In-cell architecture of the nuclear pore complex and nuclear membrane remodeling during its assembly and turnover

  • Datum: 18.12.2019
  • Uhrzeit: 13:00 - 14:00
  • Vortragende(r): Matteo Allegretti
  • EMBL Heidelberg
  • Ort: Max-Planck-Institut für biophysikalische Chemie (MPIBPC)
  • Raum: Tower IV, Seminar Room, 2nd Floor
  • Gastgeber: Prof. Dr. Patrick Cramer
  • Kontakt: office.cramer@mpibpc.mpg.de
In-cell architecture of the nuclear pore complex and nuclear membrane remodeling during its assembly and turnover
The nuclear pore complex (NPC) is an essential membrane protein complex in
eukaryotes shaping a hole in the nuclear envelope. Due to its dimensions (~100 MDa
in H. sapiens) the elected method to determine the structure of the intact complex
scaffold has been cryo electron tomography and subtomogram averaging (vonAppen,
2016) in combination with crystal structures from purified components (Hoelz, 2019).
In our study we perform extensive cryo-FIB-milling coupled to cryo-electron
tomography and subtomogram averaging to get structures of the entire NPC scaffold
in exponentially-growing cells with and without protein knock-outs. The new
structures not only largely overcomes in resolution previously determined structures
with the same method (Mahamid, 2016; Mosalaganti, 2018), but also shows significant
differences in sub-complexes architecture to previous in vitro work (Kim, 2018),
emphasizing the importance of determining structures of membrane proteins in their
native membrane context. We combine the in cellulo structures with integrative
modeling and crosslinks analysis and show that the main mRNA export platform architecture
reveals a new configuration that accommodates spatially and temporally the existent
biochemical data. The new mRNA export architecture discloses also the cytoplasmic exposure
of an NPC-intrinsic autophagic receptor (Lee et al, NCB 2019). We validate the NPC-Atg
interaction obtaining high-resolution snapshots using correlative light and
electron microscopy on plastic sections (Kukulski, 2011), and 3D-cryo CLEM (Arnold, 2016) demonstrating
that NPC degradation occurs through membrane remodeling events at the nuclear envelope and
passes through an autophagosome intermediate owning NPC-containing nuclear
vesicles in the cytoplasm. Our results highlight the power of in cell structural biology
to provide novel insights into two fundamental processes of eukaryotic life.
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