VL 32 (Ralph Gräf) Flashcards
Nuclear envelope dynamics at the G2/ M transition and NEBD
- open mitosis → nuclear membrane disassembles in prophase
- A: late G2; intact nucleus
- B: prophase; folding of nuclear envelope (dynein runs along MTs to (-)- end→centrosome);→tension; inhibit dynein→no nuclear envelope breakdown
- C: later prophase; rupture of nuclear envelope due to disassembly of
lamin network (phosphorylation by CDK1) - NPC phosphorylation → NPC dissociation before nuclear envelope breakdown → protein entry; membrane-bound complexes remain
Nuclear import and export: the nuclear pore complex (NPC)
- transport into nucleus during interphase
- 3000-5000 NPCs/cell
- 90-120 mio Da
- ca. 30 different proteins = nucleoporins = NUPs (orruring in a multiple of 8)
- 16x larger than ribosome
- Diameter: 120 nm
- Pore size: 30-40 nm
- Passive metabolite diffusion; globular protein till 60 kDa (also depends on shape, protein species)
- Disassembly into soluble subcomplexes during mitosis; re-assembly starts in telophase
- up to 1000 molecules/s
- export: RNPs, ribosome SU, t/mRNAs
- import: TFs, chromatin components, ribosome proteins, nuclear lamina components
- transport requires NLS/NES; retention: NRS
Y-complexes form cytoplasmic, nucleoplasmic ring
- import receptor (karyopherin) importin β for proteins with NLS
- importin α cooperates with importin β (downregulated) →importin β not always with importin α
- importin β binds α through NLS-like sequence
- cargo-complex interacts with FG-repeats in NPC
- import complex dissociation through Ran-GTP
- association: Ran-GEF + Chromatin
- Ran-GAP at cytosolic filaments
➔ gradient: Ran-GDP in cytoplasm; Ran-GTP in nucleus
➔ directed transport
Specific nuclear export:
Specific nuclear export:
* export receptor binds NES + Ran-GTP = export complex
* interaction: export complex + FG-repeats → shuttle
* Ran-GDP dissociates export complexes
Specific nuclear import and export
FG repeats:
* In NPC
* natively unfolded → no structure; mesh of protein chains → barrier for proteins > 60 kDa → bigger proteins have to phase separate into mesh
* other solubility environment
→ “hydrogel” structure with H2O
* →phase separation
* NTF-binding sites
Nuclear transport of nuclear envelope-associated proteins:
- Outer nuclear envelope → Inner nuclear envelope
(a) Diffusion/retention model; peripheral NPC channels
- lateral diffusion
- directed transport
- proteins secondary bound to other structures at inner nuclear membrane
(b) Ran-dependent with FG-repeats
* Membrane protein diffusion in outer nuclear membrane envelope
* Transport over cytoplasmic domain
* 8 NPC-segments are permeable between segments → lateral transport
Summary: specific nuclear transport
- Formation of the import complex within the cytosol (cargo- NLS/import receptor with or w/o adapter)
- Docking of the import complex at the cytosolic fibers of the NPC
- Import through interaction of the import receptor with NPC proteins (import requires no ATP)
- Exchange of GDP for GTP at Ran at chromatin (RCC1)
- Ran-GTP binds to import receptor within the nucleus and dissociates the import complex
- The import receptor-Ran-GTP complex travels through the NPC back to the cytoplasm
- If import adapters are involved, the import adapter-cargo- complex is dissociated upon binding of an export receptor and Ran-GTP.
- The export receptor/import adapter/Ran-GTP complex travels through the NPC back to the cytosol. Now the import adapter itself is the cargo.
- Ran-GAP at cytosolic fibers of the NPC provides Ran-GDP for import.
- Ran-GTP dissociates import complexes and forms export complexes. Ran-GDP dissociates export complexes
- Inner nuclear membrane proteins are concentrated at the INM either through Ran dependent transport or diffusion/retention
Nuclear pore complex assembly
- after mitosis
- early-telophase: membrane cisternae from ER associate with chromatin mass
- NPC incorporation
Mitosis – early telophase: encasing of nucleoporins during envelope assembly
Interphase: nucleoporin incorporation in existing nuclear envelope membranes
Cell cycle phases:
- M-phase: mitosis, cell division (cytokinesis)
- Interphase: cell growth, DNA replication; G0-phase branches off G1
- Checkpoints: biochem. regulatory circuits, regulating transitions between cell cycle phases (DNA damage checkpoints in G1/2, S, spindle checkpoint: M)
- G1/2 (gap)
- G0: fully differentiated cells; entry into cell cycle due to signals
- S: replication; centrosome duplicatio
Cell cycle regulation:
* regulation by
–> Phosphorylation and dephosphorylation
–> ubiquitylation/degradation
main regulators:
* CDKs + regulatory SU (= cyclin)→activity
Overview: cell cycle regulation
G1- Phase
- decision: exit for differentiation (= G0) or proliferation
- Cdk activity absence→regulatory protein network repressing genes for cell cycle progression
- DNA quality control: damages→stopped cell cycle→apoptosis, if repair failed
- Repressive network may be inactivated by external signals such as mitogens sent from neighboring cells
→overcome restriction point
→S-phase
G1-phase: restriction point regulation
Molecular players:
* Rb = retinoblastoma protein (mutated in retinoblastoma); tumor suppressor protein
–> E2F = TFs, association with DP1
→ form: functional dimer
–> Histone deacetylase
→3 different Rbs, 10 different E2F, 2 DP1
Absence of mitogens:
* resting state – restriction point not overcome:
TFs→no gene transcription due to Rb-inhibition
–> Rb recruits histone deacetylase→heterochromatin
Mitogens (local acting hormones) present
* Tyr-kinase receptor-binding
* Map (mitogen activating protein) signaling cascade
–> TFs phosphorylated
* Cyclin D expression
* Rb phosphorylation
→dissociation: Rb, histone deacetylase
* RNA-Pol II-binding
→gene transcription
