Journal: The Journal of Neuroscience

Article Title: MPP3 Is Required for Maintenance of the Apical Junctional Complex, Neuronal Migration, and Stratification in the Developing Cortex

doi: 10.1523/JNEUROSCI.5627-12.2013

Figure Lengend Snippet: Disruption of adherens junctions in Mpp3 cKO cortex. A–C, p120 catenin levels and apical membrane localization are essentially unaffected in E12.5 Mpp3 cKO cortex. At areas with a loss of p120 catenin, there is cellular disorganization in the ventricular zone (C). B and C are magnification of boxes 1 and 2 in A, respectively. D, E, Likewise, at areas in which Nectin1 is unaffected, cellular organization is indistinguishable from control cortex, but at areas with a loss of Nectin1, a clear disruption of cellular organization was observed (E). F, G, No difference in level and localization of adherens junction protein β-catenin between control (F) and Mpp3 cKO (G) at the level of the third ventricle in which Cre recombinase is not expressed. H, I, Whereas β-catenin displays a punctate pattern at the apical membrane in control cortex (H), in the Mpp3 cKO, there is a loss and delocalization of β-catenin (I). J–O, The same differences were observed between control and Mpp3 cKO for adherens junction proteins p120 catenin (J, K), N-cadherin (L, M), and M-cadherin (N, O). P, Q, Clear loss of Nectin1, which directly interacts with MPP3, during loss of MPP3. R, S, The PAR complex member PAR3 has a punctate distribution in the control cortex (R), but in the Mpp3 cKO cortex, there is a prominent loss of PAR3 from the apical membrane (S). Scale bars, 20 μm.

Article Snippet: Le Bivic, Developmental Biology Institute of Marseille Luminy, Marseille, France), Nectin1 (1:500; MBL), β-catenin (1:100; BD Biosciences), p120 catenin (1:100; BD Biosciences), N-cadherin (1:500; BD Biosciences), M-cadherin (1:500; BD Biosciences), PAR3 (1:200; Millipore), phospho-Histone H3 (pH3; 1:500; Millipore), BrdU (1:250, Abcam), Ki67 (1:100; BD Biosciences), CUX1 (1:200; Santa Cruz Biotechnology), CTIP2 (1:100; Abcam), Calretinin (1:500; Millipore Bioscience Research Reagents), Sox2 (1:300; Millipore Bioscience Research Reagents), Tbr1 (1:200; Abcam), Tbr2 (1:200; Abcam), and Tuj1 (1:200; Covance).

Techniques:

Journal: The Journal of Cell Biology

Article Title: GTSE1 tunes microtubule stability for chromosome alignment and segregation by inhibiting the microtubule depolymerase MCAK

doi: 10.1083/jcb.201606081

Figure Lengend Snippet: Mitotic defects after GTSE1 depletion are dependent on the activity of the MT depolymerase MCAK. (A) MCAK coimmunoprecipitates with GTSE1. Western blot after immunoprecipitation (IP) from U2OS cell lysates using either anti-GTSE1 or anti-GFP antibodies and probing with anti-GTSE1 or anti-MCAK antibodies. (B) Western blot after immunoprecipitation from U2OS NFLAP-GTSE1 cell lysates using either anti-GFP or anti–c-myc antibodies and probing with anti-GFP or anti-MCAK antibodies. (C) Immunofluorescence images of mitotic U2OS cells after control, GTSE1, MCAK, or combined GTSE1 and MCAK RNAi, stained for DNA (DAPI) and MTs (tubulin). (D) Quantification of the mean length of astral MTs in three dimensions from immunofluorescence analysis as shown in C. 10 astral MTs were measured per cell. n = 10 cells per experiment; 3 experiments per condition. (E) Quantification of the percentage of cells lacking astral MTs from immunofluorescence analysis as shown in C. n > 100 cells per experiment; 3 experiments per condition. (F) Quantification of inner spindle tubulin fluorescence intensity from immunofluorescence analysis as shown in C. n ≥ 19 per experiment; 3 experiments per condition. (G) Analysis of spindle orientation. Quantification of the percentage of metaphase cells with a spindle tilt angle of >20° is shown for each RNAi condition. n > 140 cells; 4 experiments per condition. (H) Quantification of the percentage of cells with misaligned chromosomes from immunofluorescence analysis as shown in C. n > 100 cells per experiments; 3 experiments per condition. (I) Immunofluorescence images of mitotic U2OS cells showing MCAK localization after control and GTSE1 RNAi. Cells were stained for MCAK, GTSE1, and MTs (tubulin). (J) Quantification of MCAK intensity on the inner spindle normalized to tubulin intensity in U2OS cells using immunofluorescence images after control and GTSE1 RNAi. n ≥ 105 cells; 3 independent experiments. P-values were obtained using a Mann-Whitney U test. (K) Quantification of MCAK intensity using GFP fluorescence on the inner spindle normalized to tubulin intensity in HeLa cells expressing BAC-based MCAK-GFP using immunofluorescence images after control and GTSE1 RNAi. n ≥ 89 cells; 3 independent experiments. P-values were obtained using a Mann-Whitney U test. Bars, 5 µm. All error bars represent SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Article Snippet: The following antibodies were obtained from commercial sources: mouse anti–α-tubulin (DM1α; Sigma-Aldrich), mouse anti-MCAK (clone 1G2; Abnova), human nuclear antibodies to nuclear antigens–centromere autoantibody (CREST; CS1058; Europa Bioproducts Ltd.), mouse anti–c-myc (Oncogene/EMD Millipore), rat anti-EB1 (clone KT-51; Absea Biotechnology), rabbit anti-TACC3 (H-300; Santa Cruz Biotechnology, Inc.), rabbit anti–ch-TOG (86073; Abcam), rabbit anti–Aurora B (2254; Abcam), rabbit anti-Kif2A (NB500-180; Novus Biologicals), and rabbit anti-GFP (AB6556; Abcam).

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Immunofluorescence, Control, Staining, Fluorescence, MANN-WHITNEY, Expressing

Journal: The Journal of Cell Biology

Article Title: GTSE1 tunes microtubule stability for chromosome alignment and segregation by inhibiting the microtubule depolymerase MCAK

doi: 10.1083/jcb.201606081

Figure Lengend Snippet: GTSE1 interacts directly with MCAK and inhibits its MT depolymerase activity in vitro. (A) Immunoblot (IB) showing MCAK pulled down in an in vitro GST pulldown using GST alone, GST-GTSE1 1–460, and GST-GTSE1 463–739 fragments. Input represents 2.5% of total MCAK protein used for the GST pulldown assay. (B) Kymograph depicting 50 nM MCAK depolymerizing a GMPCPP-stabilized MT (Video 5). The black dashed line represents the start of the experiment when MCAK was added. The second kymograph depicts a GMPCPP-stabilized MT maintaining constant length in the presence of 50 nM MCAK plus 250 nM GTSE1 (Video 6). The black dashed line represents the start of the experiment when MCAK and GTSE1 were added. Depolymerization rates of GMPCPP-stabilized MTs in the presence of 50 nM MCAK alone, 50 nM MCAK with a fivefold excess of GTSE1, and 50 nM MCAK with an equimolar amount of GTSE1 are shown in the box plot. n > 100 MTs; 4 experiments. (C) Kymographs of MT growth and catastrophe in the presence of 20 µM tubulin (Tb) alone or 20 µM tubulin and 250 nM GTSE1 from GMPCPP-MT seeds. Box plot shows the shrinkage rate after catastrophe of 20 µM tubulin alone and 20 µM tubulin with 250 nM GTSE1. n = 34 and 90 MTs, respectively.

Article Snippet: The following antibodies were obtained from commercial sources: mouse anti–α-tubulin (DM1α; Sigma-Aldrich), mouse anti-MCAK (clone 1G2; Abnova), human nuclear antibodies to nuclear antigens–centromere autoantibody (CREST; CS1058; Europa Bioproducts Ltd.), mouse anti–c-myc (Oncogene/EMD Millipore), rat anti-EB1 (clone KT-51; Absea Biotechnology), rabbit anti-TACC3 (H-300; Santa Cruz Biotechnology, Inc.), rabbit anti–ch-TOG (86073; Abcam), rabbit anti–Aurora B (2254; Abcam), rabbit anti-Kif2A (NB500-180; Novus Biologicals), and rabbit anti-GFP (AB6556; Abcam).

Techniques: Activity Assay, In Vitro, Western Blot, GST Pulldown Assay

Journal: Nature Metabolism

Article Title: Interaction of sortilin with apolipoprotein E3 enables neurons to use long-chain fatty acids as alternative metabolic fuel

doi: 10.1038/s42255-025-01389-5

Figure Lengend Snippet: Cortical gene expression patterns were studied in male mice (12 weeks of age) of the indicated Sort1 and human APOE genotypes using quantitative RT-PCR (n = 8-18 animals per group, depending on the gene analyzed). Individual data points as well as the mean ± s.d. of the entire genotype group are given. Statistical significance of data was tested using two-way ANOVA with Tukey’s multiple comparison test. Acadl , acyl-CoA dehydrogenase long chain; Acadm , acyl-CoA dehydrogenase medium chain; Acadvl , acyl-CoA dehydrogenase very long chain; Acsl5 , acyl-CoA synthetase long chain family member 5; Cd36 , fatty acid translocase; Cpt1a , carnitine O -palmitoyltransferase 1 A; Cpt1c , carnitine O -palmitoyltransferase 1 C; Cpt2 , carnitine O -palmitoyltransferase 2; Crat , carnitine O -acyltransferase; Slc16a1 , solute carrier family 16 member 1 encoding monocarboxylate transporter (MCT) 1; Slc16a3 , solute carrier family 16 member 3 encoding MCT4; Slc16a7 , solute carrier family 16 member 7 encoding MCT2; Slc22a5 , solute carrier family 22 member 5; Slc27a1 , solute carrier family 27 member 1encoding fatty acid transport protein 1 (FATP1); Slc27a6 , solute carrier family 27 member 6, encoding FATP6.

Article Snippet: Gene expression analyses were performed using the following probes: Acadl (Mm00599660_m1), CRAT (Mm00483985_m1), Cpt1a (Mm-01231183_m1), Cpt2 (Mm00487202_m1), Acadvl (Mm00444293_m1), Acadm (Mm00431611_m1), Slc16a1 (Mm01306379_m1) and Slc22a5 (Mm00441468_m1).

Techniques: Gene Expression, Quantitative RT-PCR, Comparison

Journal: Nature Metabolism

Article Title: Interaction of sortilin with apolipoprotein E3 enables neurons to use long-chain fatty acids as alternative metabolic fuel

doi: 10.1038/s42255-025-01389-5

Figure Lengend Snippet: Gene expression patterns were compared in neurons and astrocytes isolated from the brains of male mice (12 weeks of age) of the indicated Sort1 and human APOE genotypes using fluorescence-activated cell sorting (E3WT n = 8, E3KO n = 5; E4WT and E4KO n = 7 mice). Individual data points and the mean ± s.d. of the entire genotype group are given. Statistical significance was tested using two-way ANOVA with Tukey’s multiple-comparison test. Each cell preparation was performed as an independent experiment. a , b , RT–qPCR analyses of neuronal markers NeuN and Baf53b ( a ) as well as astrocyte-specific markers Aldh1l1 and Gfap ( b ) confirm the purity of sorted neuronal and astrocyte cell preparations. c , RT–qPCR analyses of genes encoding enzymes in transport and metabolism of fatty acids document interaction of APOE and Sort1 genotypes in neurons but not astrocytes. Acadl , acyl-CoA dehydrogenase long chain; Acadm , acyl-CoA dehydrogenase medium chain; Acadvl , acyl-CoA dehydrogenase very long chain; Cpt1a , carnitine O -palmitoyltransferase 1A; Cpt2 , carnitine O -palmitoyltransferase 2; Crat , carnitine O -acyltransferase; Slc16a1 , solute carrier family 16 member 1 encoding monocarboxylate transporter (MCT) 1; Slc22a5 , solute carrier family 22 member 5.

Article Snippet: Gene expression analyses were performed using the following probes: Acadl (Mm00599660_m1), CRAT (Mm00483985_m1), Cpt1a (Mm-01231183_m1), Cpt2 (Mm00487202_m1), Acadvl (Mm00444293_m1), Acadm (Mm00431611_m1), Slc16a1 (Mm01306379_m1) and Slc22a5 (Mm00441468_m1).

Techniques: Gene Expression, Isolation, Fluorescence, FACS, Comparison, Quantitative RT-PCR