Journal: Biomolecules

Article Title: Distinct Effects of Familial Parkinson's Disease-Associated Mutations on α-Synuclein Phase Separation and Amyloid Aggregation.

doi: 10.3390/biom13050726

Figure Lengend Snippet: Figure 1. WT and familial PD-associated mutant α-Syn undergo LLPS in vitro. (A) Illustration showing the familial PD-associated mutations. The NTD, NAC, and CTD of α-Syn are indicated in different colors. (B) Turbidity assays showing the formation of phase-separated condensates by WT and mutant α-Syn. Turbidity was evaluated by monitoring the absorbance at 405 nm. Data are presented as mean ± SD (n = 3 independent replicates). p values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. n.s., p > 0.05. **, p < 0.01. (C) Fluorescence and differential interference contrast (DIC) images showing the morphologies of Rhod-labeled WT and mutant α-Syn droplets. The α-Syn concentration is 200 µM. Scale bar, 5 µm. All the experiments were carried out in the presence of 20% PEG-10000.

Article Snippet: For each sample, 10 μL of α-Syn WT and mutant condensates were incubated with 1 μL ThS solution at 37 ◦C for 0 h, 24 h, and 48 h. ThS fluorescence was visualized with a 100 X oil immersion objective under a Nikon A1 microscope at 488 nm [48].

Techniques: Mutagenesis, In Vitro, Fluorescence, Labeling, Concentration Assay

Journal: Biomolecules

Article Title: Distinct Effects of Familial Parkinson's Disease-Associated Mutations on α-Synuclein Phase Separation and Amyloid Aggregation.

doi: 10.3390/biom13050726

Figure Lengend Snippet: Figure 2. The familial PD-associated mutations do not affect the fluidity of α-Syn condensates. (A) Representative FRAP images of WT and mutant α-Syn condensates. The fluorescence images of prebleached, bleached (0 s), and bleached after 100 s recovery are shown. Scale bar, 2 µm. (B) The normalized FRAP curves of α-Syn condensates shown in A. Data are presented as mean ± SD (n = 3 independent replicates) and normalized to the maximal prebleach and minimal postbleach fluorescence intensities. The concentration of α-Syn is 200 µM. All the experiments were carried out in the presence of 20% PEG-10000.

Article Snippet: For each sample, 10 μL of α-Syn WT and mutant condensates were incubated with 1 μL ThS solution at 37 ◦C for 0 h, 24 h, and 48 h. ThS fluorescence was visualized with a 100 X oil immersion objective under a Nikon A1 microscope at 488 nm [48].

Techniques: Mutagenesis, Concentration Assay

Journal: Biomolecules

Article Title: Distinct Effects of Familial Parkinson's Disease-Associated Mutations on α-Synuclein Phase Separation and Amyloid Aggregation.

doi: 10.3390/biom13050726

Figure Lengend Snippet: Figure 5. α-Syn undergoes a liquid-to-solid phase transition differently regulated by familial PD- associated mutations. (A) Representative FRAP images of WT and mutant α-Syn condensates after 48 h incubation. The fluorescence images of prebleached, bleached (0 s), and bleached after 100 s recovery are shown. Scale bar, 2 µm. (B) The normalized FRAP curves of α-Syn condensates shown in A. Data are presented as mean ± SD (n = 3 independent replicates) and normalized to the maximal prebleach and minimal postbleach fluorescence intensities. The concentration of α-Syn is 200 µM. All the experiments were carried out in the presence of 20% PEG-10000.

Article Snippet: For each sample, 10 μL of α-Syn WT and mutant condensates were incubated with 1 μL ThS solution at 37 ◦C for 0 h, 24 h, and 48 h. ThS fluorescence was visualized with a 100 X oil immersion objective under a Nikon A1 microscope at 488 nm [48].

Techniques: Sublimation, Mutagenesis, Incubation, Concentration Assay

Journal: Journal of Clinical Investigation

Article Title: Recombinant annexin A6 promotes membrane repair and protects against muscle injury

doi: 10.1172/jci128840

Figure Lengend Snippet: Figure 1. Ca2+-dependent annexin repair cap recruitment at the site of injury. Myofibers were generated to express the Ca2+ indicator GCaMP5G (green), and time-lapse single-slice images were assessed at time points after membrane disruption. (A) GCaMP5G fluorescence was present at the site of injury, at 2 sec- onds (arrow), indicating the presence of Ca2+ immediately after damage at the site of injury (top panel). These data were validated with a non–protein-based Ca2+ indicator, Fluo-4 AM (green, bottom panel). (B) Time-lapse images of myofibers coelectroporated with GCaMP5G and annexin A6-tdTomato (A6, red). GCaMP5G fluorescence was present at the site of injury localized around the annexin A6–free zone (arrowhead) and at the annexin A6 cap (arrow). GCaMP5G colocalized (merge, yellow, arrow) with the annexin A6 repair cap. Scale bars: 5 μm. (C) Myofibers expressing fluorescently tagged annexins A1, A2, or A6 were injured at multiple Ca2+ concentrations. Annexin A1 and A6 repair cap size was reduced at 0.1 mM Ca2+ compared with 2 mM and 0.5 mM. Annexin A2 repair cap area was significantly reduced at 0.05 mM Ca2+ compared with 2 mM, 0.5 mM, and 0.1 mM Ca2+. (D) Cap kinetics were plotted as cap Feret diameter over a range of Ca2+ concentrations. Annexin A2 had a statistically significant leftward shift in Km(1/2), followed by annexin A6 and then A1. Data are expressed as mean ± SEM. Differences were tested by 1-way ANOVA with Tukey’s multiple-comparisons test (C). *P < 0.05 (n = 5 myofibers per condition).

Article Snippet: Subcloning of annexin A1, A2, and A6 to replace the GFP tag with tdTomato (Addgene) was performed by Mutagenix.

Techniques: Generated, Membrane, Disruption, Fluorescence, Expressing

Journal: Journal of Clinical Investigation

Article Title: Recombinant annexin A6 promotes membrane repair and protects against muscle injury

doi: 10.1172/jci128840

Figure Lengend Snippet: Figure 2. Annexin expression promoted release of blebs from the site if myofiber repair. Myofibers were electroporated with the Ca2+ indicator GCaMP5G (green) with or without tdTomato-labeled annexin A1, annexin A2, or annexin A6. Ca2+ area and fluorescence were assessed after membrane damage. (A) High-magnification Z-projection images illustrate external blebs filled with the Ca2+ indicator emanating from the lesion when annexin A1, A2, or A6 was coexpressed and a corresponding reduction in Ca2+ indicator within the myofiber when compared with GCaMP5G alone (see panel B). (B) Membrane marked by FM 4-64 shows GCaMP5G-negative vesicles form in the absence of annexin overexpression. Scale bars: 5 μm. (C) Expression of annexin A6 or A2 result- ed in an increased number of GCaMP5G-positive blebs. (D) Expression of annexin A6 resulted in the formation of the largest GCaMP5G-positive blebs. Data are expressed as mean ± SEM. Differences were tested by 1-way ANOVA with Tukey’s multiple-comparisons test. *P < 0.05 (n = 16 myofibers from n = 3 mice per condition).

Article Snippet: Subcloning of annexin A1, A2, and A6 to replace the GFP tag with tdTomato (Addgene) was performed by Mutagenix.

Techniques: Expressing, Labeling, Fluorescence, Membrane, Over Expression

Journal: Journal of Clinical Investigation

Article Title: Recombinant annexin A6 promotes membrane repair and protects against muscle injury

doi: 10.1172/jci128840

Figure Lengend Snippet: Figure 3. Annexin expression reduced Ca2+ within the myofiber. Myofibers were electroporated with the Ca2+ indicator GCaMP5G (green) with or without tdTomato-labeled annexin A1, annexin A2, or annexin A6 (red imaging not shown in this image). Ca2+ area and fluorescence were assessed after membrane damage. (A) Time-lapse single-slice images reveal that coexpression of either annexin A1, A2, or A6 resulted in a significant reduction in GCaMP5G fluores- cence (green) measured inside the myofiber at the site of injury over time. (B) Expression of either annexin A1, A2, or A6 resulted in a significant reduction in GCaMP5G fluorescence measured inside the myofiber at the site of injury over 240 seconds of imaging, with annexin A6 inducing the greatest reduction in GCaMP5G fluorescence. (C) Both annexin A2 and A6 contributed to the early reduction in GCaMP5G fluorescence, as seen by imaging during the first 20 seconds after injury. (D) Initial GCaMP5G mean fluorescence was not significantly different between groups. Scale bars: 5 μm. Data are expressed as mean ± SEM. Differences were tested by 2-way ANOVA with Bonferroni’s multiple-comparisons test (B and C) or 1-way ANOVA with Tukey’s multiple-compari- sons test (D). * P < 0.05 (n = 9 myofibers from n = 3 mice per condition).

Article Snippet: Subcloning of annexin A1, A2, and A6 to replace the GFP tag with tdTomato (Addgene) was performed by Mutagenix.

Techniques: Expressing, Labeling, Imaging, Fluorescence, Membrane

Journal: Journal of Clinical Investigation

Article Title: Recombinant annexin A6 promotes membrane repair and protects against muscle injury

doi: 10.1172/jci128840

Figure Lengend Snippet: Figure 4. Annexin A6 Ca2+-binding mutant reduced annexin repair cap recruitment and decreased myofiber membrane repair capacity. (A) Myofibers were coelectroporated with wild-type-tdTomato (red labels) and either wild-type-GFP or mutant-GFP (green labels) annexin constructs, and cap size was assessed after membrane damage; only the red channel is shown to demonstrate the effect on wild-type annexin. (B) Coexpression of mutant annexin A6E233A was sufficient to reduce wild-type annexin A6 cap assembly. Cap kinetics were plotted as cap Feret diameter over a range of Ca2+ concentrations, from 0–2 mM. (C) Coexpression of annexin A6E233A was sufficient to significantly reduce the cap area of coexpressed annexin A1, A2, and A6. *P < 0.05 for WT + WT vs. WT + mutant. (D) Myofibers were electroporated with annexin A6–GFP or mutant A6E233A– GFP. Annexin A6E233A cap area (small arrow) was significantly smaller compared with annexin A6 (large arrowhead), correlating with increased FM 4-64 fluorescence area (large arrowhead). Scale bars: 5 μm. Data are expressed as mean ± SEM. Differences were assessed by 2-tailed t test (A, C, and D). *P < 0.05 (n = 4–18 myofibers from n = 3 mice per condition).

Article Snippet: Subcloning of annexin A1, A2, and A6 to replace the GFP tag with tdTomato (Addgene) was performed by Mutagenix.

Techniques: Binding Assay, Mutagenesis, Membrane, Construct, Fluorescence

Journal: Environmental Pollution (Barking, Essex : 1987)

Article Title: Molecular mechanisms underlying NLRP3 inflammasome activation and IL-1β production in air pollution fine particulate matter (PM 2.5 )-primed macrophages ☆

doi: 10.1016/j.envpol.2023.122997

Figure Lengend Snippet: ROFA induces inflammasome priming, ASC-specks formation, and IL-1β secretion in human monocytes and macrophages. (A) Representative dot plots of inflammasome-reporter THP-1-ASC-GFP cells incubated with different PM 2.5 surrogates at 100 μg/mL for 6 or 24 h. (B) Quantification of ASC-GFP fluorescence indicative of inflammasome priming and specks formation in THP-1-ASC-GFP cells incubated with increasing PM 2.5 concentrations for 6 or 24 h. (C) IL-1β levels in cell culture supernatants from THP-1-ASC-GFP cells after PM 2.5 incubation at 100 μg/mL for 6 or 24 h. (D) IL-1β release in cell culture supernatants from monocyte-derived macrophages obtained from differentiated PBMCs from healthy donors and incubated with ROFA at 100 μg/mL for 24 h. Data are presented as mean ± SEM from at least three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus RPMI (priming). § p < 0.05 and §§§ p < 0.001 versus RPMI (specks).

Article Snippet: THP-1-ASC-GFP cells and ASC-Citrine BMDMs were acquired in a FACSCanto II equipment (BD Biosciences, Franklin Lakes, NJ, US).

Techniques: Incubation, Fluorescence, Cell Culture, Derivative Assay