Journal: bioRxiv

Article Title: Inhomogeneous Tau polymerization, core–shell organization, and seed formation during Tau condensate aging

doi: 10.64898/2026.03.18.711671

Figure Lengend Snippet: (A) Schematic of seeding Tau aggregation in Tau biosensor (HEK293 expressing TauRD P301S -CFP) cells by aged Tau/RNA condensates. (B) Example images of Tau biosensor cells seeded, or not, with 24 h-old Tau/RNA condensates. Scale bars = 50 μm. (C) High-resolution imaging and 3D-reconstruction of TauRD P301S -CFP in condensate seeded Tau biosensor cells, with counterstaining of the nuclear envelope by Lamin B1 immunostaining shows subcellular positioning of seeded Tau species: Many small Tau foci form in the cytosol and some at the nuclear envelope, larger cytoplasmic Tau aggregates are positioned close to the nucleus, and some Tau clusters also form in the nucleus. Scale bars = 2 μm. (D) Confocal time course imaging of Tau biosensor cells upon seeding with 24 h-old Tau/RNA condensates. Images show sequential formation of Tau accumulation in the same cell: first, cytosolic Tau foci (CLUS) form, followed by Tau foci at the nuclear envelope (NE), larger cytoplasmic Tau aggregates (CYT) close to the nucleus, and, finally, intranuclear circular Tau aggregates (NUC) can be observed. (E) Quantification Tau accumulation types from time course imaging experiments. For analysis, cytoplasmic CLUS and CYT were combined. n=21 analyzed time course series (z-stack), data shown as mean±SEM, one-way ANOVA with Tukey post-test for percentage at 21 h for each accumulation class. (F) STED microcopy of seeded Tau biosensor cells, counter stained with SiR-tubulin (left panel) or immunostained for Lamin B1 (right panel), showing different Tau accumulation types. Zoom-ins show elongated cytosolic Tau structures adjacent to microtubules (left) and Tau foci at the outer nuclear envelope (right). Position of nuclei are indicated by white stars, inner nuclear envelope-nucleoplasm border is indicated by white, dashed lines. Scale bars = 5 μm in overview and 1 μm in zoom-ins. (G) Principle of CFP lifetime FLIM in Tau biosensor cells expressing TauRD P301S -CFP or TauRD P301S -CFP and TauRD P301S -YFP (TauRD P301S -CFP/YFP). CFP lifetime is quenched by molecular crowding in TauRD P301S -CFP accumulations and by both molecular crowding and Tau-Tau interactions in TauRD P301S -CFP/YFP accumulations. (H) Example images of seeded Tau biosensor cells (top: TauRD P301S -CFP cells; bottom: TauRD P301S -CFP/YFP cells). CFP intensity is shown, as well as CFP lifetime components, fit-free defined based on ROIs in phasor plots), superimposed on CFP intensity. Lifetime components could be defined for free soluble Tau (LT SOL , pink), Tau foci in cytosol (CLUS) and at the nuclear envelope (NE; LT CLUS+NE ), cytosolic (CYT) and nuclear (NUC; LT CYT+NUC ) Tau aggregates, and amyloid-like cytosolic Tau aggregates (AMY; LT AMY ). Scale bars = 5 μm. (I) Lifetimes of Tau accumulation types in TauRD P301S -CFP and TauRD P301S -CFP/YFP accumulations. Data shown as mean±SD, comparison of Tau accumulation types within cell type: one-way ANOVA with Tukey post-test. (J) FRET contribution to CFP lifetime quenching in seeded TauRD P301S -CFP/YFP cells, estimated by subtracting lifetimes of Tau accumulation types measured in TauRD P301S -CFP/YFP cells from that measured in TauRD P301S -CFP cells. % values give the proportion of plotted values to entire CFP lifetime quenching in TauRD P301S -CFP/YFP cells. Data shown as mean±SD. (K) Examples of ODT overlaid with correlative fluorescent image of seeded and unseeded TauRD P301S -CFP/YFP cells. (L) Quantification of densities (mg/ml) determined from RI tomograms for Tau accumulations (CYT, NUC) and subcellular compartments (cytoplasm, nucleoplasm, nuclear envelope, and nucleolus). Note, nuclear envelope density in seeded Tau biosensor cells was determined as proxy for Tau foci at the nuclear envelope. n = 15-66 measurements, box plot shows full data range (Min to Max) with all data points, line indicates median, cross indicates mean. Comparison within aggregate type and subcellular compartments: one-way ANOVA with Tukey post-test, or Student T-test for nuclear envelope.

Article Snippet: HEK293 cells stably expressing the Tau repeat domain (TauRD) containing the frontotemporal dementia (FTD)-mutation P301S and fused to CFP or YFP (Tau biosensor cells; TauRD P301S -CFP/YFP); ATCC #CRL-3275; cells provided by Marc Diamond through Erich Wanker) were grown in 8-well imaging dishes (Ibidi).

Techniques: Expressing, Imaging, Immunostaining, Staining, Comparison

Journal: iScience

Article Title: Robust calibration and quantification of FRET signals using multiplexed biosensor barcoding

doi: 10.1016/j.isci.2025.113743

Figure Lengend Snippet: Construction of FRET calibration standards (A) Construction of FRET-ON calibration standard by Y349F mutation of Cyto-FAK. (B) Plot of YFP/CFP ratio of CytoFAK and FRET-ON (mean ± SEM, n = 20 cells) in response to 100 ng/mL EGF added at 6 min. (C) Plot of YFP/CFP ratio of CytoFAK and FRET-ON (mean ± SEM, n = 20 cells) in response to FAK inhibitor VS-6063 over 24 h. (D) Construction of FRET-OFF calibration standard by removal of the H3 domain of the H3K9me9 (W45A) biosensor. (E) Plot of YFP/CFP ratio of FRET-OFF and H3K9me3 biosensor (mean ± SEM, n = 20 cells) in response to 5 μM TCP over 48 h.

Article Snippet: H3K9me3 (W45A) biosensor , Addgene , Cat#120808.

Techniques: Mutagenesis

Journal: iScience

Article Title: Robust calibration and quantification of FRET signals using multiplexed biosensor barcoding

doi: 10.1016/j.isci.2025.113743

Figure Lengend Snippet: Construction of FRET calibration standards (A) Construction of FRET-ON calibration standard by Y349F mutation of Cyto-FAK. (B) Plot of YFP/CFP ratio of CytoFAK and FRET-ON (mean ± SEM, n = 20 cells) in response to 100 ng/mL EGF added at 6 min. (C) Plot of YFP/CFP ratio of CytoFAK and FRET-ON (mean ± SEM, n = 20 cells) in response to FAK inhibitor VS-6063 over 24 h. (D) Construction of FRET-OFF calibration standard by removal of the H3 domain of the H3K9me9 (W45A) biosensor. (E) Plot of YFP/CFP ratio of FRET-OFF and H3K9me3 biosensor (mean ± SEM, n = 20 cells) in response to 5 μM TCP over 48 h.

Article Snippet: H3K9me3 biosensor , Addgene , Cat#120802.

Techniques: Mutagenesis

Journal: Cell death & disease

Article Title: RhoA balances microglial reactivity and survival during neuroinflammation.

doi: 10.1038/s41419-023-06217-w

Figure Lengend Snippet: Fig. 5 Inflammation disrupts Ca2+, pH, and mitochondrial homeostasis in RhoA-deficient microglia. A Lifeact fluorescence labeling in RhoA WT, RhoA T19N, or RhoA KO HMC3 microglia (n = 3 independent experiments). B Cell counting in RhoA WT, RhoA Q63L, RhoA T19N, or RhoA KO HMC3 microglia (n = 8 independent experiments). Graph displays mean with SD. ****p < 0.0001 (Two-way ANOVA). RhoA WT, RhoA T19N, or RhoA KO HMC3 microglia expressing a global Ca2+ biosensor (C), mitochondrial Ca2+ biosensor (D), endoplasmic reticulum Ca2+ biosensor (E), pH biosensor (F), MitoTimer biosensor (G), or mitophagy biosensor (H) and exposed to LPS (1 µg/ml; 20 min (C-F) or 60 min (G and H)). Graphs (mean and SD) display fluorescence changes (n = 15 cells per group from 3 independent experiments for each biosensor). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). Scale bars: 20 µm.

Article Snippet: Raichu-RhoA (provided by M. Matsuda [27]), pTriEx-RhoA FLARE.sc Biosensor WT (RRID:Addgene_12150), pTriEx-RhoA FLARE.sc Biosensor Q63L (RRID:Addgene_12151), pTriEx-RhoA FLARE.sc Biosensor T19N (RRID:Addgene_12152), pRK5-myc-RhoA WT (RRID:Addgene_12962), pRK5-mycRhoA Q63L (RRID:Addgene_12964), pRK5-myc-RhoA T19N (RRID:Addgene_12963), EGFP-p65 (RRID:Addgene_111190), GW1-pHRed (RRID:Addgene_31473), GW1CMV-Perceval (RRID:Addgene_21737), Laconic/pcDNA3.1 (+) (RRID:Addgene_118627), Pyronic /pcDNA3.1 (+) (RRID:Addgene_51308), pcDNA3.1 FLII12Pglu-700uDelta6 (RRID:Addgene_17866), Cyto-ABKAR (RRID:Addgene_61510), pLentiEKAR2G2 (RRID:Addgene_40178), Kras-Src FRET biosensor (RRID:Addgene_78302), pFRET-HSP33 cys (RRID:Addgene_16076), pGP-CMV-GCaMP6F (RRID:Addgene_40755), mCherry-Lifeact-7 (RRID:Addgene_54491), pMitoTimer (RRID:Addgene_52659), pCLBW cox8 EGFP mCherry (RRID:Addgene_78520).

Techniques: Labeling, Cell Counting, Expressing

Journal: Cell death & disease

Article Title: RhoA balances microglial reactivity and survival during neuroinflammation.

doi: 10.1038/s41419-023-06217-w

Figure Lengend Snippet: Fig. 2 RhoA regulates microglial metabolic reprogramming during inflammation. HMC3 microglia expressing the ATP biosensor (A), Glucose biosensor (B), Lactate biosensor (C), or Pyruvate biosensor (D) were transfected with RhoA Q63L (red) or RhoA WT (blue) and exposed to LPS (1 µg/ml; 20 min) (n = 15-30 cells per group from 3 independent experiments for each biosensor). Primary cortical microglia expressing the ATP biosensor (E) or Lactate biosensor (F) were transfected with the RhoA Q63L (red) or RhoA WT (blue) and exposed to LPS (1 µg/ml; 20 min) (n = 6 cells per group from 3 independent experiments for each biosensor). Panels are time-lapse ratio images coded according to the pseudocolor ramps. Graphs (means and SD) display F490/F435 (A and E), FRET/Donor (B), and Donor/FRET (C, D, and F) ratio change at 0 (CT) and 20 min. G Seahorse measurements of bioenergetic parameters in HCM3 microglia expressing RhoA Q63L or RhoA WT. The parameters were calculated based on the OCR following the sequential addition of LPS, oligomycin, FCCP, rotenone, and antimycin A. Results are from at least 3 independent experiments. Graphs show the mean with SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). Scale bars: 20 µm.

Article Snippet: Raichu-RhoA (provided by M. Matsuda [27]), pTriEx-RhoA FLARE.sc Biosensor WT (RRID:Addgene_12150), pTriEx-RhoA FLARE.sc Biosensor Q63L (RRID:Addgene_12151), pTriEx-RhoA FLARE.sc Biosensor T19N (RRID:Addgene_12152), pRK5-myc-RhoA WT (RRID:Addgene_12962), pRK5-mycRhoA Q63L (RRID:Addgene_12964), pRK5-myc-RhoA T19N (RRID:Addgene_12963), EGFP-p65 (RRID:Addgene_111190), GW1-pHRed (RRID:Addgene_31473), GW1CMV-Perceval (RRID:Addgene_21737), Laconic/pcDNA3.1 (+) (RRID:Addgene_118627), Pyronic /pcDNA3.1 (+) (RRID:Addgene_51308), pcDNA3.1 FLII12Pglu-700uDelta6 (RRID:Addgene_17866), Cyto-ABKAR (RRID:Addgene_61510), pLentiEKAR2G2 (RRID:Addgene_40178), Kras-Src FRET biosensor (RRID:Addgene_78302), pFRET-HSP33 cys (RRID:Addgene_16076), pGP-CMV-GCaMP6F (RRID:Addgene_40755), mCherry-Lifeact-7 (RRID:Addgene_54491), pMitoTimer (RRID:Addgene_52659), pCLBW cox8 EGFP mCherry (RRID:Addgene_78520).

Techniques: Expressing, Transfection

Journal: Cell death & disease

Article Title: RhoA balances microglial reactivity and survival during neuroinflammation.

doi: 10.1038/s41419-023-06217-w

Figure Lengend Snippet: Fig. 3 RhoA regulates microglial proinflammatory reactivity. HMC3 microglia expressing a ROS biosensor (A), AMPK biosensor (B), ERK biosensor (C), Src biosensor (D), and GFP-tagged p65 NFkB subunit (E) were transfected with the RhoA Q63L construct or with RhoA WT and then exposed to LPS (1 µg/ml for 20 min) (n = 18–100 cells per group from 3 independent experiments for each biosensor). Panels show time- lapse ratio images or mean fluorescent intensity (MFI) coded according to the pseudocolor ramps. F ELISA (TNF-α or IL-1ß) from culture supernatants of primary cortical microglia transfected with RhoA Q63L or RhoA WT and exposed to LPS (1 µg/ml) for 3 h (n = 4 independent experiments). Graphs are means with SD. *p < 0.05, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). Scale bars: 20 µm.

Article Snippet: Raichu-RhoA (provided by M. Matsuda [27]), pTriEx-RhoA FLARE.sc Biosensor WT (RRID:Addgene_12150), pTriEx-RhoA FLARE.sc Biosensor Q63L (RRID:Addgene_12151), pTriEx-RhoA FLARE.sc Biosensor T19N (RRID:Addgene_12152), pRK5-myc-RhoA WT (RRID:Addgene_12962), pRK5-mycRhoA Q63L (RRID:Addgene_12964), pRK5-myc-RhoA T19N (RRID:Addgene_12963), EGFP-p65 (RRID:Addgene_111190), GW1-pHRed (RRID:Addgene_31473), GW1CMV-Perceval (RRID:Addgene_21737), Laconic/pcDNA3.1 (+) (RRID:Addgene_118627), Pyronic /pcDNA3.1 (+) (RRID:Addgene_51308), pcDNA3.1 FLII12Pglu-700uDelta6 (RRID:Addgene_17866), Cyto-ABKAR (RRID:Addgene_61510), pLentiEKAR2G2 (RRID:Addgene_40178), Kras-Src FRET biosensor (RRID:Addgene_78302), pFRET-HSP33 cys (RRID:Addgene_16076), pGP-CMV-GCaMP6F (RRID:Addgene_40755), mCherry-Lifeact-7 (RRID:Addgene_54491), pMitoTimer (RRID:Addgene_52659), pCLBW cox8 EGFP mCherry (RRID:Addgene_78520).

Techniques: Expressing, Transfection, Construct, Enzyme-linked Immunosorbent Assay

Journal: Cell death & disease

Article Title: RhoA balances microglial reactivity and survival during neuroinflammation.

doi: 10.1038/s41419-023-06217-w

Figure Lengend Snippet: Fig. 5 Inflammation disrupts Ca2+, pH, and mitochondrial homeostasis in RhoA-deficient microglia. A Lifeact fluorescence labeling in RhoA WT, RhoA T19N, or RhoA KO HMC3 microglia (n = 3 independent experiments). B Cell counting in RhoA WT, RhoA Q63L, RhoA T19N, or RhoA KO HMC3 microglia (n = 8 independent experiments). Graph displays mean with SD. ****p < 0.0001 (Two-way ANOVA). RhoA WT, RhoA T19N, or RhoA KO HMC3 microglia expressing a global Ca2+ biosensor (C), mitochondrial Ca2+ biosensor (D), endoplasmic reticulum Ca2+ biosensor (E), pH biosensor (F), MitoTimer biosensor (G), or mitophagy biosensor (H) and exposed to LPS (1 µg/ml; 20 min (C-F) or 60 min (G and H)). Graphs (mean and SD) display fluorescence changes (n = 15 cells per group from 3 independent experiments for each biosensor). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). Scale bars: 20 µm.

Article Snippet: Raichu-RhoA (provided by M. Matsuda [27]), pTriEx-RhoA FLARE.sc Biosensor WT (RRID:Addgene_12150), pTriEx-RhoA FLARE.sc Biosensor Q63L (RRID:Addgene_12151), pTriEx-RhoA FLARE.sc Biosensor T19N (RRID:Addgene_12152), pRK5-myc-RhoA WT (RRID:Addgene_12962), pRK5-mycRhoA Q63L (RRID:Addgene_12964), pRK5-myc-RhoA T19N (RRID:Addgene_12963), EGFP-p65 (RRID:Addgene_111190), GW1-pHRed (RRID:Addgene_31473), GW1CMV-Perceval (RRID:Addgene_21737), Laconic/pcDNA3.1 (+) (RRID:Addgene_118627), Pyronic /pcDNA3.1 (+) (RRID:Addgene_51308), pcDNA3.1 FLII12Pglu-700uDelta6 (RRID:Addgene_17866), Cyto-ABKAR (RRID:Addgene_61510), pLentiEKAR2G2 (RRID:Addgene_40178), Kras-Src FRET biosensor (RRID:Addgene_78302), pFRET-HSP33 cys (RRID:Addgene_16076), pGP-CMV-GCaMP6F (RRID:Addgene_40755), mCherry-Lifeact-7 (RRID:Addgene_54491), pMitoTimer (RRID:Addgene_52659), pCLBW cox8 EGFP mCherry (RRID:Addgene_78520).

Techniques: Labeling, Cell Counting, Expressing