Journal: Journal of Cell Science

Article Title: Selective disruption of microtubule formation at the nuclear envelope impairs the bone resorption capacity of osteoclasts

doi: 10.1242/jcs.264166

Figure Lengend Snippet: MTOC proteins and Golgi are recruited to the nuclear envelope in RAW264.7-derived osteoclasts. (A) Immunostaining of AKAP6 (green) and GM130 (magenta) together with DNA (DAPI) in RAW264.7-derived osteoclasts after 4 days of RANKL addition. Scale bars: 10 µm. Immunostaining of (B) PCNT (green), (C) CDK5RAP2 (green) and (D) PCM1 (green) and γ-tubulin (magenta) together with DNA (DAPI) in RAW264.7-derived osteoclasts after 4 days of RANKL addition. Arrows indicate centrosomes. Scale bars: 10 µm. Images representative of three experimental repeats.

Article Snippet: RAW264.7 cells were obtained from ATCC and maintained in high glucose DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, 61965059) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140079), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, 15140-122) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Derivative Assay, Immunostaining

Journal: Journal of Cell Science

Article Title: Selective disruption of microtubule formation at the nuclear envelope impairs the bone resorption capacity of osteoclasts

doi: 10.1242/jcs.264166

Figure Lengend Snippet: RAW264.7-derived osteoclasts exhibit a nuclear envelope MTOC alongside a functional centrosome. (A) Immunostaining of α-tubulin (red), AKAP6 (green), GM130 (magenta) and DNA (DAPI) in RAW264.7-derived osteoclasts differentiated for 4 days. (B) Immunostaining of EB-1 (green), γ-tubulin (magenta) and DNA (DAPI) in RAW264.7-derived osteoclasts treated with nocodazole (upper row) and after 1 min (middle row) or 2 min (bottom row) recovery at 37°C. In A and B, asterisks indicate the perinuclear microtubule cage and arrows denote centrosomal asters. (C) Immunostaining of α-tubulin (red), PCNT (green), GM130 (magenta), and DNA (DAPI) in 4 day RAW264.7-derived osteoclasts after 1.5 min of recovery from nocodazole-induced microtubule depolymerization. Asterisks denote the NE nucleation, arrows denote centrosomal nucleation and arrowheads denote Golgi nucleation. Scale bars: 10 µm. Images representative of three experimental repeats.

Article Snippet: RAW264.7 cells were obtained from ATCC and maintained in high glucose DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, 61965059) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140079), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, 15140-122) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Derivative Assay, Functional Assay, Immunostaining

Journal: Journal of Cell Science

Article Title: Selective disruption of microtubule formation at the nuclear envelope impairs the bone resorption capacity of osteoclasts

doi: 10.1242/jcs.264166

Figure Lengend Snippet: Nesprin-1α and AKAP6β isoforms predominate in RAW264.7-derived osteoclasts and form a functional LINC complex. (A) Immunostaining of AKAP6 (green), nesprin-1 (magenta) and DNA (DAPI) in RAW264.7 cells either non-differentiated (upper panels) or differentiated into osteoclasts for 4 days with 50 ng ml −1 of RANKL (lower panels). Non-differentiated cells lack detectable expression of both AKAP6 and nesprin-1 (also denoted with an arrow). Scale bars: 10 µm. (B) Heatmap of qPCR-derived RNA expression levels for Emr1 , Akap6 , Syne1a , Trap and Ctsk during osteoclast differentiation (days 1–4 of RANKL treatment) relative to non-differentiated precursor cells ( n =3). Expression values are normalized and represented as a percentage of maximal expression per gene using the viridis color map. (C) qPCR analysis of Syne1a (gray circles) and Syne1 giant (black triangles) mRNA expression during osteoclast differentiation (D1, D2, D3, D4, day 1–4). (D) qPCR analysis of Akap6a (gray circles) and Akap6b (black triangles) transcript levels at the indicated differentiation stages. Data in C and D are presented as mean±s.d. from three independent experiments, normalized to the non-differentiated condition. (E) Representative images of RAW264.7-derived osteoclasts treated with vehicle (water) or 5 μM DTT and co-stained with Sun2C (green), laminB1 (magenta) antibodies and DAPI. The Sun2C antibody recognizes an epitope which is masked when nesprin-1α is upregulated during osteoclast differentiation. DTT treatment restores epitope accessibility. Non-differentiated cells, indicated with an arrow, exhibit Sun2C reactivity. Scale bars: 10 µm. Images in A and E representative of three experimental repeats.

Article Snippet: RAW264.7 cells were obtained from ATCC and maintained in high glucose DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, 61965059) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140079), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, 15140-122) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Derivative Assay, Functional Assay, Immunostaining, Expressing, RNA Expression, Staining

Journal: Journal of Cell Science

Article Title: Selective disruption of microtubule formation at the nuclear envelope impairs the bone resorption capacity of osteoclasts

doi: 10.1242/jcs.264166

Figure Lengend Snippet: AKAP6 knockdown disrupts NE-MTOC formation in RAW-derived osteoclasts. (A) qPCR analysis of mRNA expression in siControl- and siAKAP6-treated osteoclasts. Bars represent mean fold change (normalised to siControl) for the indicated genes, with individual experimental values shown as dots (three independent experiments). Akap6 expression was significantly reduced following AKAP6 depletion, whereas Syne1a , Syne1 giant , the macrophage marker Emr1 , and the osteoclast markers Ctsk , Nfatc1 and Trap transcript levels were not significantly affected. ***P <0.001; ns, not significant (two-way ANOVA followed by Bonferroni's multiple comparisons test). (B–F) Immunostaining in RAW264.7-derived osteoclasts transfected with control siRNA (siControl) or AKAP6 siRNA (siAKAP6) of (B) AKAP6 (green) and GM130 (magenta), and DNA (DAPI); (C) nesprin-1 (green), CDK5RAP2 (magenta), and DNA (DAPI); (D) PCM1 (green), GM130 (magenta), and DNA (DAPI); (E) PCNT, (green), γ-tubulin (magenta), and DNA (DAPI); and (F) CDK5RAP2 (green), γ-tubulin (magenta), and DNA (DAPI). Note that although perinuclear localization of PCNT and CDK5RAP2 is lost upon AKAP6 knockdown, centrosomal localization is unaffected (arrows). Scale bars: 10 μm. Images in B–F representative of three experimental repeats.

Article Snippet: RAW264.7 cells were obtained from ATCC and maintained in high glucose DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, 61965059) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140079), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, 15140-122) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Knockdown, Derivative Assay, Expressing, Marker, Immunostaining, Transfection, Control

Journal: Journal of Cell Science

Article Title: Selective disruption of microtubule formation at the nuclear envelope impairs the bone resorption capacity of osteoclasts

doi: 10.1242/jcs.264166

Figure Lengend Snippet: AKAP6 Depletion disrupts NE-MTOC function, but not centrosomal-MTOC. (A) Immunostaining of α-tubulin (red), PCNT (green), GM130 (magenta) and DNA (DAPI) in RAW264.7-derived osteoclasts transfected with control siRNA (siControl) or AKAP6-targeting siRNA (siAKAP6). All stainings were performed in cells subjected to nocodazole washout (1.5 min). The yellow arrow indicates centrosomal microtubule organization and the arrowhead indicates Golgi-derived nucleation. (B) Quantification of fluorescence intensity profiles for α-tubulin, PCNT and GM130 in 0.2 µm wide concentric bands relative to distance from the nuclear edge (0.0). For each biological replicate ( n =3), 33–36 nuclei were analyzed per condition, and graphs show the mean±s.d. of the three independent biological experiments. The outward shift in the α-tubulin peak position is indicated by the red bracket, and the reduction in peak intensity is indicated by the green bracket. Statistical comparisons of peak position and peak intensity (amplitude) were performed at the experiment level with an unpaired two-tailed t -test; corresponding P -values are shown in the graphs. (C) Immunostaining of α-tubulin (red), EB1 (green), γ-tubulin (magenta) and DNA (DAPI) in RAW264.7-derived osteoclasts transfected with siControl or siAKAP6 performed after 1 min of nocodazole washout. Insets (2.5× magnification) highlight γ-tubulin-positive centrosomes exhibiting normal centrosomal microtubule outgrowth in both conditions. (D) Quantification of tubulin intensity in a 3 μm area surrounding each centrosome in siControl cells and siAKAP6 centrosomes from three independent experiments. SuperPlots display the overall mean (black line) and biological-replicate mean (color-coded triangles) from n =3, with the individual centrosomes (30–35 per condition) shown as matching color-coded dots superimposed beneath them. ns, not significant (paired two-tailed t -test). Scale bars: 10 μm.

Article Snippet: RAW264.7 cells were obtained from ATCC and maintained in high glucose DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, 61965059) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140079), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, 15140-122) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Immunostaining, Derivative Assay, Transfection, Control, Fluorescence, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Selective disruption of microtubule formation at the nuclear envelope impairs the bone resorption capacity of osteoclasts

doi: 10.1242/jcs.264166

Figure Lengend Snippet: AKAP6 depletion disrupts microtubule-actin cytoskeletal crosstalk in osteoclasts. (A) Representative images of RAW264.7-derived osteoclasts transfected with siControl or siAKAP6, stained for F-actin (phalloidin, green) and microtubules (α-tubulin, red). Corresponding BIOP JACoP analyses are shown in grayscale, illustrating the spatial overlap between F-actin and microtubules (lower panel). Scale bars: 10 μm. Dotted white lines indicate the boundary between the actin-dense sealing zone and the cytoplasm. (B) Quantification of microtubule-actin overlap in siControl and siAKAP6-treated osteoclasts. The actin-microtubule overlap was quantified in arbitrary units (a.u.) as the ratio of actin that colocalized with microtubules normalized against the total actin pixel count. SuperPlots display the overall mean (black line) and biological-replicate mean (color-coded triangles) from n =3, with the individual cells (12–25 per condition) shown as matching color-coded dots superimposed beneath them. ** P =0.0043 (paired two-tailed t -test). (C) Representative immunostaining images of RAW264.7-derived osteoclasts transfected with siControl or siAKAP6 co-stained for actin (phalloidin, green) and microtubules (α-tubulin, red). The right panels show higher magnification images of the boxed regions, highlighting actin ring structure. Images in C representative of three experimental repeats. Scale bars: 10 μm.

Article Snippet: RAW264.7 cells were obtained from ATCC and maintained in high glucose DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, 61965059) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140079), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, 15140-122) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Derivative Assay, Transfection, Staining, Two Tailed Test, Immunostaining

Journal: Journal of Cell Science

Article Title: Selective disruption of microtubule formation at the nuclear envelope impairs the bone resorption capacity of osteoclasts

doi: 10.1242/jcs.264166

Figure Lengend Snippet: AKAP6 depletion impairs osteoclast resorptive activity in RAW264.7-derived osteoclasts. (A) Representative images of RAW264.7-derived osteoclasts transfected with siControl- and siAKAP6 cultured on CaP-coated wells. Cells were removed, and the CaP substrate was stained with 2.5% AgNO 3 to visualize calcium deposits; resorption pits appear as unstained areas. Scale bars: 250 µm. (B) Relative resorbed area of siControl and siAKAP6-treated osteoclasts. Data represent mean±s.d. of three independent experiments, with three wells per experiment. **P =0.0081 (paired two-tailed t -test). (C) Representative images of RAW264.7-derived osteoclasts transfected with siControl or siAKAP6, and stained for F-actin (phalloidin, red), CaP (calcein, green) and nuclei (DAPI, blue). Note, calcein stains CaP, meaning resorption pits with lower calcein intensity indicate higher osteoclast resorption. Scale bars: 10 μm. (D) Quantification of osteoclast resorptive activity across sealing zone size categories (<500 μm 2 , 500–2000 μm 2 , >2000–5000 μm 2 and >5000 μm 2 ). SuperPlots display individual resorption pits as color-coded dots (siControl, blue; siAKAP6, red), with biological replicate means ( n =3; 35–80 pits per experiment, equal numbers per condition) superimposed as colored triangles, each color representing a different replicate. Error bars show overall mean±s.d. Resorption was quantified using the calcein ratio (calcein intensity inside the sealing zone divided by the intensity outside), where values <1 indicate active resorption. *P <0.05; **P <0.01 (two-way ANOVA with Bonferroni's post-hoc test).

Article Snippet: RAW264.7 cells were obtained from ATCC and maintained in high glucose DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, 61965059) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140079), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, 15140-122) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Activity Assay, Derivative Assay, Transfection, Cell Culture, Staining, Two Tailed Test