Background: Macrophages robustly secrete MMP-9 upon activation, and mechanisms for active delivery of MMP-9 vesicles to the cell surface have not been described. Results: MMP-9 is packaged into unique ER protein-containing vesicles that associate with microtubule motors in activated macrophages. Conclusion: Macrophage activation requires enhanced microtubule stabilization for rapid secretion of up-regulated MMP-9. Significance: Understanding the nature and mechanisms of intracellular trafficking of MMP-9 is relevant to both immunology and cancer biology fields.

Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly up-regulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and diseases involving defects in bone protein secretion and deposition. Protein trafficking along the exocytic and endocytic pathways is aided by many molecules, with Rab GTPases being master regulators of vesicle targeting. In this study, we used microarray analysis to identify the Rab GTPases that are up-regulated during a 5-day AA differentiation of OBs, namely Rab1, Rab3d, and Rab27b. Further, we investigated the role of identified Rabs in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane utilizing Rab dominant negative (DN) expression. We also observed that experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs partially alleviated this negative feedback mechanism and resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels was also observed implicating the functional importance of Rab1, Rab3d and Rab27b in these major collagen-producing cells.

Background: EB1 is a microtubule (MT) plus-end-binding protein known to influence MT stability. Results: EB1 is up-regulated in osteoblasts and is required for bone cell differentiation. Conclusion: EB1 affects ␤-catenin stability and cooperates at cell-cell adhesion sites to influence gene expression. Significance: Learning how peripherally targeted proteins interact at cell-cell contact sites is crucial for understanding developmental processes.