Title: The Influence of Microtubules and Microtubule-Based Structures on Osteoclast and CD4+ T Cell Function
Description: The burden of osteoporosis and low bone mass is unrelenting, affecting over 50% of the U.S. population over the age of 50. In a similar reach but different clinical realm, nearly 40% of all men and women will be diagnosed with cancer at some point during their lifetimes. The impact of both of these diseases is compounded by the limited knowledge of cellular mechanisms and the insufficiency of effective treatment options. At the microscopic level of the cell cytoskeleton, increasing evidence has led researchers to further explore microtubules (MTs) and MT-based structures, such as primary cilia, as potential keys to unlocking improved treatment options. However, the way in which microtubules regulate the processes giving rise to these diseases remains a critical gap in knowledge. The works outlined here aimed to elucidate mechanisms that may be used to combat diseases attacking the skeletal and immune systems. In order to characterize the influence of primary cilia with respect to osteoclast differentiation, we implemented a series of treatments to an immortalized macrophage cell line: cilia lengthening (using Fenoldopam) and mechanical stimulation (using oscillatory fluid flow).

The results were analyzed by a combination of immunocytochemistry and quantitative PCR. Our first result showed definitively that while osteoclasts do not possess primary cilia, their macrophage precursors do. We also discovered that these macrophage primary cilia are dynamic and can be modulated; cells whose cilia had been lengthened showed a significant decrease in osteoclast formation, indicating that macrophage cilia resorption may be a necessary step for osteoclast differentiation to occur. Combined with findings from previous studies, there is increasing evidence that the primary cilium, as a therapeutic target for bone diseases, may offer a dual beneficial approach to both promote bone formation and downregulate osteoclast activity. We then explored the possibility of directional MT translocation during T-cell activation being linked to Rho GTPases, which regulate actin polymerization. WASp and WAVE2, known to have functional roles in T-cell activation, were identified as primary candidates.

In order to investigate this relationship, we implemented a stepwise micropatterning procedure by which PDMS was used to transfer local areas of activation (presenting fluorescently-tagged antibodies against CD3 and CD28) which, upon T-cell receptor (TCR) triggering, could mimic immune synapse (IS) formation. We showed that, although there was no correlation between the spatial organization of MTs and WASp, MTs and WAVE2 location were highly correlated, providing strong evidence for a link between these two systems. In addition, MT disruption via nocodazole resulted in a significant decrease in T-cell activation and mechanosensing capabilities. Given the role of WAVE2 in promoting cell spreading and adhesion during IS formation, this result provides additional evidence that this cytoskeletal filament is in fact connected to proteins involved in actin nucleation and elongation. We anticipate the work in Aim 1 to help reveal a previously unexplored therapeutic target for osteoporosis, a disease that currently has no clinical manifestations prior to a fracture event.

Further investigation has the potential to contribute to diagnosis and prevention techniques, as well as new treatments. Similarly, given the emergence of adoptive T-cell immunotherapy for immune-related disorders, the findings of Aim 2 will advance our understanding of both the biological and mechanical influence of the cytoskeleton and motivate microtubules as one component of a more comprehensive armamentarium of treatment approaches.