Fig 1: MARK4 deficiency affects MTOC and speck nucleation.(a) Differentiated THP-1 cells were stimulated with nigericin (10 µ? for 1.5 h) or left untreated (control). Endogeneous levels of NLRP3 and MARK4 were co-stained with ?-tubulin. (b,c) HEK293T cells were co-overexpressed with GFP-MARK4, Cherry-NLRP3 and ASC-Flag (indicated as MARK4 GFP o.e.); or co-overexpressed with MARK4 shRNA (shown by green GFP), Cherry-NLRP3 and ASC-Flag (indicated as MARK4 shRNA). co-overexpression with MARK4-GFP drove NLRP3 to MTOC, and knock down of MARK4 by shRNA (indicated by GFP) led to a dilated ring structure of NLRP3. Quantification of speck size was shown. Scale bar, 40 µm. Mean±s.e.m. for all the cells taken from five different views at × 20 magnification for each group. Comparisons of the two different groups were analysed by unpaired t-test. ****P<0.0001 was considered as statistically significant (b). Mitochondria distribution was shown after speck formation (c). See also Supplementary Movie 6. (d) In differentiated THP-1 cells, upon nigericin stimulation (10 µ? for 1.5 h), NLRP3 was translocated to MTOC, indicated by ?-tubulin. NLRP3 remained in cytoplasm distribution in MARK4 shRNA cell. Experiments were repeated at least three times. Scale bar, 10 µm.
Fig 2: Loss of MARK4 affects IL-1ß production under NLRP3 inflammasome activation.(a) Mark4 expression under unprimed and LPS primed conditions in mouse bone marrow-derived macrophages (BMDM). Cytoplasm proximity ligation assay (PLA) signal per cell cytoplasm is used for quantification. Mean±s.e.m. for all the cells taken from 5 to 8 different views at × 40 magnification for each group. Experiments have been repeated three times. Scale bar, 10 µm. (b) siRNA of MARK4, NLRP3 or ASC caused reduction of ATP induced IL-1ß production in THP-1 cells. (c) shRNA of MARK4 caused reduction of IL-1ß induced by NLRP3 stimuli, including monosodium urate (MSU) and cholesterol crystals (CC) in THP-1 cells. Mock represents macrophages without further stimulation. (d) BMDM derived from Mark4 KO mice exhibited selectivity towards NLRP3 activating stimuli. Mean±s.e.m., three to four independent experiments combined (b–d), comparisons of the two different groups were analysed by unpaired t test. NS was considered as not statistically significant. *P<0.05, **P<0.01 and ***P<0.001 were considered as statistically significant (a–d). (e) Representative western blots of caspase-1 (Casp1) and IL-1ß in the supernatant (Sup.) in Mark4+/+ and Mark4-/- macrophages activated with indicated stimuli. Cell lysates were used as controls to indicate equal amount of cells for analysis.
Fig 3: Mapping of interacting domains between MARK4 and NLRP3.(a) Western blots of co-immunoprecipitated full-length NLRP3 with truncated MARK4 (KD: kinase domain; CD: catalytic domain; UBA: Ubiquitin-associated domain), respectively. (b) Western blots of coimmunoprecipitated truncated NLRP3 (Pyrin Del: pyrin domain deletion; Pyrin only: pyrin domain only; NACHT: Nucleotide-binding oligomerization domain; LRR: Leucine-rich repeat) with full-length MARK4, or co-immunopreciptated MARK4 with truncated NLRP3, respectively. (c) Schematic diagram showing that MARK4 catalytic kinase domain and NLRP3 Pyrin &NACHT domain were essential for their interaction. (d) Western blots of co-immunoprecipitated NLRP3 point mutants with MARK4 respectively. Whole cell lysates were analysed as indication of transfection. (e) Purified recombinant NLRP3 (1-291aa) immobilized on the glutathione sepharose can pull down purified recombinant full-length MARK4 directly. Western blots are representatives of three independent experiments.
Fig 4: MARK4 expression, a-tubulin post-translational modifications, and changes in the inflammatory response post-myocardial infarction. a, Representative confocal images of primary cardiomyocytes (CMs) isolated from Mark4 -/- or control mice at baseline (BL) or at day 3 post-MI (MI), scale bar= 20 µm. b-c, Levels of pro-inflammatory cytokines at day 3 post-MI (n=6 per group) (b). Left ventricular ejection fraction (LVEF) at day 3 post-MI (n=4 per group) (c). d-e, Western blots (WBs) of detyrosinated a-tubulin (dTyr-tub) in cell lysates of CMs isolated from wild-type mice at day 3 post-MI or post-sham surgery (S), with the lysates of the remaining cells from the same hearts used as control. Representative WBs (d). Ratio of dTyr-tubulin over total a-tubulin quantified using western blot data from biologically independent samples (S group: n=4 mice; MI group: n=5 mice) (e). f-g, Western blots of cell lysates from the isolated cardiomyocytes of Mark4 -/- or control mice at day 3 post-MI, to detect detyrosinated a-tubulin (dTyr-tub), polyglutamylated a-tubulin (Polyglu-tub), acetylated a-tubulin (Ace-tub), and a–tubulin (a-tub). Representative images (f). Ratio of dTyr-tub, or polyglu-tub, or ace-tub over total a-tubulin quantified using western blot data from biologically independent samples (n=3 mice per group) (g). The box bounds represent the 25th and 75th percentiles, the middle line shows the median, and the whiskers show the minimum and maximum (b). Mean±s.e.m.; two-tailed unpaired t-test (c, e, g). P values are indicated on the graphs.
Fig 5: VASH2 status in cardiomyocytes pre- and post-myocardial infarction, and the schematic summary of the results. a-b, STED images of VASH2 and a-tubulin (a-tub) in wild-type (WT) cardiomyocytes (CMs) at baseline (BL) or post-myocardial infarction (MI). Representative images, scale bar=2 µm (a). Pearson Correlation Coefficient (PCC) of VASH2 and a-tub signals, percentage (%) of VASH2 signals on the polymerized microtubules (MTs), and percentage of VASH2 signals off the MTs, in the following groups (b): WT BL (n=4 mice / n=38 CMs examined over 2 independent experiments), and WT MI (n=38 CMs of n=6 mice / n=38 CMs examined over 3 independent experiments). c, Real-time PCR on post-MI CMs, from the following groups: Mark4 +/+ MI (n= 5 mice), and Mark4 -/- MI (n=6 mice). d, Quantification of VASH2 mean fluorescence intensity (MFI) within cell area (region of interest, ROI) using the STED images from the following groups: Mark4 +/+ MI (n=6 mice / n= 38 CMs examined over 3 independent experiments), and Mark4 -/- MI (n= 6 mice/ n= 47 CMs examined over 3 independent experiments). Mean ± s.e.m.; two-tailed unpaired t-test (b, c, d). P values are indicated on the graphs. e, A working model for MARK4-dependent regulation of microtubule detyrosination after MI: Upon ischaemic injury, increased MARK4 phosphorylates MAP4 at its KXGS motifs. Phosphorylated MAP4 either changes its conformation on the polymerized microtubules, or detaches itself from the polymerized microtubules to form oligomerized MAP4 structures in the cytosol. The phosphorylation of MAP4 by MARK4 allows for space access of VASH2 to the polymerized microtubules, thereby promoting a-tubulin detyrosination. As a consequence, the increased level of detyrosinated microtubules causes a reduction in contractile function of the cardiomyocyte.
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