Fig 1: AKAP9-dependent Golgi MTOC in AKAP6-depleted cells.(A) Immunostaining of a-tubulin (green), cardiac troponin I (red, cardiomyocyte-specific), and DNA (DAPI) in siControl- or siAKAP6-treated P3 cardiomyocytes. Arrows denote the perinuclear microtubule cage in siControl cells, while arrowheads denote disorganized microtubules. (B) Quantification of A as a-tubulin intensity in concentric bands around the nucleus normalized to the total intensity of a-tubulin in the cell, 119 siControl cells and 131 siAKAP6 cells were quantified per condition from four independent experiments. Error bars represent the SD. (C) Immunostaining of a-tubulin (green), GM130 (red), AKAP9 (magenta), and DNA (DAPI) in siControl-, siAKAP6, and siAKAP6 + siAKAP9-treated P3 cardiomyocytes after 2 min of recovery from nocodazole-induced microtubule depolymerization, indicating a switch of MTOC activity from nuclear envelope to Golgi upon AKAP6 depletion (arrowheads). (D) Quantification of a-tubulin intensity in 0.5 µm wide concentric bands in AKAP6-depleted cells, as in C. 37 siControl cells and 48 siAKAP6 cells were quantified per condition from three independent experiments. Error bars represent the SD. (E) Staining of ?-tubulin (magenta) in siControl- or siAKAP6-treated cells. (F–G) Immunostaining of (F) ninein (magenta) and AKAP9 (green) in AKAP6-depleted cells or (G) ninein (magenta) and Pcnt (green) in AKAP9-depleted cells. Scale bars: 10 µm. Figure 4—source data 1.Underlying data for graphs in panels B and D.
Fig 2: AKAP6 is sufficient to anchor the PACT domain of Pcnt and AKAP9 to the nuclear envelope of nesprin-1a expressing epithelial cells.(A) ARPE19 epithelial cells co-transfected with mCherry-nesprin-1a, FLAG-Pcnt-PACT, and the corresponding GFP construct, were immunostained with anti-FLAG (magenta) and DNA (DAPI). (B) Immunostaining of FLAG (magenta) in ARPE19 cells co-transfected with tdTomato-Farnesyl (tdTomato-F) or tdTomato-AKAP6-SR1-Farnesyl (tdTomatoSR1-F) together with FLAG-Pcnt-PACT indicating that farnesylated SR1 is sufficient to recruit PACT to the plasma membrane. (C) ARPE19 cells co-transfected with mCherry-nesprin-1a, FLAG-PcntS-Myc with or without AKAP6-GFP, were immunostained with anti-Myc (magenta), and DNA (DAPI). Scale bars: 10 µm.
Fig 3: AKAP6 is a key component of nuclear envelope MTOCs.(A) Immunostaining of AKAP6 (green) and GM130 (red) in human osteoclasts after 3, 6 or 7 days of differentiation. Scale bars: 10 µm. (B) Immunostaining of nesprin-1 (green), AKAP6 (magenta), a-tubulin (red), and DNA (DAPI) in control or AKAP6-depleted osteoclasts. Scale bars: 10 µm. (C) Quantification of B as nuclear intensity normalized to shControl. Error bars represent the SD. n = 48, 48, 34, 34 (from left to right). Data are pooled from three independent donors. Statistical test: two-way ANOVA with post-hoc Bonferroni comparison. ****: p<0.0001, n.s.: no significance. (D–E) Immunostaining of AKAP9 (green), Pcnt (magenta), a-tubulin (red), and DNA (DAPI) in control and AKAP6-depleted osteoclasts. Scale bars: 10 µm. (E) Quantification of nuclear intensity normalized to shControl. Error bars represent the SD. n = 77, 39, 77, 39, 58, 76 (from left to right). Data were pooled from three independent donors. Statistical test: two-way ANOVA with post-hoc Bonferroni comparison. ****p<0.0001. (F) Immunostaining of a-tubulin (green), EB1 (magenta), GM130 (red), and DNA (DAPI) in control and AKAP6-depleted osteoclasts, after 15 s of recovery from cold-induced microtubule depolymerization. Scale bars: 20 µm. Figure 6—source data 1.Underlying data for graphs in panels C and E.
Fig 4: AKAP6 is sufficient to recruit endogenous Pcnt and AKAP9 to the nuclear envelope of nesprin-1a expressing epithelial cells.(A–B) ARPE19 cells co-transfected with mCherry-nesprin-1a and AKAP6-GFP and immunostained for endogenous (A) Pcnt (magenta) or (B) AKAP9 (magenta) indicating that AKAP6 can recruit endogenous centrosomal proteins to the nuclear envelope of nesprin-1a-expressing cells. Note the partial nuclear localization of Pcnt or AKAP9 near the centrosome (asterisks). (C–D) Percentage of Cherry-nesprin-1a-AKAP6-GFP expressing cells showing non, partial, crescent or full recruitment of Pcnt (C) or AKAP9 (D) to the nuclear envelope. Error bars represent the SD. Data are pooled from four independent experiments. Total number of Cherry-nesprin-1a/AKAP6-GFP expressing cells analyzed are indicated. (E) ARPE19 cells co-transfected with mCherry-nesprin-1a and AKAP6-GFP and immunostained for endogenous GM130 (magenta). Transfected cell is labeled with a yellow arrowhead. (F) Percentage of Cherry-nesprin-1a/AKAP6-GFP expressing cells showing non, partial, crescent or full recruitment of GM130 to the nuclear envelope. Data are represented as individual biological replicates, together with mean ± SD from three independent experiments. Total number of Cherry-nesprin-1a/AKAP6-GFP expressing cells analyzed are indicated. Scale bars: 10 µm. Figure 8—source data 1.Underlying data for graphs in panels C, D and F.
Fig 5: AKAP6 regulates two pools of microtubules at the nuclear envelope.(A) Immunostaining of a-tubulin (green), GM130 (red), AKAP9 (magenta), and DNA (DAPI) in control, as well as in the indicated siRNA-transfected cells, after 2 min of recovery from nocodazole-induced microtubule depolymerization. Asterisks indicate centrosomal MTOC and arrowheads indicate nuclear envelope MTOC. (B) Quantification of A as a-tubulin intensity in concentric bands around the nucleus normalized to the total intensity of a-tubulin in the cell. 37 siControl cells, 53 siAKAP9 cells, 34 siPcnt cells and 43 siAKAP9+siPcnt cells were quantified per condition, from two independent experiments. Error bars represent the SD. (C) Immunostaining of ?-tubulin (magenta), a-tubulin (green), troponin I (red), and DNA (DAPI) in siRNA-treated cardiomyocytes after 2 min of recovery from nocodazole-induced microtubule depolymerization. (D) Quantification of C, as ?-tubulin intensity at the nuclear envelope normalized to siControl-treated cells. Statistical test: one-way ANOVA with post-hoc Bonferroni’s comparison. ****: p<0.0001, n.s.: no significance, n = 127, 63, 136, 115 (from left to right), data are pooled from three independent experiments. Error bars represent the SD. (E) Model representing the two pools of microtubules regulated by AKAP6. AKAP6 orchestrates MTOC activity at the nuclear envelope by anchoring both ?-TuRC biding proteins, Pcnt and AKAP9. While Pcnt and AKAP9 cooperate at the nuclear envelope for the nucleation of microtubules, Golgi-dependent microtubule nucleation depends on AKAP9. Scale bars: 10 µm. Figure 5—source data 1.Underlying data for graphs in panels B and D.
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