cytoskeletal reorganisation  (Cytoskeleton Inc)

Journal: Journal of Cell Science

Article Title: Paclitaxel compromises nuclear integrity in interphase through SUN2-mediated cytoskeletal coupling

doi: 10.1242/jcs.264494

Figure Lengend Snippet: Paclitaxel-induced cytoskeletal reorganisation around the nucleus in interphase. (A) Confocal micrographs of cells fixed after 16 h incubation in control medium or 5 nM paclitaxel. DNA was stained using Hoechst 33342 (cyan), and microtubules using α-tubulin immunofluorescence (magenta). MTOCs are marked with arrowheads. Scale bars: 20 μm. (B) STORM imaging of α-tubulin immunofluorescence in cells fixed after 16 h incubation in control medium or 5 nM paclitaxel. Lower panels show α-tubulin clusters generated with HDBSCAN analysis. Different colours distinguish individual α-tubulin clusters, representing individual microtubule filaments or filament bundles. Scale bars: 10 μm. (C) Violin plot comparing the diameter of α-tubulin clusters from B between control (blue) and paclitaxel-treated cells (red). The mean is shown in black with error bars showing the s.e.m. from three biological repeats ( n =3). The mean of each biological repeat is marked with a triangle and consists of >300 measurements from five or more cells. *** P =0.00012 (unpaired two-tailed t -test). (D,E) Confocal micrographs of control and paclitaxel-treated cells stained for DNA using Hoechst 33342 (cyan), microtubules using α-tubulin immunofluorescence (magenta), and either (D) F-actin using Alexa Fluor 488–phalloidin (green) or (E) vimentin using immunofluorescence (green). Scale bars: 20 μm. (F) Left, 2D slice of a reconstructed tomogram of the NE region in a paclitaxel-treated cell. Bundled vimentin filaments (green arrowheads) and microtubules (magenta arrowheads) are seen closely apposed to the NE (cyan arrowheads). Right, segmentation of the NE (cyan), microtubules (magenta) and vimentin filaments (green) from the tomogram. Scale bars: 100 nm. Images in A and D–F are representative of three biological repeats each with >50 cells.

Article Snippet: The extent of the cytoskeletal reorganisation correlated with the concentration of paclitaxel, as microtubules became increasingly bundled and the actin cytoskeleton more condensed with increasing concentrations (0 to 10 nM) of paclitaxel ( ).

Techniques: Incubation, Control, Staining, Immunofluorescence, Imaging, Generated, Two Tailed Test

Journal: Bioactive Materials

Article Title: Geometry-driven immunomodulation in 3D-printed bioceramics: Negative curvature promotes macrophage M2 polarization via Ras-MAPK/HIF-1α signaling for vascularized osteogenesis

doi: 10.1016/j.bioactmat.2026.01.001

Figure Lengend Snippet: (A) Immunofluorescence images of β-Tubulin /CD206 and β-Tubulin /HIF-1α of macrophages in different Gaussian curvature groups before and after treatment with Adezmapimod . (B, C) Protein quantification and Western blotting image of ERK1/2, p-ERK1/2, β-tubulin, HIF-1α and CD206 proteins in all groups before and after treatment with Adezmapimod and Paclitaxel. (D) Mechanism summary of Gaussian curvature-driven macrophage polarization.

Article Snippet: The reagents used in the experiment included: H-DMEM(11965118, Gibco, USA.), α-DMEM medium(12571063, Gibco, USA.), TritonX-100(ST1723, Beyotime, China), 4 % paraformaldehyde (BL539A, Biosharp, China),FBS(A5256701, Gibco, USA.),ECM medium (Science Cell, USA.),and DAPI staining solution (C1006, Beyotime, China),BCIP/NBT(C3206, Beyotime, China), reactive oxygen species kit (S0033S, Beyotime, China), BSA (B2064, ≥98 %, Sigma-Aldrich, USA.),CD31 antibody (ab28364, Abcam, USA.), secondary anti-IGg (ab175773, Alexa Fluor® 680, Abcam, USA.), Phalloidin-iFluor 488(ab176753, Abcam, USA.), CCR7(AF5293, Bioss, China), CD206 (bsm-60761R, Bioss, China), iNOS (bs-22924R, Bioss, China), RIPA (P0013, Beyotime, China), p-ERK1/2 (AF3687, Affinity, USA.) and ERK1/2 (#AF0155, Affinity, USA), luminol detection reagent (sc-2048, Santa Cruz, USA.), GAPDH (Cat#KC-5G5, Kangchen Biotechnology, China), Trizol(15596026CN, Invitrogen, USA.), DEPC(R0601, Thermo Scientific, USA.), TBST(R017R.0000, Thermo Scientific, USA.), HIF-1a(GTX127309, GeneTex, USA.), β-Tubulin(10094-1-AP, Proteintech, UK) Adezmapimod (SB 203580, MCE, USA.) medium and Paclitaxel (99.88 %, HY-B0015R, MCE), ELISA Arg-1(E-EL-M3092, ELabSci@, China), TNF-α (E-EL-M3063, ELabSci@, China), OPN(22952-1-AP, Proteintech, UK.), F4/80 (GB11027-100, Servicebio, China) Alkaline phosphatase activity kit (P0321S, Beyotime, China), Matrigel (CLS356234, Corning, USA), Microfill MV120 (Flow tech, USA), EDTA(17892, Thermo Scientific, USA.) xylene(X112051, AR,99 %, Aladdin, China), ethanol (107-21-1, AR,99 %, Aladdin, China).

Techniques: Immunofluorescence, Western Blot

Journal: bioRxiv

Article Title: N-terminal phosphorylation inhibits Arabidopsis katanin and affects vegetative and reproductive development in opposite ways

doi: 10.64898/2026.01.20.700631

Figure Lengend Snippet: The KTN1 DDD version has decreased microtubule severing activity in vitro . (A) Diagram showing the location of the three experimentally validated serine phosphorylation sites in Arabidopsis KTN1. (B-E) Time course of rhodamine-labeled, taxol-stabilized microtubules incubated with 2mM ATP and 25nM of the indicated p60 katanin versions. See also Supplementary Videos 1-3. (B) KTN1 (n = 6), phosphomimetic and phosphonull mutants of either S92 (n = 5 each), S147 (n = 6 each), or S199 (n = 5 each). (C) KTN1 (n = 6) and double phosphomimetic mutations at S92 and S147 (n = 6), S92 and S199 (n = 6 movies) and S147 and S199 (n = 5). (D) KTN1 (n = 16), triple phosphonull (AAA, n = 15), and triple phosphomimetic (DDD, n = 13) mutant. (E) Extended duration of the DDD time course shown in (D) . Scale bar = 10 µm. ( F, G ) Plots of microtubule fluorescence signal over time from experiments in (C) and (D) . Each image in a series is normalized to the fluorescence signal of the first frame of that series. Error bars represent SEM of at least three separate protein preparations.

Article Snippet: The ATPase activity of 25 nM of KTN1, DDD, or AAA in the presence of either 25nM or 250nM of taxol-stabilized microtubules was evaluated using a commercially available ATPase ELIPA Kit (Cytoskeleton #BK051) according to the manufacturer’s instructions.

Techniques: Activity Assay, In Vitro, Phospho-proteomics, Labeling, Incubation, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: N-terminal phosphorylation inhibits Arabidopsis katanin and affects vegetative and reproductive development in opposite ways

doi: 10.64898/2026.01.20.700631

Figure Lengend Snippet: DDD has lower microtubule binding affinity and ATPase activity in vitro . (A) Representative Coomassie Blue-stained SDS-PAGE gels of microtubule co-sedimentation assays. 1 µM KTN1 (n = 3), DDD (n = 3), or AAA (n = 6) was incubated with the indicated concentration of taxol-stabilized microtubules. S, supernatant; P, pellet. Arrows identify the different protein bands. (B) Binding curves of KTN1, DDD, and AAA proteins corresponding to (A) . Each data point represents the mean ± SEM. Data were fit to a one-site saturation binding model to obtain the microtubule binding affinity and maximum amount of protein binding. (C-E) Plots of ATPase activity over time of 25 nM of KTN1 (C) , DDD (D) , and AAA (E) proteins. ATPase activity was measured as the amount of Pi release in the absence (black) or presence of either 25 nM (blue) or 250 nM microtubules (red). Each data point represents the mean ± SEM from 9 independent experiments.

Article Snippet: The ATPase activity of 25 nM of KTN1, DDD, or AAA in the presence of either 25nM or 250nM of taxol-stabilized microtubules was evaluated using a commercially available ATPase ELIPA Kit (Cytoskeleton #BK051) according to the manufacturer’s instructions.

Techniques: Binding Assay, Activity Assay, In Vitro, Staining, SDS Page, Sedimentation, Incubation, Concentration Assay, Protein Binding