Journal: bioRxiv

Article Title: Mutant Prion Protein Endoggresomes are Hubs for Local Axonal Organelle-Cytoskeletal Remodeling

doi: 10.1101/2023.03.19.533383

Figure Lengend Snippet: a Membrane-extracted (ME) correlative light and scanning electron microscopy (CLSEM) image of a neuron transfected with PrP PG14 -mCh (red). Arrowheads indicate axons. Green Inset shown on middle panel. White arrows point to MT tracks. Orange inset shown on right panel. Blue arrows point to actin. Red arrowheads point to disrupted MT tracks. b Images of an axon from a neuron transfected with PrP PG14 -mTagBFP2 (red) imaged using highly-inclined and laminated optical sheet (HILO) microscopy, stained with Phalloidin-AF647 and imaged with direct STORM (dSTORM). Insets I-II of axonal swellings shown on the right. Arrowheads point to actin accumulation around the axonal swellings. c Super-resolution (SR) Airyscan confocal microscopy maximum projection images of PrP PG14 -mCh, immunostained with alpha tubulin antibody. Round Arrows indicate MT tracks at NON-AGG sites; Arrowheads indicate MT at AGG site. d SR-Airyscan images of PrP WT - or PrP PG14 -mTagBFP2 axons, co-immunostained with acetylated (Ac) and tyrosinated (Tyr) tubulin antibodies. Arrows indicate intact/straight tracks; Arrowheads indicate broken MT. e Percent Ac- or Tyr-MT track architecture (straight, bend, or break) at WT (in PrP WT -mTagBFP2 axons) and NON-AGG or AGG sites (in PrP PG14 -mTagBFP2 axons). e HILO microscopy images of PrP PG14 -mCh axons immunostained against either acetylated and tyrosinated tubulin, and corresponding Super-resolution radial fluctuation (SRRF)-processed images at the AGG sites (i-ii; insets). Insets: Arrowheads point to MT tracks bending; Arrows point to MT tracks breaking. See also figure S3.

Article Snippet: Mito-mTagBFP2 was cloned by replacing EGFP in mito-EGFP with mTagBFP2 sequence in pcDNA3.1 from mTagBFP2-Lysosome-20 (Addgene #55308).

Techniques: Membrane, Electron Microscopy, Transfection, Microscopy, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: Mutant Prion Protein Endoggresomes are Hubs for Local Axonal Organelle-Cytoskeletal Remodeling

doi: 10.1101/2023.03.19.533383

Figure Lengend Snippet: a Membrane-extracted CLSEM image of a neuron transfected with PrP PG14 -mCh (red). White arrowheads indicate axons. Blue inset (right) shows an AGG site. White arrows point to MT tracks. Blue arrows point to actin surrounding PrP PG14 aggregates. b Membrane-extracted SEM image of an axonal region of a neuron transfected with PrP PG14 -mCh. Insets (i-ii) show AGG sites. Red arrowheads and pseudo-color highlights show broken (i) or bending (ii) MT tracks. Insets (iii-iv) show NON-AGG sites. c HILO image of an axon from a neuron co-transfected with PrP PG14 -mTagBFP2 and mito-EGFP, co-immunostained with alpha tubulin and antibodies against tyrosinated (Tyr) tubulin. Inset (i) shows AGG site and SRRF processed images of alpha tubulin and Tyr-tubulin channels. Inset (ii) shows a HILO image of PrP PG14 -mTagBFP2 and mito-EGFP at AGG site and SRRF processed images of alpha tubulin and Tyr-tubulin channels. Arrows point to straight/intact MT tracks. Arrowheads point to broken MT tracks.

Article Snippet: Mito-mTagBFP2 was cloned by replacing EGFP in mito-EGFP with mTagBFP2 sequence in pcDNA3.1 from mTagBFP2-Lysosome-20 (Addgene #55308).

Techniques: Membrane, Transfection

Journal: bioRxiv

Article Title: Mutant Prion Protein Endoggresomes are Hubs for Local Axonal Organelle-Cytoskeletal Remodeling

doi: 10.1101/2023.03.19.533383

Figure Lengend Snippet: a Images of axons expressing PrP WT -mCh or PrP PG14 -mCh, immunostained with KHC and KLC1 or with KIF1A antibodies. Orange boxes show AGG, NON-AGG, and WT ROIs. b Normalized intensity distribution frequencies (F) of KHC, KLC1, and KIF1A signal at AGG, NON-AGG, and WT sites plotted as box plots with kernel density estimates from histogram. c Representative inverted images of axons expressing PrP WT -mTagBFP2 or PrP PG14 -mTagBFP2 and mito-EGFP, and immunostained with KHC and KIF1A antibodies. Insets (below) shown as overlay of sub-pixel localization detection (magenta spots; arrowheads). d Quantitation of percent mitochondria (mean ± SEM) colocalizing with 0, 1, 2, and ≥ 3 KHC or KIF1A motor clusters. ***p<0.001, **p<0.01, *p<0.05, ns = not significant; b Kruskal-Wallis test, d Student’s t-test.

Article Snippet: Mito-mTagBFP2 was cloned by replacing EGFP in mito-EGFP with mTagBFP2 sequence in pcDNA3.1 from mTagBFP2-Lysosome-20 (Addgene #55308).

Techniques: Expressing, Quantitation Assay

Journal: bioRxiv

Article Title: Mutant Prion Protein Endoggresomes are Hubs for Local Axonal Organelle-Cytoskeletal Remodeling

doi: 10.1101/2023.03.19.533383

Figure Lengend Snippet: a Schematic (top) and representative inverted images of axons (middle) of neurons expressing soluble EGFP, PrP PG14 -mTagBFP2, and KIF5C-2xmCh for 2 days, and corresponding line scan intensity profiles (bottom) (n axons = 9). b Schematic (top) and representative inverted images of axons (middle) from neurons expressing soluble EGFP, PrP PG14 -mTagBFP2, and KIF5C(1-560)-2xmCh for 2 days, and corresponding line scan intensity profiles (bottom) (n axons = 13). c Schematic (top), timeline of sequential transfection of EGFP and PrP PG14 -mTagBFP2 for 2 days and KIF5C(1-560)-2xmCh for 6 hours (bottom left), and images at the axon terminus (bottom right). Arrowheads point to endoggresomes. Arrows point to EGFP and KIF5C(1-560)-2xmCh accumulation at the axonal growth cone. d Timeline of FRAP assay (top) of axon terminals induced with PrP WT - or PrP PG14 -mCh AAV for 5 days, sequentially transfected with KIF5C(1-560)-mCit for 12 hours prior to live imaging. (bottom) FRAP profile of KIF5C(1-560)-mCit.

Article Snippet: Mito-mTagBFP2 was cloned by replacing EGFP in mito-EGFP with mTagBFP2 sequence in pcDNA3.1 from mTagBFP2-Lysosome-20 (Addgene #55308).

Techniques: Expressing, Transfection, FRAP Assay, Imaging

Journal: bioRxiv

Article Title: Mutant Prion Protein Endoggresomes are Hubs for Local Axonal Organelle-Cytoskeletal Remodeling

doi: 10.1101/2023.03.19.533383

Figure Lengend Snippet: a First-frame images of axons co-expressing PrP PG14 -mCh and mito-EGFP for 2 d. The yellow dotted line indicates an imaging region of time-lapse (bottom left) and kymograph (right). b Quantitation of mito-EGFP length at AGG vs NON-AGG sites at 5 days post-PrP PG14 -mCh expression. c First-frame images of axons co-transfected with mito-EGFP and PrP PG14 -mCh for 5 days (top two panels) and corresponding kymographs (bottom panel) generated from time-lapse movies (5 minutes, 300 frames, 1 frame/sec). d Quantitation of mito-EGFP length co-transfected with PrP WT -mCh for 2 days or PrP PG14 -mCh for 12 hours, 2 days, and 5 days. e Axons co-expressing PrP PG14 -mCh, Rab7-EGFP, and mito-mTagBFP2. Insets and highlights indicate AGG sites. f Quantitation of percent mito-mTagBFP2 colocalize with Rab7(WT/Q67L)-EGFP at WT, NON-AGG, or AGG sites. g First-frame images of axons co-expressing PrP PG14 -mCh, Rab7 WT -EGFP, and mito-mTagBFP2 and representative frames from time-lapse videos at WT or AGG sites (insets). h First-frame images of axons co-expressing PrP PG14 -mCh, Rab7 Q67L -EGFP, and mito-mTagBFP2 and representative frames from time-lapse videos at WT or AGG sites (insets). i Quantitation of mitochondrial fission frequency index (% of fission frequency / mitochondrial length). Box plots were shown with Tukey definition; (+) indicates the mean. All bar graphs are shown as mean ± SEM. ***p<0.001, **p<0.01, *p<0.05, ns = not significant; b, d Kruskal-Wallis test. f, i Tukey’s multiple comparison test. See also figure S4.

Article Snippet: Mito-mTagBFP2 was cloned by replacing EGFP in mito-EGFP with mTagBFP2 sequence in pcDNA3.1 from mTagBFP2-Lysosome-20 (Addgene #55308).

Techniques: Expressing, Imaging, Quantitation Assay, Transfection, Generated, Comparison

Journal: bioRxiv

Article Title: Mutant Prion Protein Endoggresomes are Hubs for Local Axonal Organelle-Cytoskeletal Remodeling

doi: 10.1101/2023.03.19.533383

Figure Lengend Snippet: a First-frame images of axons co-expressing PrP WT -mCh, Rab7 WT -EGFP, and mito-mTagBFP2 and representative frames from time-lapse videos at WT or AGG sites (insets). b First-frame images of axons co-expressing PrP WT -mCh, Rab7 Q67L -EGFP, and mito-mTagBFP2 and representative frames from time-lapse videos at WT or AGG sites (insets). Arrows point to corresponding organelles in the first-frame images and time-lapse insets.

Article Snippet: Mito-mTagBFP2 was cloned by replacing EGFP in mito-EGFP with mTagBFP2 sequence in pcDNA3.1 from mTagBFP2-Lysosome-20 (Addgene #55308).

Techniques: Expressing

Journal: bioRxiv

Article Title: Mutant Prion Protein Endoggresomes are Hubs for Local Axonal Organelle-Cytoskeletal Remodeling

doi: 10.1101/2023.03.19.533383

Figure Lengend Snippet: a Representative inverted color first frames of axons (top two panels) and kymographs (bottom panel) generated from time-lapse movies (5 minutes, 300 frames, 1 frame/sec) of PrP WT - or PrP PG14 -mTagBFP2, co-transfected with SNAP(JF646)-Rab7 and mito-EGFP and treated with 25 nM of TMRE. Arrowheads point to TMRE-negative mito-EGFP. b TMRE intensities per mito-EGFP in WT, NON-AGG, or AGG regions. c Percent mito-EGFP that are TMRE-positive. All bar graphs are shown as mean ± SEM. ***p<0.001, **p<0.01, *p<0.05, ns = not significant; b, c Tukey’s multiple comparison test.

Article Snippet: Mito-mTagBFP2 was cloned by replacing EGFP in mito-EGFP with mTagBFP2 sequence in pcDNA3.1 from mTagBFP2-Lysosome-20 (Addgene #55308).

Techniques: Generated, Transfection, Comparison