Single Molecule Imaging of the Calcium Ion Regulation of Nuclear Pore Passive Permeability

Abstract

Nuclear pore complex (NPC) is the sole pathway for direct communication between the cytoplasm and the nucleoplasm of eukaryotic cell. Based on the criteria of the molecular size exclusion, the NPC allows unregulated passive diffusion of small molecules (< 40 kDa) and facilitated translocation of larger molecules (up to 50 MDa). While recent evidence suggests a third transport mode: Ca2+ regulated transport. In details, the nuclear pore permeability can be regulated by Ca2+ stored in the lumen of nuclear envelope and endoplasmic reticulum. However, the mechanism of Ca2+ regulated transport remains poorly understood. Here we applied a speedy single molecule fluorescence microscopy to characterize the dependence of the nuclear pore passive permeability on the Ca2+ store concentration by snapshots of real-time transient movements of 3 - 40 kDa dextran molecules through the NPCs. We observed novel features under real-time trafficking conditions that escape detection by ensemble measurements: decreased amount of Ca2+ in the store induced restricted passive diffusion of dextran molecules with longer diffusion times and lower transport efficiencies through the NPCs. Dextran molecules cannot penetrate deeply into the nuclear pore and the majority were rejected or trapped by likely barriers formed on both sides of NPCs when the stored calcium was significantly depleted. Our results suggest that a filamentous structure that occludes the NPC may be altered by the depletion of calcium in the store.