Conformational states of the nuclear pore complex induced by depletion of nuclear Ca2+ stores

Science

Volumn 273, Issue 5283, 1996, Pages 1875-1877

  Perez Terzic, Carmen ^a   Pyle, Jason ^a   Jaconi, Marisa ^a   Stehno Bittel, Lisa ^a,b   Clapham, David E ^a  

^a MAYO CLINIC   (United States)

^b UNIVERSITY OF KANSAS MEDICAL CENTER   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CALCIUM ION; NUCLEAR PROTEIN;

ANIMAL CELL; ARTICLE; ATOMIC FORCE MICROSCOPY; CALCIUM TRANSPORT; CELL NUCLEUS MEMBRANE; CHANNEL GATING; CONTROLLED STUDY; DIFFUSION; NONHUMAN; OOCYTE; PRIORITY JOURNAL; PROTEIN CONFORMATION; SCANNING ELECTRON MICROSCOPY; XENOPUS LAEVIS;

EID: 0029744799     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5283.1875     Document Type: Article

References (23)

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13. 2+ store as it does the endoplasmic reticulum store, we were unable to use it as a pharmacological tool. The nuclei were then fixed for 4 hours in a buffer solution containing 1% glutaraldehyde and 4% formaldehyde (pH 7.2). The nuclear envelope was spread manually on an electron microscope carbon grid and prepared for critical-point drying. The specimen was rinsed twice in 0.1 M phosphate buffer (pH 7.2) and in buffer supplemented with 1% osmium for 10 min. Samples were dehydrated with incremental concentrations of ethanol before being dried in a critical-point dryer (Ted Pella, Inc., Tustin, CA). Fixed critical point-dried intact nuclei and nuclear envelopes directly adhered to double-sided carbon tape on brass mounts and were coated with a discontinuous layer of platinum (∼1 nm thick) by use of an Ion Tech indirect argon ion-beam sputtering system (VCR Group, San Francisco, CA) operating at an accelerating voltage of 9.5 KV and 4.2 mA. Samples were examined at various accelerating voltages (1.0, 2.4, 3.5, and 5.0 kV) in a Hitachi S-900 FESEM in the secondary electron mode, and images were recorded on Polaroid Type 52 or Type 55 film. Results were expressed as means ± SEM. Transmission electron microscopy of NPCs gave similar results (data not shown). All experiments were performed at room temperature (22° ± 2°C).
14. 2+ store as it does the endoplasmic reticulum store, we were unable to use it as a pharmacological tool. The nuclei were then fixed for 4 hours in a buffer solution containing 1% glutaraldehyde and 4% formaldehyde (pH 7.2). The nuclear envelope was spread manually on an electron microscope carbon grid and prepared for critical-point drying. The specimen was rinsed twice in 0.1 M phosphate buffer (pH 7.2) and in buffer supplemented with 1% osmium for 10 min. Samples were dehydrated with incremental concentrations of ethanol before being dried in a critical-point dryer (Ted Pella, Inc., Tustin, CA). Fixed critical point-dried intact nuclei and nuclear envelopes directly adhered to double-sided carbon tape on brass mounts and were coated with a discontinuous layer of platinum (∼1 nm thick) by use of an Ion Tech indirect argon ion-beam sputtering system (VCR Group, San Francisco, CA) operating at an accelerating voltage of 9.5 KV and 4.2 mA. Samples were examined at various accelerating voltages (1.0, 2.4, 3.5, and 5.0 kV) in a Hitachi S-900 FESEM in the secondary electron mode, and images were recorded on Polaroid Type 52 or Type 55 film. Results were expressed as means ± SEM. Transmission electron microscopy of NPCs gave similar results (data not shown). All experiments were performed at room temperature (22° ± 2°C).
15. 2+ store as it does the endoplasmic reticulum store, we were unable to use it as a pharmacological tool. The nuclei were then fixed for 4 hours in a buffer solution containing 1% glutaraldehyde and 4% formaldehyde (pH 7.2). The nuclear envelope was spread manually on an electron microscope carbon grid and prepared for critical-point drying. The specimen was rinsed twice in 0.1 M phosphate buffer (pH 7.2) and in buffer supplemented with 1% osmium for 10 min. Samples were dehydrated with incremental concentrations of ethanol before being dried in a critical-point dryer (Ted Pella, Inc., Tustin, CA). Fixed critical point-dried intact nuclei and nuclear envelopes directly adhered to double-sided carbon tape on brass mounts and were coated with a discontinuous layer of platinum (∼1 nm thick) by use of an Ion Tech indirect argon ion-beam sputtering system (VCR Group, San Francisco, CA) operating at an accelerating voltage of 9.5 KV and 4.2 mA. Samples were examined at various accelerating voltages (1.0, 2.4, 3.5, and 5.0 kV) in a Hitachi S-900 FESEM in the secondary electron mode, and images were recorded on Polaroid Type 52 or Type 55 film. Results were expressed as means ± SEM. Transmission electron microscopy of NPCs gave similar results (data not shown). All experiments were performed at room temperature (22° ± 2°C).
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21. AFM uses deflection of a fine probe to gauge the force, and hence physical boundary, of structures. The resolution of AFM is limited primarily by probe tip size and shape, but it is generally possible to resolve nanometer structures on biological surfaces. Repetitive scanning of the probe across the surface to be imaged defines a three-dimensional topographic map [G. Binnig, C. F. Quate, C. Gerber, Phys. Rev. Lett. 129, 930 (1986); M. Radmacher, R. W. Tillmann, M. Fritz, H. E. Gaub, Science 257, 1900 (1992)]. The nuclear envelope was prepared and fixed as described for FESEM except that after fixation, the nuclear envelope was spread on a clean coverslip glass and air-dried before sampling. All AFM images were obtained with AutoProbe LS (Park, Scientific Instruments, Sunnyvale, CA) modified for biological samples, and analyzed by ProScan software, version 1.1 (Park, Scientific Instruments). Both pyramidal silicon nitride and sharpened silicon nitride tips with chrome coating were used (spring constant, 0.08 N/m). Images were obtained with an average scanning speed of 3 Hz. Sample scanning was performed in constant-force mode with probe sample force maintained ≤1 nN. Results were expressed as means ± SEM. Experiments were performed at 22° ± 2°C.
22. AFM uses deflection of a fine probe to gauge the force, and hence physical boundary, of structures. The resolution of AFM is limited primarily by probe tip size and shape, but it is generally possible to resolve nanometer structures on biological surfaces. Repetitive scanning of the probe across the surface to be imaged defines a three-dimensional topographic map [G. Binnig, C. F. Quate, C. Gerber, Phys. Rev. Lett. 129, 930 (1986); M. Radmacher, R. W. Tillmann, M. Fritz, H. E. Gaub, Science 257, 1900 (1992)]. The nuclear envelope was prepared and fixed as described for FESEM except that after fixation, the nuclear envelope was spread on a clean coverslip glass and air-dried before sampling. All AFM images were obtained with AutoProbe LS (Park, Scientific Instruments, Sunnyvale, CA) modified for biological samples, and analyzed by ProScan software, version 1.1 (Park, Scientific Instruments). Both pyramidal silicon nitride and sharpened silicon nitride tips with chrome coating were used (spring constant, 0.08 N/m). Images were obtained with an average scanning speed of 3 Hz. Sample scanning was performed in constant-force mode with probe sample force maintained ≤1 nN. Results were expressed as means ± SEM. Experiments were performed at 22° ± 2°C.
23. Supported by an NIH grant to D.E.C. We thank D. Braddock and V. Parpura for technical assistance with the AFM, C. Frethem (University of Minnesota) for the use of the FESEM, J. Charlesworth (Mayo Foundation) for use of the critical-point dryer, and J. Chong and A. Terzic for helpful comments on the manuscript.