Journal: bioRxiv

Article Title: Phosphoarginine modulates oligomerization and repressor activity of mycobacterial ClpC2

doi: 10.64898/2026.06.30.735635

Figure Lengend Snippet: A) Diagram of the DNA template used for in vitro transcription studies, including a σ70-dependent E. coli promoter, clpC2 operator, and sequence encoding a F30-broccoli RNA aptamer. B) Transcription of the broccoli aptamer was monitored by the increase in fluorescence over time (green trace). Inclusion of 6 µM Mtb ClpC2 decreased transcription (red trace). DFHBI-1T alone produced minimal fluorescence (gray trace). C) Transcription from a template incorporating a scrambled operator sequence was similar in the absence (cyan) or presence (blue) of 6 µM Mtb ClpC2. By contrast, 2 µM rifampin completely inhibited transcription (orange trace). D) Transcriptional rate was measured as a function of Msm ClpC2 concentration, using templates with scrambled or intact operator sequences. E) Transcription was measured in the presence of the indicated concentration of Mtb ClpC2, or with the additional inclusion of 500 µM pArg, 10 µM pArg casein, 10 µM unphosphorylated casein, 1.2-fold molar excess Ruf or 1.2-fold molar excess CymA. F). Transcriptional repression is shown for the indicated concentration of wild-type Mtb ClpC2, and for Mtb ClpC2 variants incorporating R56A or T256A mutations. Error bars reflect standard deviation of ≥3 replicates. Statistical significance was determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test to compare each condition to ClpC2 alone (blue bar) at a given concentration. Multiplicity-adjusted p -values are indicated as follows: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Non-significant comparisons are not shown.

Article Snippet: In a separate tube, 200 nM of template DNA was incubated in buffer with or without ClpC2, pArg casein (10 μM), casein (10 μM), pArg (500 μM), Ruf (1.2-fold molar excess), or CymA (1.2-fold molar excess) at 37°C for 15 min. DNA mixtures and holoenzyme were mixed and incubated at 37°C for 15 min, followed by addition of 100 nM of DFHBI-1T (MedChemExpress).

Techniques: In Vitro, Sequencing, Fluorescence, Produced, Concentration Assay, Standard Deviation, Comparison

Journal: Glia

Article Title: Drosophila tweety facilitates autophagy to regulate mitochondrial homeostasis and bioenergetics in Glia.

doi: 10.1002/glia.24484

Figure Lengend Snippet: FIGURE 4 Elevated mitochondrial oxidative stress in tweety mutant glia. (a) Representative fluorescence images and ratio-images of single live wild-type (WT) and tty1 mutant (tty1) glial cells expressing the ratiometric mitochondrial oxidative stress sensor Mito-Grx1-roGFP2. (b) Elevated mitochondrial oxidative stress upon loss of tty in glia. Mito-Grx1-roGFP2 fluorescence ratios of primary glial cells of the indicated genotypes are normalized to the mean of the wild-type (WT) ratios. Higher ratio values indicate elevated oxidative stress. ***p < .0001; Mann– Whitney test. (c) Glial (repo-GAL4) overexpression of tty (+ tty) or human TTYH1 (+ TTYH1) significantly reduced mitochondrial oxidative stress in both wild-type and tty1 mutant glia. Data points for “WT” are the same as in Figure 4b. ***p < .0001; one-way ANOVA. (d) Loss of tweety sensitizes flies to oxidative stress inducers. Climbing ability of tty1 mutant flies with (tty1+GR1) or without (tty1) genomic rescue was assessed. Boxplots display median climbed height normalized to the mean of the control (tty1+GR1 with DMSO). Flies were fed with food containing vehicle (0.1% DMSO), rotenone (50 μM), or paraquat (1 μM) for 3 days prior to climbing assay. n.s., not significant, *p < .05; t-test compared to “DMSO.” (e) Antioxidant partially restores mitochondrial turnover in tty1 mutant glia. Boxplots show MitoTimer fluorescence ratio of primary glial cells of the indicated genotypes. Primary cells were treated with or without 40 ng/mL AD4 in culture medium for 3 days prior to imaging experiments. **p < .01, *p < .05; Mann–Whitney test. (f and g) Exogenous antioxidant alleviates locomotor phenotypes of tweety mutant flies. Climbing ability (f) and locomotor activity (g) of wild-type and tty1 flies were assessed and quantified after feeding with food containing 40 μg/mL AD4 (N-acetyl cysteine amide) for 1 week after eclosion. ***p < .001, **p < .01, *p < .05; t-test.

Article Snippet: AD4 (HY-110256) and UK-5099 (HY-15475) were obtained from Med Chem Express.

Techniques: Mutagenesis, Fluorescence, Expressing, MANN-WHITNEY, Over Expression, Control, Climbing Assay, Imaging, Activity Assay

Journal: Glia

Article Title: Drosophila tweety facilitates autophagy to regulate mitochondrial homeostasis and bioenergetics in Glia.

doi: 10.1002/glia.24484

Figure Lengend Snippet: FIGURE 6 Loss of tweety shifts glial bioenergetics toward glycolysis-dependent ATP production. (a) Representative ratio-images of single live wild-type and tty1 glial cells expressing ratiometric ATP/ADP sensor PercevalHR. (b) Steady-state PercevalHR fluorescence ratios of primary glial cells of the indicated genotypes. n.s. not significant; one-way ANOVA. (c) Time-lapse traces of PercevalHR fluorescence ratio of primary glial cells measured by live-cell imaging. Data points are mean ± SEM of ratios from the primary glial cells of the indicated genotypes, and are normalized to ratio values at 1 min before 2-DG (10 mM) application. Dashed line indicates 10-min time-point after 2-DG application. (d and e) ATP:ADP levels are more sensitive to glycolytic blockage upon loss of tweety in glia. Quantification of PercevalHR fluorescence at 10 min after 2-DG application to the glial cells of the indicated genotypes. Boxplots display percentage change of ratio values (d) and ratio values (e). ***p < .0001, **p < .01; Mann–Whitney test. (f and g) Quantification of PercevalHR fluorescence before (g) and 10 min after (h) 2-DG application to the glial cells treated with 40 ng/mL AD4 in culture medium for 3 days prior to imaging experiments. Boxplots display steady-state ratio values (g) and percentage change of ratio values (h). n.s. not significant, *p < .05; Mann–Whitney test. (h and i) Genetic inhibition of autophagy phenocopies loss of tweety. Changes in PercevalHR fluorescence ratio upon 2-DG application to primary glial cells expressing short hairpin RNAs against Atg5 (Atg5RNAi), Atg8a (Atg8aRNAi), and mCherry (mCherryRNAi; as control) are shown as time-lapse traces (i) and percentage change of ratio values (j). ***p < .001, **p < .01; t-test. (j and k) Autophagy induction does not restore sensitivity to glycolytic blockage in tweety mutant glia. PercevalHR fluorescence ratio at steady-state (k) and 10 min after 2-DG application (l) to wild-type glial cells and Atg1-overexpressing (repo > Atg1) wild-type and tty1 glial cells. Data points for “WT” are the same as in Figure 6b,e. ****p < .0001, ***p < .001; Mann–Whitney test. (l) Schematic showing the function of tweety in autophagy, mitochondrial turnover, and bioenergetics in glia. Old or damaged mitochondria are engulfed in autophagosomes to be delivered to endolysosomes for enzymatic degradation. Loss of tweety impedes fusion between autophagosomes and endolysosomes, causing a slow mitochondrial turnover. Old mitochondria which exhibit elevated oxidative stress accumulate. Mitochondrial metabolism such as pyruvate oxidation diminishes. Consequently, loss of tweety stipulates glial cells to ramp up glycolysis for ATP production.

Article Snippet: AD4 (HY-110256) and UK-5099 (HY-15475) were obtained from Med Chem Express.

Techniques: Expressing, Fluorescence, Live Cell Imaging, MANN-WHITNEY, Imaging, Inhibition, Control, Mutagenesis