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. 2009 Feb 3;106(5):1451-6.
doi: 10.1073/pnas.0804464106. Epub 2009 Jan 21.

Src-dependent repression of ARF6 is required to maintain podosome-rich sealing zones in bone-digesting osteoclasts

Tobias Heckel  1 , Cornelia Czupalla, Ana Isabel Expirto Santo, Mihaela Anitei, Maria Arantzazu Sanchez-Fernandez, Kerstin Mosch, Eberhard Krause, Bernard Hoflack
Affiliations

Src-dependent repression of ARF6 is required to maintain podosome-rich sealing zones in bone-digesting osteoclasts

Tobias Heckel et al. Proc Natl Acad Sci U S A. .

Abstract

Bone digestion occurs when osteoclasts adhere onto bone surfaces and polarize to form acidic, hydrolase-rich resorption lacunae. For this process, they condense their actin-rich podosomes in tight belts to establish sealing zones, which segregate their basal membranes from those facing resorption lacunae. This polarization process remains poorly understood. Here, we combined quantitative proteomics and gene silencing to identify new substrates of the Src tyrosine kinase, a key regulator of osteoclast function. We now report that a depletion of the ARF GTPase-activating protein GIT2, which localizes to sealing zones upon Src phosphorylation, or a lack of GTP hydrolysis on ARF6 impairs sealing zone formation and polarized membrane traffic. Surprisingly, the Rho guanine nucleotide exchange factors alpha and beta PIX, which usually coordinate ARF and Rho signaling, were found to be dispensable. We conclude that the Src-dependent localization of GIT2 is essential for down-regulating ARF6 activity at sealing zones, and thus for maintaining osteoclast polarity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification of candidate Src targets in osteoclasts. (A) Raw 264.7 cell-derived osteoclasts were seeded on osteologic discs and infected with recombinant adenoviruses encoding for Src-GFP (green). Twenty-four hours after viral infection, cells were fixed, stained with phalloidin (red) and DAPI (blue), and analyzed by confocal microscopy. (B) Osteoclasts grown on osteologic discs were fixed, double-stained with anti-phosphotyrosine antibody (green) and phalloidin (red), and analyzed by confocal microscopy. Note that after treatment with the Src inhibitor SU6656 (10 μM), both the sealing zone itself and the tyrosine-phosphorylated proteins enriched at the sealing zone disappear. (Scale bars, 20 μm.) (C) Osteoclasts were treated with 10 μM SU6656 (+) or left untreated (−), cell lysates were prepared, and tyrosine-phosphorylated proteins were immunoprecipitated with anti-phosphotyrosine antibody. Cell lysates (Left) and immunoprecipitates (Right) were probed by Western blotting for tyrosine-phosphorylated proteins. (D) Strategy. Osteoclasts were differentially labeled by growing them in medium containing either light (Arg-0) or heavy (Arg-6) arginine during their RANKL-induced differentiation before treatment of one sample with SU6656. After immunoprecipitation with anti-phosphotyrosine antibodies, precipitated proteins were digested with trypsin and analyzed by mass spectrometry. (E) Abundance ratios of proteins identified in the anti-phosphotyrosine immunoprecipitate. An isotope ratio of 0.8 or below was considered to be decreased protein abundance. (F) Cell lysates (Left) and immunoprecipitates (Right) were prepared as in C and probed by Western blotting for the indicated proteins.
Fig. 2.
Fig. 2.
Src-dependent localization of GIT proteins to the sealing zone of osteoclasts. Osteoclasts were seeded on osteologic discs; treated with Src inhibitor or left untreated; fixed; and stained with phalloidin (red), DAPI (blue), and with antibodies for the indicated proteins (green). Cells were analyzed by confocal microscopy. (Scale bars, 20 μm.)
Fig. 3.
Fig. 3.
RNAi-mediated depletion of GIT2 and Src impairs sealing zone formation in osteoclasts. (A) Osteoclasts were either mock-electroporated or electroporated with stealth RNAi duplexes targeting the indicated genes or with scrambled RNAi duplexes. Electroporated osteoclasts were grown for an additional 43 h, and mRNA was extracted, reverse transcribed, and analyzed by quantitative PCR using the comparative ct method. Shown are relative mRNA levels of Src and GIT2 normalized to GAPDH mRNA levels. (B). Electroporated osteoclasts were grown for an additional 43 h, and total cell lysates were prepared and probed by Western blotting for Src, GIT2, and Tubulin. (C and D) Osteoclasts were electroporated as in A. Electroporated osteoclasts were grown for an additional 43 h on osteologic discs, fixed, stained with phalloidin (red) and DAPI (blue), and analyzed by confocal microscopy. Sealing zones were counted (C). Values are mean ± SD from 3 different experiments (n = 300 osteoclasts/experiment). Representative images are shown (D). *, P < 0.001. (Scale bars, 20 μm.)
Fig. 4.
Fig. 4.
Active ARF6 impairs sealing zone formation and osteoclasts polarization. (A and C) Osteoclasts were seeded on osteologic discs and infected with recombinant adenoviruses encoding for HA-tagged ARF6, ARF6Q67L, and ARF6T27N or mock-infected (control). Forty-two hours after viral infection, cells were fixed; stained with anti-HA or with anti-LAMP1 antibody (green), phalloidin (red), and DAPI (blue); and analyzed by confocal microscopy. (Scale bars, 20 μm.) (B) Sealing zones were counted; values are mean ± SD from 3 different experiments (n = 250 osteoclasts/experiment). *, P < 0.001.
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References

    1. Linder S, Aepfelbacher M. Podosomes: Adhesion hot-spots of invasive cells. Trends Cell Biol. 2003;13:376–385. - PubMed
    1. Luxenburg C, et al. The architecture of the adhesive apparatus of cultured osteoclasts: From podosome formation to sealing zone assembly. PLoS ONE. 2007;2:e179. - PMC - PubMed
    1. Jurdic P, Saltel F, Chabadel A, Destaing O. Podosome and sealing zone: Specificity of the osteoclast model. Eur J Cell Biol. 2006;85:195–202. - PubMed
    1. Vaananen HK, Zhao H, Mulari M, Halleen JM. The cell biology of osteoclast function. J Cell Sci. 2000;113(Pt 3):377–381. - PubMed
    1. Saltel F, Destaing O, Bard F, Eichert D, Jurdic P. Apatite-mediated actin dynamics in resorbing osteoclasts. Mol Biol Cell. 2004;15:5231–5241. - PMC - PubMed

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