Abstract
Osteoclasts are bone resorbing cells that participate in the maintenance of bone health. Pathological increase in osteoclast activity causes bone loss, eventually resulting in osteoporosis. Actin cytoskeleton of osteoclasts organizes into a belt of podosomes, which sustains the bone resorption apparatus and is maintained by microtubules. Better understanding of the molecular mechanisms regulating osteoclast cytoskeleton is key to understand the mechanisms of bone resorption, in particular to propose new strategies against osteoporosis. We reported recently that β-tubulin isotype TUBB6 is key for cytoskeleton organization in osteoclasts and for bone resorption. Here, using an osteoclast model CRISPR/Cas9 KO for Tubb6, we show that TUBB6 controls both microtubule and actin dynamics in osteoclasts. Osteoclasts KO for Tubb6 have reduced microtubule growth speed with longer growth life time, higher levels of acetylation, and smaller EB1-caps. On the other hand, lack of TUBB6 increases podosome life time while the belt of podosomes is destabilized. Finally, we performed proteomic analyses of osteoclast microtubule-associated protein enriched fractions. This highlighted ARHGAP10 as a new microtubule-associated protein, which binding to microtubules appears to be negatively regulated by TUBB6. ARHGAP10 is a negative regulator of CDC42 activity, which participates in actin organization in osteoclasts. Our results suggest that TUBB6 plays a key role in the control of microtubule and actin cytoskeleton dynamics in osteoclasts. Moreover, by controlling ARHGAP10 association with microtubules, TUBB6 may participate in the local control of CDC42 activity to ensure efficient bone resorption.
Highlights
Osteoclasts are giant multinucleated cells involved in bone resorption
Similar to what we described in primary osteoclasts treated with Tubb6 siRNAs (Guérit et al, 2020), we found that osteoclasts derived from RAW264.7 Tubb6 KO cells presented several defects when plated on glass: increase of abnormal podosome belts and podosome rings, which are immature podosome belts (Figures 1C,D and Supplementary Figure 1B)
Tubulin, microtubule, and cytoskeleton as keywords, we found that all Gene Ontology (GO) terms associated with the cytoskeleton in molecular functions, cellular components, and biological process showed a significant enrichment in the S2 fraction (Supplementary Table 2)
Summary
Osteoclasts are giant multinucleated cells involved in bone resorption Their counterparts, osteoblasts, participate in new bone formation. Both cell types are crucial for bone homeostasis which is highly dynamic despite bone stiffness. The sealing zone surrounds a highly convoluted plasma membrane domain: the ruffled border, which is involved in bone resorption being the siege of acid and protease secretions respectively dissolving mineral matrix and digesting bone proteins. The dynamic of this podosomal structure allows osteoclast sliding and generates series of resorption pits at the bone surface (Søe and Delaissé, 2017)
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