Journal: Life Science Alliance

Article Title: Tyrosine phosphorylation of lamin A by Src promotes disassembly of nuclear lamina in interphase

doi: 10.26508/lsa.202101120

Figure Lengend Snippet: (A) MCF7 and MDA-MB-231 cells were treated with (+) or without (−) 50 nM dasatinib for 1 h and then lysed. Equal amounts of the whole cell lysates (WCLs) were incubated with anti-lamin A/C or pre-immune serum (IgG) as the control. The immunonocomplexes were analyzed by immunoblotting (IB) with anti-PY or anti-lamin A/C antibodies. An equal amount of WCLs was analyzed by immunoblotting with anti-Src, anti-Src pY416, or anti-actin. The tyrosine phosphorylation of lamin A was quantified and expressed as -fold relative to the level of MCF7 without dasatinib. (B) MCF7 and MDA-MB-231 cells were treated with (+) or without (−) 50 nM dasatinib for 1 h. The cells were fixed and stained for lamin A, lamin B1, and DNA. Representative images are shown. Scale bars, 10 μm. The nuclear circularity (4π × area/perimeter 2 ) was determined. The P -values were calculated from at least 150 cells pooled from three independent experiments. The percentage of the cells with nuclear lobulation was measured (n ≥ 400). The values (mean ± SD) are from three experiments. *** P < 0.001. (C) MDA-MB-231 cells were infected with lentivirues capable of expressing FLAG-lamin A or the mutants (Y45F and Y45D) and selected in the medium with neomycin (neo). An equal amount of WCLs was analyzed by immunoblotting (IB) with antibodies as indicated. (D) The cells as described in panel C were fixed and stained for FLAG-lamin A, lamin B1, and DNA. Representative images are shown. Scale bars, 10 μm. The percentage of the cells with nuclear lobulation was measured (n ≥ 900). Values (means ± SD) are from three experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are available for this figure.

Article Snippet: The rabbit polyclonal anti-Src pY416 (MAB2685) antibody was purchased from R&D Systems.

Techniques: Incubation, Control, Western Blot, Phospho-proteomics, Staining, Infection, Expressing

Journal: Nature Communications

Article Title: Cell metabolism regulates integrin mechanosensing via an SLC3A2-dependent sphingolipid biosynthesis pathway

doi: 10.1038/s41467-018-07268-w

Figure Lengend Snippet: Depletion of DES2 recapitulates most of defects observed upon depletion of CD98hc. a Depletion of DES2 by siRNA impairs RhoA activation by mechanical force application on integrins. Right panel, quantification of RhoA activation, means are plotted with s.e.m. as error bars from n = 2 experiments, * P < 0.05 in a two-way ANOVA. b Depletion of DES2 by shRNA impairs SFK phosphorylation at Y416. One experiment representative of n = 2. c Depletion of DES2 impairs mechanically induced GEF-H1 activation. One experiment representative of n = 2. d Depletion of DES2 triggers GEF-H1 sequestration in cell membrane. s supernatant, p membrane pellet. One experiment representative of n = 2. e DES2 depletion partially inhibits mechanically coupled YAP/Taz activation as measured by RT-qPCR of ANKRD1 transcript. Means are plotted with s.e.m. as error bars from n = 6 with * P < 0.05 and **** P < 0.0001 in a two-way ANOVA. f Depletion of DES2 impairs generation of traction forces. Traction force microscopy was performed on subconfluent cells. Scale bar is 50 µm

Article Snippet: The rabbit monoclonal anti-GEF-H1 55B6 (1/500), anti-Src 36D10 (1/500), anti-Grp94 D6X2Q (1/500), anti-Grp78/BiP C50B12 (1/1000) and anti-Vimentin D21H3 (1/500) antibodies, and the rabbit polyclonal anti-phospho Y416 Src (1/500) and anti-flotillin-1 (1/1000) (#3253) antibodies were from Cell Signaling Technology.

Techniques: Activation Assay, shRNA, Phospho-proteomics, Membrane, Quantitative RT-PCR, Microscopy

Journal: Nature Communications

Article Title: Cell metabolism regulates integrin mechanosensing via an SLC3A2-dependent sphingolipid biosynthesis pathway

doi: 10.1038/s41467-018-07268-w

Figure Lengend Snippet: Loss of CD98hc impairs GEF-H1and Src family kinase activation, and integrin dynamics. a Loss of CD98hc impairs LARG and GEF-H1 activation by mechanical force application on integrins. One experiment representative of n = 2. b Loss of CD98hc impairs Src activity but does not affect ERK1/2 phosphorylation by mechanical force application on integrins. Src activity was monitored by tracking substrate FAK Y576 phosphorylation and SFK phosphorylation at Y416. c Loss of CD98hc induces constitutive recruitment of GEF-H1 to the membrane. s supernatant, p membrane pellet. One experiment representative of n = 3. d Control or CD98hc-null dermal fibroblasts were stimulated with FN-coated magnetic beads. Cells were lysed and lysates were fractionated into cytosolic (s) and membrane (p) fractions by centrifugation. Active RhoGEFs was pulled-down from each fraction with GST-RhoA17A beads. One experiment representative of n = 2. e , f Diffusion rate ( t- half) of integrin α5 and β3 as calculated from the curve fit generated from the FRAP measurements performed on n = 27, 41, and 67; and 73, 36, and 57 adhesions, respectively, for control, CD98hc-null and C330S cells on integrin α5 and β3. Error bars are 95% CI calculated from that fit. g , h Integrin α5 and β3 mobile fraction as calculated from the curve fit generated from the FRAP measurements. Error bars are 95 CI calculated from that fit. i Trafficking of integrin β1 in control or CD98hc-null cells. Integrin β1 was labeled with Alexa 488 coupled antibody then integrin trafficking was chased for indicated time. Extracellular staining was quenched and only intracellular labeled integrin is observed. Scale bar is 50 µm

Article Snippet: The rabbit monoclonal anti-GEF-H1 55B6 (1/500), anti-Src 36D10 (1/500), anti-Grp94 D6X2Q (1/500), anti-Grp78/BiP C50B12 (1/1000) and anti-Vimentin D21H3 (1/500) antibodies, and the rabbit polyclonal anti-phospho Y416 Src (1/500) and anti-flotillin-1 (1/1000) (#3253) antibodies were from Cell Signaling Technology.

Techniques: Activation Assay, Activity Assay, Phospho-proteomics, Membrane, Control, Magnetic Beads, Centrifugation, Diffusion-based Assay, Generated, Labeling, Staining

Journal: The Journal of Experimental Medicine

Article Title: Fyn kinase regulates misfolded α-synuclein uptake and NLRP3 inflammasome activation in microglia

doi: 10.1084/jem.20182191

Figure Lengend Snippet: Fyn and CD36 contribute to aggregated αSyn uptake into microglia . (A) Immunoblot analysis of aggregated αSyn-treated WT and Fyn −/− microglial lysates reveals a rapid induction of SFK activity in WT, but not Fyn −/− , microglia. Error bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 3). Con, control. (B) ICC reveals rapid increase in p-Y416 SFK levels in αSyn-treated Iba-1–positive WT microglial cells. Scale bars, 15 µm. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 4). (C) IHC analysis to show microglial Fyn activation in the AAV-SYN model at 30 d. Schematic of AAV injection on the side. Scale bars, 15 µm. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 4 mice per group). (D) Upon its application to microglial cells, αSyn associates with TLR2 and CD36, as evidenced by coIP analysis. Upon αSyn treatment, Fyn associates with CD36, but not TLR2. Error bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 3). IP, immunoprecipitation. (E) Immunoprecipitation of FLAG-tagged human CD36 in αSyn-treated HEK cells shows a transient interaction of CD36 and αSyn. Error bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 3). IB, immunoblotting. (F) Immunoblot for p-Y416 SFK reveals that Fyn activation occurs downstream of CD36. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 3). (G) Immunoblot analysis showing diminished αSyn uptake by CD36 −/− BMDMs. Error bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 3). (H) Immunoblot analysis also reveals that reduced αSyn was taken up by Fyn −/− microglia. Error bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 5). (I and J) ICC for human αSyn revealed diminished uptake of the protein in Fyn-deficient microglia. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 5). Scale bars, 15 µm. (K) Whole-cell lysate analysis from αSyn-treated, WT-transfected, and activation loop–deficient (Y417A) Fyn-FLAG–transfected cells showed diminished αSyn uptake in inactive Fyn mutant–expressing cells. Error bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 3). Asterisks indicate the level of statistical significance: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Article Snippet: Antibodies against the rabbit p-Y416 SFK and native p65 were purchased from Cell Signaling.

Techniques: Western Blot, Activity Assay, Control, Two Tailed Test, Activation Assay, Injection, Immunoprecipitation, Transfection, Mutagenesis, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Fyn kinase regulates misfolded α-synuclein uptake and NLRP3 inflammasome activation in microglia

doi: 10.1084/jem.20182191

Figure Lengend Snippet: Microglial Fyn activation in human PD brains. (A) Significantly increased Fyn expression and activation were observed in AAV-SYN–injected nigral lysates, as assessed by immunoblots. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 4 mice per group). (B) IHC analysis revealed Fyn induction in Iba-1–positive microglia in AAV-SYN–injected nigral brain sections. Scale bars, 30 µm. (C) Significant increase in Fyn expression and activation in PD ventral midbrain lysates, when compared with age-matched control lysates. Representative immunoblot is shown. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 12). (D) IHC analysis of PD brain sections revealed strongly increased p-Y416 SFK expression within Iba-positive microglia when compared with age-matched non-PD brain sections. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 6). Scale bars, 15 µm. (E) IHC shows increased microglial Fyn expression in PD ventral midbrain sections. Scale bars, 15 µm. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 6). Asterisks indicate the level of statistical significance: *, P ≤ 0.05; ***, P ≤ 0.001.

Article Snippet: Antibodies against the rabbit p-Y416 SFK and native p65 were purchased from Cell Signaling.

Techniques: Activation Assay, Expressing, Injection, Western Blot, Two Tailed Test, Control