Quantifying nucleoporin stoichiometry inside single nuclear pore complexes in vivo.

Area of Science:

• Cell biology
• Molecular biology
• Biophysics

Background:

• The nuclear pore complex (NPC) is a massive cellular machine regulating transport between the nucleus and cytoplasm.
• NPCs comprise approximately 30 distinct nucleoporins (Nups), with estimated copy numbers of 8, 16, or 32 per complex.
• Previous estimates lacked direct confirmation in live cells due to technical challenges in counting proteins within single NPCs.

Purpose of the Study:

• To directly count the copy number of 24 different nucleoporins (Nups) within individual nuclear pore complexes (NPCs) in live yeast cells.
• To validate or refine existing models of NPC stoichiometry and composition.
• To establish a method for in situ molecular counting of supramolecular structures.

Main Methods:

• Utilized single-molecule SPEED microscopy for direct, quantitative analysis of Nup copy numbers in live yeast.
• Focused on counting 24 specific nucleoporins within individual NPCs.
• Applied advanced imaging techniques to resolve protein counts at the single-molecule level within a complex biological structure.

Main Results:

• Confirmed 8 copies for four peripheral Nups and 16 copies for fourteen scaffold Nups, aligning with prior estimates.
• Discovered a maximum of 16 copies for Nsp1 and Nic96, deviating from the previously estimated 32 copies.
• Quantified 10-15 copies for six other Nups, differing from expected 8 or 16 copies.

Conclusions:

• Single-molecule SPEED microscopy provides accurate in situ quantification of protein copy numbers in NPCs.
• The study reveals significant deviations from established Nup stoichiometry, necessitating revisions to current NPC models.
• This methodology offers a powerful tool for investigating the structure-function relationships of NPCs and other large molecular assemblies.