ARAP1 Bridges Actin Dynamics and AP-3-Dependent Membrane Traffic in Bone-Digesting Osteoclasts

Abstract

Bone-resorbing osteoclasts play a central role in bone remodeling and its pathology. To digest bone, osteoclasts re-organize both F-actin, to assemble podosomes/sealing zones, and membrane traffic, to form bone-facing ruffled borders enriched in lysosomal membrane proteins. It remains elusive how these processes are coordinated. Here, we show that ARAP1 (ArfGAP with RhoGAP domain, ankyrin repeat and PH domain-containing protein 1) fulfills this function. At podosomes/sealing zones, ARAP1 is part of a protein complex where its RhoGAP domain regulates actin dynamics. At endosomes, ARAP1 interacts with AP-3 adaptor complexes where its Arf-GAP domain regulates the Arf1-dependent AP-3 binding to membranes and, consequently lysosomal membrane protein transport to ruffled borders. Accordingly, ARAP1 or AP-3 depletion in osteoclasts alters their capacity to digest bone in vitro. and AP-3δ-deficient mocha mice, a model of the Hermansky-Pudlak storage pool syndrome, develop osteoporosis. Thus, ARAP1 bridges F-actin and membrane dynamics in osteoclasts for proper bone homeostasis.

Keywords: Cell Biology; Functional Aspects of Cell Biology; Organizational Aspects of Cell Biology.

Graphical abstract

Figure 1

Expression of ARAP1 during Osteoclastogenesis (A) Transcript variants of validated murine ARAP1 isoforms. The different domains are indicated. The target sequence of the stealth siRNA and AB, the epitope recognized by the anti-murine ARAP1 antibody, are situated at the C terminus of all isoforms. (B) Immunoblotting of ARAP1 in osteoclasts. (C) ARAP1 expression in osteoclasts and its precursors. RAW264.7 cells were grown on plastic in the absence or presence of RANKL and harvested at the indicated times. Undifferentiated RAW264.7 cells (“Day 0”) were grown without RANKL, and cell extracts were prepared. Protein expression was determined by western blotting and quantified using the Fiji software. GAPDH was used as a control. The figures presented are representative of at least 3 independent experiments (mean ± SD; **p < 0.01, ***p < 0.001). See also Figure S1.

Figure 2

Localization of Endogenous and GFP-Tagged ARAP1 in Osteoclasts (A–E) Osteoclasts were grown on glass or apatite-collagen-coated surfaces (ACCs), fixed, and stained for (A, B, D, and E) ARAP1 (green) and AP-3 (magenta) actin (red) and DAPI (blue) and (C) AP-3s1 (blue), actin (magenta), ARAP1 (red), and LAMPI (green). White arrows are pointing towards AP-3 positive endosomes. Outlines of single osteoclasts are depicted by white circles in the actin channel. Scale bar: 10 μm. (F) Enlargement of rectangle in B. Fluorescence intensities across the indicated white lines (at sealing zones) were plotted. Images were analyzed using the Fiji software. Scale bar: 10 μm. (G) Enlargement of rectangle in E. Fluorescence intensities across the white lines (at sealing zones) as indicated were plotted. Images were analyzed using the Fiji software. Scale bar: 10 μm. (H) Descriptive statistics to identify ARAP1/AP-3-positive endosomes. The global Pearson correlation coefficients for endogenous and overexpressed ARAP1, AP-3ɗ, EEA1, Rab7a, LAMPI, GM-130, and VPS35 were determined using the Volocity software with automated thresholding (Costes et al., 2004). (n = 3, mean ± SD). See also Figure S2.

Figure 3

ARAP1 Affects Podosomal Belt Organization (A) Effect of ARAP1 and AP-3 knockdown on podosomes in overview (scale bar: 100 μm) and detail (scale bar: 10 μm). (A) ARAP1 and AP-3 were depleted in osteoclast using stealth siRNA as described. Cells were fixed and stained on glass coverslips for DAPI (blue) and phalloidin (red). The outline of single osteoclasts were marked in white. Scale bar in overview: 100 μm; scale bar in detail: 10 μm. (B) ARAP1 knockdown cells were subsequently transfected with GFP-tagged ARAP1 isoform 2 (green) using a recombinant adenovirus. Fluorescence intensities across the white lines (at podosomes) as indicated were plotted. Scale bar in overview: 100 μm; scale bar in detail: 10 μm. (C) The number of osteoclasts in A and B with an intact and altered (not intact) podosomal belt were counted on an 11-mm glass coverslip and plotted. The intactness of the podosomal belt was judged based on the presence of a clear belt on the periphery of an osteoclast. If osteoclasts had dispersed podosomes or arbitrary rings that were phalloidin-positive within the cell, they were judged as non-intact. (n = 3, mean ± SD). (D) The knockdown efficiencies were assessed by immunoblotting (ARAP1) or by qPCR (AP-3) and plotted. (mean ± SD).

Figure 4

ARAP1 and AP-3 Depletion Affects Osteoclast Resorption (A) Effect of siRNA-mediated ARAP1 depletion on the podosomal belt and sealing zones. Osteoclasts were treated with siRNA targeting the ARAP1 genes or mock siRNA and then grown for 48 hr on osteological disks as indicated in Methods. Cells were then fixed and stained with phalloidin (red) and DAPI (blue). (Scale bar: 10 μm). The knockdown efficiency was determined by western blot. The number of osteoclasts with intact and altered (not intact) podosomal belts was counted on an 11-mm glass coverslip and plotted. (B) Effect of siRNA-mediated ARAP1 and AP-3μ1 depletion on sealing zone dynamics. Osteoclasts were treated with siRNAs targeting the corresponding genes or mock siRNA and then plated on apatite-collagen-coated (ACC) multiwell dishes. After 24 hr, they were infected with a recombinant adenovirus encoding the mRFP-ezrin actin-binding domain. After 32 hr, osteoclasts were imaged by time-lapse microscopy (200 ms per frame, 1 frame per minute for 30 min, scale bar: 10 μm). See also Videos S3, S4, and S5. Sealing zone diameter was measured using the Fiji software. The relative sealing zone diameter was plotted for each sealing zone assessed. The change of the sealing zone diameter per minute was plotted and tested using Student's t test. (n = 3, mean ± SD; ****p < 0.0001) The size of the sealing zones above 20 μm in control and siARAP1- and siAP-3-treated osteoclasts was counted on an 11-mm ACC-coated glass coverslip (n = 3, mean ± SD; **p < 0.01). The knockdown efficacy of AP-3μ1 was determined with qPCR. (C) Effect of siRNA-mediated ARAP1 and AP-3μ1 depletion on resorption. The resorptive capacity of osteoclasts treated with siARAP1 or mock siRNA was measured using Corning Osteo Assay Surfaces. The resorbed area from siARAP1 and siAP-3μ1 osteoclasts on 24-well Osteo Assay dishes was plotted using Student's t test. (n = 3, mean ± SD; ****p < 0.0001).

Figure 5

ARAP1 Regulates the Association of AP-3 with Membranes and LAMPI Trafficking to the Ruffled Border (A–F) Effect of siRNA-mediated depletion of ARAP1 or AP-3μ1 on AP-3 and LAMPI fluorescent signals. Osteoclasts treated with siRNAs targeting ARAP1 or AP-3μ1 or mock siRNA were grown on glass or apatite-collagen-coated (ACC) coverslips, fixed, and stained with DAPI and phalloidin and with anti-ARAP1 or anti-AP-3s1 antibodies or anti-LAMP1 antibodies. (scale bar: 10 μm). (G) AP-3s1 fluorescence intensities in siARAP1-treated osteoclasts were measured from individual stacks and relative values were plotted (n = 3, mean ± SD; ****p < 0.0001). (H) The relative LAMPI fluorescence intensity at the ruffled border in AP-3μ1 knockdown (KD) was measured and plotted (n = 3, mean ± SD; Student's t test, ****p < 0.0001). The large error bars can be explained by variations in the KD efficiency in individual osteoclasts. (I) Effect of siRNA-mediated depletion of ARAP1 and AP-3μ1 depletion on steady-state LAMPI. Osteoclasts treated with siARAP1, siAP-3μ1, or mock siRNA were grown on plastic, harvested, and immunoblotted with anti-LAMPI, ARAP1, and GAPDH antibodies. AP-3 knockdown efficiency was determined by qPCR. The intensity of LAMPI in immunoblotting was analyzed using the Fiji software and plotted.

Figure 6

Bone Phenotype of Mocha Mice (A–E) Mocha mice aged 4–6 weeks were analyzed for trabecular bone parameters at their right tibias. (A–D) Bone volume, trabecular thickness (Tb.Th.), trabecular separation (Tb.Sp.), and trabecular number were analyzed with the CTan and CTvol software after microCT. Statistical analysis was done in the GraphPad Prism 7 Software using ordinary one-way ANOVA (n = 8 [WT], 11 [HET], and 4 [HOM]; mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001). (E) 3D projections of the trabecular bone of mocha mice.