Bone Abstracts

The ArfGAP with RhoGAP domain, ankyrin repeat and PH domain containing protein 1 (ARAP1) is a multi-domain protein that binds to phosphatidylinositol lipids within different biological membranes and integrates Rho and Arf signalling pathways. It is ubiquitously expressed in many cells including osteoclasts and its overexpression is known to affect Golgi and induce filopodia by activation of Cdc42. We therefore hypothesized that ARAP1 might also play an important role in osteoclasts by controlling both osteoclast polarity and post-Golgi transport to the ruffled border, two key events necessary for bone degradation.

We found ARAP1 at two different localizations in osteoclasts, on actin-rich podosomes that condense to sealing zones and at vesicles in the peri-nuclear region, rich in Lamp1, actin and the adaptor protein complex 3 (AP-3). This is in agreement with previous studies where ARAP1 was found in the network of AP-3 vesicles. AP-3 is responsible for targeting selected transmembrane proteins as LAMP1 to lysosomes and lysosome-related organelles; its role in vesicular transport to the ruffled border however remains elusive. Live-cell imagining revealed that ARAP1 dynamics follow those of actin at podosomes and sealing zones. Osteoclasts with overexpressed ARAP1 were also AP-3 positive but remarkably those compartments were enlarged, pointing to a key role of ARAP1 in AP-3 endosome maturation.

Knockdown of ARAP1 results in the disruption of the podosomal belt organization, changes in the classical osteoclast cell morphology and strikingly significant reduction in resorption capacity.

In addition knockdown of the AP3-μ1 subunit, which results in the dissociation of the AP-3 complex, leads to significantly increased resorption of targeted osteoclasts, possibly by disrupting AP-3-dependent lysosomal membrane protein trafficking to the ruffled border. Based on this data we suggest that ARAP-1 is important for osteoclast function but also for AP-3 dependent trafficking, which appears to be a key machinery in osteoclast activity.