Structural heterogeneity of nuclear pores in yeast.

Abstract

Proteins are the workhorses of the cell; they rarely work in isolation but rather in assemblies of multiple proteins. Those that mediate interactions and material flow across membranes, such as the nuclear pore complex (NPC), are particularly fascinating. The NPC is a >50 MDa complex embedded in the nuclear envelope composed of ∼550 proteins of 30 different types (Nups) that regulates nucleocytoplasmic transport between the cytoplasm and nucleoplasm and play critical roles in gene regulation via many functions, including the structuring of chromatin. What is fascinating is how the cell pieces hundreds of Lego-like Nups to form these giants, how these Legos work in concert to enable this giant's function, and provide structural plasticity (i.e., different states). Because NPCs cannot be fully reconstituted in vitro and have in-cell plasticity, studying them in situ is critical. We used cryo-focused ion beam (cryo-FIB) milling to create sections (lamellae) out of snap-frozen yeast cells, cryo-electron tomography (cryo-ET) for 3D visualization of complexes in situ, and subtomogram analysis for high-resolution mapping to reveal the native architecture and structural heterogeneity of the nuclear pores in S. cereviseae. NPC assembly can take up to an hour and mature NPCs exist as different isoforms, each of which may perform specialized functions in nucleocytoplasmic transport and moderating chromatin interactions. Despite evidence pointing to structural heterogeneity, the structural mapping of NPC isoforms remains elusive. Recent advancements in cryo-FIB milling and cryo-ET, as well as the development of our analysis pipelines, have allowed us to perform this mapping. For the first time, we show different isoforms of mature NPCs in situ, including structures of its parts that have remained elusive and reveal a first peak into intermediate stages of NPC assembly.