Quantifying Nucleoporin Stoichiometry Inside Single Nuclear Pore Complexes In vivo

Lan Mi,Andre Lindquist,Alexander Goryaynov,Michael Rexach,Weidong Yang

doi:10.1038/srep09372

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.

Highlights

The nuclear pore complex (NPC) is one of the largest supramolecular structures in eukaryotic cells
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
For fourteen of twenty four other Nups analyzed, the maximum copy-number per NPC measured here was in agreement with previous estimates of 16 copies of Gle[2], Nup[49], Nup[53], Nup[57], Nup[82], Nup[84], Nup[116], Nup[133], Nup145C, Nup[170], Nup[188]; and 8 copies of Nup[1], Nup[60] and Nup1591,11

Summary

Introduction

The nuclear pore complex (NPC) is one of the largest supramolecular structures in eukaryotic cells. Due to the inherent difficulty of counting proteins within individual supramolecular complexes in live cells, all of the above reports of Nup stoichiometry per NPC represent best estimates of the average, rather than direct, eye-witness counts from individual NPCs. A more accurate account of Nup copy-number within individual NPCs of live cells should produce better and more realistic 3D-architecture map of NPCs. Precise information on subunit stoichiometry could change our understanding of the function, biogenesis and compositional dynamics of NPCs. Here, we utilized a singlemolecule fluorescence imaging approach called single-point edge-excitation sub-diffraction (SPEED) microscopy[17,18] to directly count the copy-number of GFP-Nup molecules within individual NPCs of live yeast. This novel approach permitted a real-time visualization of Nups within single NPCs, and their quantitation produced a significantly-revised report of subunit stoichiometry for the S. cerevisiae NPC

Methods

Results

Conclusion