MSc Exit Seminar-Reuben Philip

Characterizing the Microtubule Organizing Centres in Osteoclasts

The skeleton is a metabolically active organ that undergoes continuous remodeling in order to uphold structural integrity and to repair bone following injury. Osteoclasts are highly specialized, multinucleated cells responsible for the selective resorption of bone matrix components, however, they are also responsible for the pathological bone destruction found in microgravity environments, periodontitis, and osteoporosis. Our study investigates the origin of the microtubule cytoskeleton during differentiation and bone resorption. Microtubule nucleation is generally restricted to specific subcellular sites called microtubule organizing centres (MTOCs) and is primarily fulfilled by the centrosome in mitotic animal cells. While mononuclear osteoclast precursor cells contain centrosomal MTOCs, previous research has suggested that functional centrosomal MTOCs do not exist in osteoclasts. To revisit and characterize the MTOCs in osteoclasts, both cell line and primary cell-derived murine osteoclasts were subjected to high-resolution imaging to track centrosome behaviour and their ability to organize microtubules. Through live-cell imaging, fixed immunofluorescence, and ultrastructural analyses, we observed that most, if not all centrosomes donated from precursor cells clustered early in osteoclastogenesis and persisted post-differentiation in non-resorbing and resorbing osteoclasts. Drug-induced microtubule regrowth assays revealed that centrosomes remained individually functional post-differentiation but clustered in a microtubule-dependent manner in order to organize microtubules. Quantification of microtubules emanating from centrosome clusters showed that they were capable of nucleating more microtubules compared to lone centrosomes. Finally, by visualizing Golgi reorganization and the nucleation of Golgi-derived microtubules, we identified the Golgi as a possible non-centrosomal MTOC that potentially facilitates the production of polarized microtubule arrays in osteoclasts. Together these findings show that multinucleated osteoclasts employ unique centrosomal and non-centrosomal MTOCs to organize microtubules.

Supervisor: Prof. Rene Harrison