Journal: Neural Regeneration Research

Article Title: Drosophila models used to simulate human ATP1A1 gene mutations that cause Charcot-Marie-Tooth type 2 disease and refractory seizures

doi: 10.4103/1673-5374.391302

Figure Lengend Snippet: Most Atpα -CMT2 flies have motor and sensory defects and an abnormal circadian neuron dendritic morphology. (A) Images of control, Atpα -CMT2, and mutant flies expressing nSybGFP in the neuromuscular junction of third instar larvae. Staining with anti-GFP antibody revealed the morphology of neuromuscular junctions. All Atpα -CMT2 and mutant flies showed a decrease in synapse number and length. We utilized an online post hoc power calculator (https://clincalc.com/stats/Power.aspx) to assess statistical power, with a false positive rate set at 0.05. Except for the statistical power of Atpα mut/+ in the synapse length group, which is 56.4%, the statistical power of the other significantly different groups exceed 83.5%. (B) Plots of synapse number (left) and length (right) in control, Atpα -CMT2, and mutant flies ( n = 6–10). One-way analysis of variance with Tukey’s multiple comparison test were used. (C) Images of control, Atpα -CMT2, and mutant brains with Class IV multidendritic sensory neurons in the third instar larval body wall visualized by ppk -GAL4-driven mCD8-GFP. Except for Atpα D580F/+ and Atpα mut/+ , all other heterozygous Atpα -CMT2 flies showed significantly reduced dendritic branching. (D) Plots of dendritic branch number in control, Atpα -CMT2, and mutant flies ( n = 15–20). Dendritic branch number was compared using the nonparametric Kruskal-Wallis test with Dunn’s multiple comparison test. The statistical powers of the significantly different groups are between 94.7% and 100%. (E) Left: Image of a circadian circuit in a control brain, visualized by staining with anti-PDF antibodies. sLNvs and lLNvs indicate small and large ventral lateral neurons. Right: The region analyzed. (F) Images of representative PDF dorsal axonal projections from control, Atpα -CMT2, and mutant flies taken during the early day (ZT2) and early night (ZT14). Apart from Atpα mut/+ , there was a significant decrease in axonal arbor complexity of the PDF circuit in Atpα -CMT2 flies. (G) Quantification of the total number of intersections between concentric rings and axonal projections at ZT2 and ZT14 ( n = 8–11). Data were analyzed by one-way analysis of variance with Tukey’s multiple comparison test. The statistical powers of the significantly different groups are between 76.9% and 100%. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars represent SEM. Atpα : Na pump α subunit; CMT2: Charcot-Marie-Tooth disease type 2; Gal4: galactose-responsive transcription factor GAL4; GFP: green fluorescent protein; lLNv: large ventral lateral clock neurons; ns: not significant; PDF: pigment-dispersing factor; ppk: polyphosphate kinase; sLNv: small ventral lateral clock neurons; ZT: zeitgeber time.

Article Snippet: To visualize neuromuscular junctions in third instar larvae, D42 -Gal4,UAS-nSyb-GFP flies (Bloomington Drosophila Stock Center, Bloomington, IN, USA, Cat# 9263) were prepared and stained overnight at 4°C with a primary antibody against green fluorescent protein (GFP; 1:1000, Thermo Fisher Scientific, Waltham, MA, USA, Cat# A11120, RRID: AB_221568).

Techniques: Control, Mutagenesis, Expressing, Staining, Comparison

Journal: Science Advances

Article Title: Cl − -dependent amplification of excitatory synaptic potentials at distal dendrites revealed by voltage imaging

doi: 10.1126/sciadv.adj2547

Figure Lengend Snippet: ( A ) An image of a direct gramicidin patch recording from a dendrite. Magnified view of the patched branch in yellow (left). ( B ) V m trace recorded from a dendritic branch [shown in (A)]. ( C ) V m rest measured by gramicidin patch from dendrites ( n = 8 cells) and soma ( n = 10 cells) is plotted against the distance from the soma. ( D ) Somatic (blue, n = 19 cells) and dendritic (green, n = 11 cells) V m rest with or without 10 μM VU0463271 (soma: yellow, n = 9 cells; dendrite: red, n = 11 cells). Dunnett’s test, ** P < 0.01; n.s., not significant. ( E and F ) V m rest (E) and leak conductance (F) plotted against distance from the soma. VU, VU0463271. ( G ) Representative gramicidin patch currents recorded from the soma (left) or a dendritic branch (right) in response to spot uncaging of DPNI-caged GABA at the recording sites, with altered holding V m indicated by different colors. ( H ) GABA currents at a dendrite (red) or at the soma (blue) plotted against the holding V m . ( I ) Images of immunostained KCC2 (magenta) in green fluorescent protein (GFP)–labeled hippocampal neurons (green). Three rectangular areas (a to c) were expanded with merged colors (middle), and the fluorescent signals for KCC2 and GFP along a line in each area are plotted on the right. ( J ) Relative fluorescent intensity of GFP (green) and KCC2 (red) plotted against the distance from the soma.

Article Snippet: The following antibodies were used: rabbit polyclonal antibody (pAb) against KCC2 (1:1000; Sigma-Aldrich, catalog no. 07-432) and ClC-2 (1:5000; Alomone Labs, Jerusalem, Israel, catalog no. ACL-002), chicken pAb against green fluorescent protein (GFP) (1:2000; Sigma-Aldrich, catalog no. AB16901), Alexa Fluor 568–conjugated pAb against rabbit immunoglobulin G (IgG) (1:400, Thermo Fisher Scientific, catalog no. A-11011), and Alexa Flour 488–conjugated pAb against chicken IgG (1:400; Thermo Fisher Scientific, catalog no. A-11039).

Techniques: Labeling

Journal: bioRxiv

Article Title: Phase separation promotes Atg8 lipidation for autophagy progression

doi: 10.1101/2024.08.29.610189

Figure Lengend Snippet: (A) Fluorescence images of the early PAS droplets supplemented with each Atg protein. Atg12–5–16 images are the same as those used in . Scale bar = 10 μm. (B) Quantification of fluorescence intensity of SNAP–Atg16 in the droplets in (A). * P = 1.04 × 10 −2 , **** P = 6.3 × 10 −6 (Atg12-5-16 vs. Atg5-16), 4.0 × 10 −5 (Atg12–5–16 vs. Atg12ΔIDR–5–16), using a two-sided Tukey’s multiple comparisons test. (C) Fluorescence images of the early PAS droplets supplemented with GB1– Atg12 17BH . Scale bar = 10 μm. (D) Fluorescence images of GFP–Atg5 and mCherry–Atg17 in yeast cells. Scale bar = 5 μm. (E) Quantification of the colocalization of GFP–Atg5 with mCherry–Atg17 at the PAS puncta in (D). Number of PAS puncta: n = 1,548 (WT), 1,990 (V43A), 1,815 (L47A), 1,710 (L54A), and 1,829 (L57A). **** P < 1.0 × 10 −15 , using a two-sided Dunn’s multiple comparisons test. See also Figure S3.

Article Snippet: For immunoblotting, antibodies against GFP (11814460001; Roche) and Atg5 were used.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Toss GERALT into chloroplast to make it green: an age-dependent regulator of chloroplast biogenesis and chlorophyll biosynthesis

doi: 10.1101/2024.08.16.608208

Figure Lengend Snippet: (A) SIM2 images of pavement chloroplast of WT and WT-OE_3E overexpressing GERALT-GFP fusion protein. Chlorophyll channel - excitation 470 nm, emission 650-750 nm. Green channel - excitation 470 nm, emission 490-530 nm, DAPI channel - excitation 405 nm, emission 440-480 nm. Overlay – superimposition of all three channels. Images are cropped from bigger pictures. Scale bar = 1 μm. (B) Western blot analysis of GERALT-GFP subchloroplast localization. Proteins isolated from chloroplasts purified from WT-OE3E 5 week old soil grown plants were separated into stroma and thylakoid fractions, separated by SDS PAGE (12 % polyacrylamide gels) and electroblotted. Proteins were probed with anti-GFP, anti-RbcL or anti-Lhcb2 antibodies.

Article Snippet: Immunodetections were performed using specific antibodies against GFP (sc-9996, Santa Cruz Biotechnology, USA) at dilution of 1:1000, against LHCII type chlorophyll a/b binding proteins (anti-Lhcb2, AS01 003, Agrisera, Sweden) at dilution of 1:5000 and against Rubisco large subunit (anti-RbcL, AS03 037, Agrisera, Sweden) at dilution of 1:10000.

Techniques: Western Blot, Isolation, Purification, SDS Page