row of graphs) and after (lower row) exposure to far-red light at the incremental total energy doses shown. Each pair of graphs shows analysis of the same animals under basal and post-exposure conditions. ( B ) Mean swimming speed during the dark phase of the visual motor response was calculated. The scatter plot shows values for individual zebrafish (gray data points) with group mean ± SE superimposed (red bars). Basal dark swimming speed did not differ between the groups, which are combined here for clarity. Swimming speed post-exposure was progressively reduced in the 30, 60 and 120 J/cm 2 groups (****p<0.00001, compared with baseline, 1-way ANOVA, with Dunnett’s post hoc test; comparison of pre- and post-exposure data for each group yielded identical results). Together, these data show that the severity of the motility phenotype during the dark phase of the visual motor response in NeuMitoFAP zebrafish treated with MG2I is determined by the amount of light energy delivered.

Article Snippet: To generate the responder construct, a 1.74 kb Pme I/ Xho I restriction fragment was released from pcDNA3.1-cox4-cox8-dL5-2xG4S-mCerulean3 ( ; ) (Addgene 73208) and ligated into the Xho I and Klenow-blunted Eco R1 sites of pT2-5UASMCS to yield pTol2-5UAS:cox4-cox8-dL5-2xG4S-mCerulean3 (2xG4S encodes a flexible GGGGSGGGGS linker between dL5** and mCerulean3 to allow correct folding of both protein domains).

Techniques: Comparison

Journal: Developmental Cell

Article Title: Sestrin2 drives ER-phagy in response to protein misfolding

doi: 10.1016/j.devcel.2024.07.004

Figure Lengend Snippet:

Article Snippet: pHAGE mKeima-COX8 , Addgene , #131626.

Techniques: Virus, Plasmid Preparation, Retroviral, Recombinant, Cloning, Transfection, Bicinchoninic Acid Protein Assay, Mutagenesis, Control, Western Blot, Stable Transfection, Expressing, Isolation, Sequencing, Software, Microscopy, Imaging, Diagnostic Assay