Movement of vault particles visualized by GFP-tagged major vault protein
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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.
KeywordsRibonucleoprotein particles Green fluorescent protein Major vault protein Microtubule association Vault motility Rat phaeochromocytoma cells
The authors thank Dr. Valerie Kickhoefer and Prof. Herbert Zimmermann for reading the manuscript and making valuable suggestions.
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