Welcoming Next Gen 7 - MYO9B Regulation of Cytoskeletal Rearrangement

Poster Number: Welcoming Next Gen 7
Publication Number: WNG 7.117

Background: Macrophages are innate immune cells that act as first responders by sampling their environment via cytoskeletal rearrangement allowing for cell migration and particle uptake. Podosomes are dynamic, actin-rich structures of the cytoskeleton imperative for adhesion and migration. Their constant turnover is dependent on the spatiotemporal regulation of the Rho family of GTPases which coordinate actin polymerization via their activation by GEFs and inactivation by GAPs. Myosin 9B (MYO9B) is an unconventional myosin protein most highly expressed in macrophages containing a Rho-specific GAP domain. The role of MYO9B in migration has previously been described, however, its’ role in other cytoskeletal-dependent processes such as podosome formation remains unknown.

Objective: This project aims to develop a model system for investigating the role of MYO9B in podosome formation and function.

Design/Methods: To address this, we isolated bone marrow-derived macrophages (BMDMs) from Wildtype (WT) and MYO9B knockout (MYO9B -/-) mice. G-LISA small G-protein Activation Assays were completed to determine levels of active Rho in the cells. Additionally, immunostaining and confocal microscopy were used to visualize podosome formation in the cells with and without phorbol 12-myristate 13-acetate (PMA) treatment to elicit podosome formation (as murine BMDMs do not regularly form podosomes).

Results: G-LISA results demonstrated a substantial increase in Rho activity in MYO9B -/- cells compared to WT cells although no associated morphology change was apparent. Additionally, we found that 15 min of PMA treatment resulted in inconsistent podosome formation in both WT and MYO9B -/- BMDMs. Thus, we increased treatment to 24 hours to determine if this would improve the regularity of podosome formation. We did not see more definitive results with this treatment time and therefore will investigate other models for podosome formation.

Conclusion(s): In conclusion, this project verified MYO9B as a regulator of Rho activity in macrophages as we observed a marked increase in Rho activity in MYO9B -/- cells. Additionally, we explored the use of BMDMs treated with PMA as a model for podosome formation but found this was not a reliable model moving forward. We have since transitioned to use bone marrow-derived dendritic cells (BMDCs) which spontaneously and reliably form podosomes without stimulation. We are also investigating the specificity of MYO9B for particular members of the Rho family in an effort to better understand the precise role of MYO9B in the basic cellular processes of innate immune cells.