TY  - GEN
KW  - nuclear pore complex; Schizosaccharomyces pombe; nucleoporin; nup; cryo-EM; cryo-ET; electron tomography ;subtomogram averaging; cryo-FIB milling; NPC; dilation
N2  - Nuclear pore complexes (NPCs) reside at the nuclear envelope (NE) where they mediate
nucleocytoplasmic exchange. They are large macromolecular assemblies built by several
hundred protein building blocks, so-called nucleoporins (Nups), which form numerous
subcomplexes to fuse the inner and outer nuclear membranes and form massive 8-fold
rotational symmetric nuclear pore. Solving the structure of NPCs as a whole imposes a
tremendous challenge. Yet, integrated structural analysis approaches led to the determination of the human, S. cerevisiae (sc) and the green algae C. reinhardtii NPC scaffold
architecture, in situ, in vitro and in cellulo, respectively. It is apparent that NPCs across
species, although varying in their subcomplex composition and stoichiometry, follow a
common architectural building concept. Three distinct rings stacked across the NE form
the NPC scaffold. The inner ring forms a central channel, and together with the flanking,
cytoplasmic and nuclear rings, ensures the nuclear pore stability. The outer rings are built
by a head-to-tail arrangement of the so-called Y-complex. In humans, two concentric rings
form each of the two outer rings while in S. cerevsiaie only one Y-complex ring on each
side of the NPC is found. In algae, two Y-rings form the nuclear ring while only one ring is
present on the cytoplasmic side. The metazoan-specific Nup358 mediates the cytoplasmic
Y-complex dimerization in human, however, the corresponding factor mediating the nuclear
ring dimerization remains elusive in all species to date. It is suspected that NPCs might
adapt their central channel diameter in response to a large variety of cues to alter nuclear
transport. However, what exactly causes such a conformational change within actively
transporting cells and how it is mediated on a molecular level is not known.
Here, I solved the structure of the S. pombe (sp) NPC within intact cells. I show
how a split Y-complex interface on the cytoplasmic face of the spNPC breaks the outer
ring conformation and thus challenges the long-standing dogma of a three-ringed NPC
architecture. I found two concentric Y-rings on the nuclear side of the spNPC. A candidate
protein to mediate this interface was the nucleoporin Ely5, which is only found in species
showing two concentric nuclear rings. However, upon analyzing the NPC architecture of an
ely5? knockout strain, I could rule out Ely5 as a sufficient Y-complex dimerization factor.
In collaboration with Matteo Allegretti, we solved an in cellulo scNPC structure and show
how it differs from the previously proposed in vitro derived architecture. We re-orient
the previously proposed mRNA export platform model anchored at the cytoplasmic ring
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to be more in line with the spatiotemporal events coordinating mRNP nuclear export. I
further present an in-depth comparison between the two yeast to the human and algal
NPC architectures revealing that while the inner ring remains highly conserved, the
architecture of the outer rings, the mRNA export platform, as well as the connections
between subcomplexes varies more than previously anticipated. To understand how NPCs
adopt their conformation during active nuclear transport on a molecular level, I solved
a spNPC structure under energy depletion conditions where active nuclear transport is
thought to be completely halted. This analysis exhibits a constricted NPC conformation,
revealing how the NPC scaffold undergoes a conformational change on a molecular level in
response to a well-defined cue. Finally, I investigate how the transmembrane nucleoporin
Pom152 (hsGP210), a protein known to form a ring surrounding the NPC within the NE
lumen, might be involved in NPC diameter control. By analyzing a human gp210? cell
line I could show that while GP210 is dispensable for proper NPC biogenesis, the missing
luminal ring did not influence the human NPC diameter observed under the conditions
described here.
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/28356/
ID  - heidok28356
Y1  - 2020///
TI  - In cellulo architecture of the nuclear pore complex
AV  - public
CY  - Heidelberg
A1  - Zimmerli, Christian Eugen
ER  -