Journal: Pflugers Archiv : European journal of physiology

Article Title: Calcium, ATP and nuclear pore channel gating

doi:

Figure Lengend Snippet: A–C Transport properties of nuclei isolated from Dunning G prostate cancer cells. A Nuclear pore complex (NPC) functional diameter and passive diffusion limit were estimated with FITC-labeled dextrans. Dextran molecules smaller than 20 kDa translocated to the nucleus. Note that probe washout was not required with confocal microscopy. B NPC capacity for macromolecular import was determined with the fluorescent phycobiliprotein B-phycoerythrin (240 kDa) conjugated to the SV40 large T antigen nuclear localization signal (NLS). The image shown was obtained after a 15-min exposure. No washout was required to assess nuclear translocation. The NPC capacity for macromolecular export was estimated by pulse-chase application of FITC-labeled ribonucleotides under conditions that support transcription. The centered panel shows an image obtained after washout of the probe mixture and exposure of the nuclei to macromolecular transport substrates. The NPC capacity for simultaneous import, export, transcription and translation was tested with the plasmid for enhanced green fluorescence protein (EGFP, pEGFP-C1 of Mol.Wt.≅3.1 MDa). C The presence of Ca2+ in the nuclear envelope (NE) cisterna was determined with the membrane-permeant Ca2+-indicator fluo-3/AM. Data in C illustrate the common [Ca2+]NE response in 16 of the 16 nuclei studied. Under control conditions, only a weak signal was detected. However, upon the addition of Ca2+-loading substrates, the signal increased. When 1 µM inositol 1,4,5-trisphosphate (IP3) or 1 mM BAPTA/AM was added to a control or Ca2+-loaded nucleus, no signal was detected

Article Snippet: The NPC sieving and transport properties were studied with laser scanning confocal fluorescence microscopy (MRC-600, Bio-Rad, Hercules, Calif., USA; LSM 410, Carl Zeiss, Oberkochen, Germany).

Techniques: Isolation, Functional Assay, Diffusion-based Assay, Labeling, Confocal Microscopy, Translocation Assay, Pulse Chase, Plasmid Preparation, Fluorescence, Membrane, Control