Biophysical Journal 70: 2067-2077 (1996)
© 1996 the Biophysical Society

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kubitscheck, U Articles by Peters, R Search for Related Content PubMed PubMed Citation Articles by Kubitscheck, U Articles by Peters, R

Single nuclear pores visualized by confocal microscopy and image processing.

Institut für Medizinische Physik und Biophysik, Westfälische Wilhelms-Universität, Munster, Germany.

ABSTRACT

How nuclear pore complexes, mediating the transport of nucleic acids, proteins, and metabolites between cell nucleus and cytoplasm, are arranged in the nuclear envelope is essentially unknown. Here we describe a method combining high-resolution confocal imaging with image processing and pattern recognition to visualize single nuclear pore complexes (120 nm diameter), determine their relative positions with nanometer accuracy, and analyze their distribution in situ. The method was tested by means of a model system in which the very same sample areas could be imaged by confocal and electron microscopy. It was thus found that single fluorescent beads of 105 nm nominal diameter could be localized with a lateral accuracy of <20 nm and an axial accuracy of approximately 20 nm. The method was applied to digitonin-permeabilized 3T3 cells, whose nuclear pore complexes were fluorescently labeled with the anti-nucleoporin antibody mAb414. Stacks of optical sections were generated by confocal imaging at high resolution. Herein the nuclear pore complexes appeared as bright diffraction-limited spots whose centers were localized by fitting them by three-dimensional gaussians. The nearest-neighbor distribution function and the pair correlation function were calculated and found to agree well with those of randomly distributed hard cylinders of 138 +/- 17 nm diameter, but not with those of randomly distributed points or nonrandomly distributed cylinders. This was supported by a cluster analysis. Implications for the direct observation of the transport of single particles and molecules through individual nuclear pore complexes are discussed.

This article has been cited by other articles:

				C. Hoppener, J. P. Siebrasse, R. Peters, U. Kubitscheck, and A. Naber High-Resolution Near-Field Optical Imaging of Single Nuclear Pore Complexes under Physiological Conditions Biophys. J., May 1, 2005; 88(5): 3681 - 3688. [Abstract] [Full Text] [PDF]

				U. Kubitscheck, D. Grunwald, A. Hoekstra, D. Rohleder, T. Kues, J. P. Siebrasse, and R. Peters Nuclear transport of single molecules: dwell times at the nuclear pore complex J. Cell Biol., January 17, 2005; 168(2): 233 - 243. [Abstract] [Full Text] [PDF]

				N. Daigle, J. Beaudouin, L. Hartnell, G. Imreh, E. Hallberg, J. Lippincott-Schwartz, and J. Ellenberg Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells J. Cell Biol., July 9, 2001; 154(1): 71 - 84. [Abstract] [Full Text] [PDF]

				O. Keminer, J.-P. Siebrasse, K. Zerf, and R. Peters Optical recording of signal-mediated protein transport through single nuclear pore complexes PNAS, October 12, 1999; 96(21): 11842 - 11847. [Abstract] [Full Text] [PDF]

				R Hartig, R. Shoeman, A Janetzko, G Tolstonog, and P Traub DNA-mediated transport of the intermediate filament protein vimentin into the nucleus of cultured cells J. Cell Sci., June 14, 1999; 111(24): 3573 - 3584. [Abstract] [PDF]

				N. Belgareh and V. Doye Dynamics of Nuclear Pore Distribution in Nucleoporin Mutant Yeast Cells J. Cell Biol., February 24, 1997; 136(4): 747 - 759. [Abstract] [Full Text] [PDF]