Single nuclear pores visualized by confocal microscopy and image processing - PubMed (original) (raw)

Single nuclear pores visualized by confocal microscopy and image processing

U Kubitscheck et al. Biophys J. 1996 May.

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.

PubMed Disclaimer

Cited by

Pathogenic variants in nucleoporin TPR (translocated promoter region, nuclear basket protein) cause severe intellectual disability in humans.
Van Bergen NJ, Bell KM, Carey K, Gear R, Massey S, Murrell EK, Gallacher L, Pope K, Lockhart PJ, Kornberg A, Pais L, Walkiewicz M, Simons C; MCRI Rare Diseases Flagship; Wickramasinghe VO, White SM, Christodoulou J. Van Bergen NJ, et al. Hum Mol Genet. 2022 Feb 3;31(3):362-375. doi: 10.1093/hmg/ddab248. Hum Mol Genet. 2022. PMID: 34494102 Free PMC article.
Hydrophobic free energy eigenfunctions of pore, channel, and transporter proteins contain beta-burst patterns.
Selz KA, Mandell AJ, Shlesinger MF. Selz KA, et al. Biophys J. 1998 Nov;75(5):2332-42. doi: 10.1016/S0006-3495(98)77677-5. Biophys J. 1998. PMID: 9788928 Free PMC article.
Kinetic analysis of translocation through nuclear pore complexes.
Ribbeck K, Görlich D. Ribbeck K, et al. EMBO J. 2001 Mar 15;20(6):1320-30. doi: 10.1093/emboj/20.6.1320. EMBO J. 2001. PMID: 11250898 Free PMC article.
Nuclear Import of Adeno-Associated Viruses Imaged by High-Speed Single-Molecule Microscopy.
Junod SL, Saredy J, Yang W. Junod SL, et al. Viruses. 2021 Jan 22;13(2):167. doi: 10.3390/v13020167. Viruses. 2021. PMID: 33499411 Free PMC article. Review.
Nuclear export of mRNA molecules studied by SPEED microscopy.
Li Y, Junod SL, Ruba A, Kelich JM, Yang W. Li Y, et al. Methods. 2019 Jan 15;153:46-62. doi: 10.1016/j.ymeth.2018.08.005. Epub 2018 Aug 18. Methods. 2019. PMID: 30125665 Free PMC article. Review.

References

1. Cell. 1986 Jun 6;45(5):699-709 - PubMed
1. Cytobios. 1985;43(174S):273-83 - PubMed
1. J Cell Biol. 1989 Sep;109(3):971-82 - PubMed
1. J Cell Biol. 1990 Apr;110(4):883-94 - PubMed
1. J Cell Biol. 1990 Sep;111(3):807-16 - PubMed

Single nuclear pores visualized by confocal microscopy and image processing - PubMed (original) (raw)