Movement of vault particles visualized by GFP-tagged major vault protein.

Vaults are abundant large ribonucleoprotein particles. They frequently colocalize with microtubules and accumulate in filamentous actin-rich lamellipodia. To examine the movement of vaults in living cells, a chimera between the green fluorescent protein and the major vault protein was created. This fusion protein assembled into vault particles as assayed by biochemical fractionation and direct observation of living or fixed cells. By fluorescence recovery after photobleaching, we analyzed the bulk transport of vault particles into neuritic tips of PC12 cells treated with nerve growth factor. Confocal laser scanning microscopy demonstrated co-localization of the major vault protein and microtubules. Video microscopy indicated that, whereas the majority of vault particles were stationary, some individual vault particles moved rapidly, consistent with the action of a microtubule-based or actin-based molecular motor.

Nuclear localization of the major vault protein in U373 cells.

The major vault protein (MVP) is the predominant member of a large ribonucleoprotein particle, named vault. Vaults are abundant in the cytosol of mammalian cells. Mammalian MVP has previously been reported to be associated with the nucleus, particularly its cytosolic surface on which vaults are thought to dock at or near the nuclear pore complex. To date the presence of vault particles inside the nucleus has been convincingly reported only for sea urchin cells. We have addressed the potential nuclear localization of MVP in mammalian cells by employing confocal laser microscopy and cryo-immunoelectron microscopy. As revealed by immunostaining and by analysis of cells transfected with a construct encoding MVP and green fluorescent protein, MVP is present in both the cytosol and in the nucleus. Cryo-electron microscopy of human astroglioma U373 cells reveals clusters of immunogold particles at nuclear pores and in the nucleoplasm suggesting that nuclear MVP is associated with particulate structures. Quantification of the fluorescence observed in the cytosol and in the nuclei reveals that about 5% of the MVP in U373 cells is localized inside the nucleus. Our results further support the notion that part of the cellular MVP can enter the nucleus.

Fibrin-based biomaterials to deliver human growth factors.

Fibrin-based biomaterial preparations can be used as provisional growth matrices for cells important in tissue repair during wound healing in vivo. Their efficacy can be enhanced by including bioactive agents that promote specific cellular responses. This study examined the controlled delivery of the angiogenic growth factors bFGF, VEGF(165), and VEGF(121) using biomatrix preparations prepared from Fibrin Sealant product components. The growth factors were added prior to formation of the Fibrin Sealant clots, and the release kinetics of the proteins from the clots measured. The results indicated that the proteins were released from the clots more slowly in the order bFGF << VEGF(165) < VEGF(121). The biologic activity of the growth factors delivered from Fibrin Sealant clots was established by assaying growth stimulation of human microvascular endothelial cells (HMVEC) and angiogenesis in the chicken embryo chorioallantoic membrane (CAM) model of neovascularization. In the latter assay, clots containing bFGF, VEGF(165), or VEGF(121) all displayed angiogenic activity. However, delivery of either bFGF, VEGF(165), or VEGF(121) alone resulted in a significant percentage of clots becoming filled with blood, indicating that the newly developing vessels invading the clots were leaky and immature. In contrast, this hemorrhaging behavior did not occur with delivery of combinations, e.g., (VEGF(165) + VEGF(121)) or (VEGF(165) + bFGF), indicating that the vessels were more mature than those produced in response to single growth factors. Thus, delivering a combination of growth factors constituted an improvement over the delivery of individual growth factors for enhancing neovascularization.