Abstract 25

OSTEOSCLEROSIS IN MICE LACKING RAB3D IS RELATED TO DISRUPTIONS IN POSTTGN VESICLE TRAFFICKING IN OSTEOCLASTS

Nathan J. Pavlos¹, Jiake Xu¹, Dietmar Riedel³, Steven L. Teitelbaum², Reinhard Jahn³, F. Patrick Ross², Ming H. Zheng¹

^1. Unit of Orthopaedics, School of Surgery and Pathology, University of Western Australia, Perth, Western Australia
^2. Department of Pathology, Washington University School of Medicine, St.
^3. Louis, USA
^4. Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

Intracellular membrane trafficking is essential to osteoclast function however little is known about the nature and regulation of the transport pathways that govern its structural and functional polarisation. We have recently reported the existence of the small exocytic-related Rab3D GTPase in osteoclasts. Here, to shed light on the possible involvement of Rab3D in osteoclast physiology we examined Rab3D-deficient mice for skeletal anomalies. Strikingly, we identify an osteosclerosis phenotype in these mice; with bones from these animals exhibiting increased total volume, increased trabecular number and reduced trabecular separation. Basal osteoclast numbers were normal, however, the total eroded surface was significantly reduced, suggesting that the resorptive defect was due to attenuated osteoclast activity rather than a disruption in osteoclast formation. Consistently, ultrastructural analyses revealed that Rab3D-/- osteoclasts exhibit irregular regulated borders. To further delineate the molecular mechanism(s) underlying this resorption deficiency, we expressed a series of enhanced yellow fluorescent (EYFP)-tagged wild-type and mutant Rab3D fusion chimeras in osteoclasts and examined their subcellular localisations and affects on bone resorption. Rab3D was found to associate with the trans-Golgi network (TGN) and an as yet undefined subset of post-TGN vesicles of non-endosomal/lysosomal origin. Moreover, while overexpression of Rab3D wild-type and its mutants had no affect osteoclastogenesis or cellular attachment, expression of the GTP-binding deficient Rab3DN135I mutant, which was largely restricted to the TGN, profoundly impaired osteoclastic bone resorption. These data document the existence of a novel Rab3D-mediated post-TGN trafficking pathway that is required for the maintenance of the ruffled border membrane during physiological bone metabolism.

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