Quantifying nucleoporin stoichiometry inside single nuclear pore complexes in vivo

Scientific Reports
Lan MiWeidong Yang

Abstract

The nuclear pore complex (NPC) is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring-scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. NPCs are composed of ~30 different nucleoporins (Nups), averaged at 8, 16 or 32 copies per NPC. This estimate has not been confirmed for individual NPCs in living cells due to the inherent difficulty of counting proteins inside single supramolecular complexes. Here we used single-molecule SPEED microscopy to directly count the copy-number of twenty-four different Nups within individual NPCs of live yeast, and found agreement as well as significant deviation from previous estimates. As expected, we counted 8 copies of four peripheral Nups and 16 copies of fourteen scaffold Nups. Unexpectedly, we counted a maximum of 16 copies of Nsp1 and Nic96, rather than 32 as previously estimated; and found only 10-15 copies of six other Nups, rather than 8 or 16 copies as expected. This in situ molecular-counting technology can test structure-function models of NPCs and other supramolecular structures in cells.

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Citations

Feb 24, 2016·Nucleus·Monika ZwergerOhad Medalia
Mar 6, 2016·Journal of Molecular Biology·Siegfried M Musser, David Grünwald
Oct 5, 2016·The Journal of Cell Biology·Benjamin L TimneyMichael P Rout
Dec 22, 2016·Nature Reviews. Molecular Cell Biology·Martin Beck, Ed Hurt
Oct 8, 2016·Integrative Biology : Quantitative Biosciences From Nano to Macro·Alberto GarciaManuela T Raimondi
Feb 17, 2017·Journal of Cell Science·Jiong MaWeidong Yang
Jul 3, 2019·Proceedings of the National Academy of Sciences of the United States of America·Pascal VallottonKarsten Weis
Apr 11, 2018·Proceedings of the National Academy of Sciences of the United States of America·Sasikumar RajooKarsten Weis
Sep 11, 2021·Proceedings of the National Academy of Sciences of the United States of America·Yichen LiWeidong Yang
Sep 30, 2021·Molecular Biology of the Cell·Johan HummertDirk-Peter Herten

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Methods Mentioned

BETA
flow cytometry
confocal microscopy
super-resolution microscopy
fluorescence imaging
electrophoresis
ion exchange chromatography

Software Mentioned

Slidebook

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