nuclear envelope Flashcards
open mitosis
nearly all higher eukaryotes do this
NE disassembles completely each mitosis when condensing/segregating chromosomes
need to reassemble at end of mitosis
NE reassembly
requires ESCRT complex
comtains CHMP4B
not seen earlier on when segregation occurring
late-anaphase/telophase - ESCRT proteins begin interacting
transient - gone in late telophase
important in sealing NE back together
KO proteins involved - get breaks and leakage when assembling nucleus
huge complexity
kinases
phosphatases
ESCRT in yeast
ESCRT proteins trace at least back to yeast (lower eukaryotes)
undergo closed mitosis
but
S. japonicus undergoes intermediate version
holes in nucleus
seen via NLS-GST mCherry reporter not localising to nucleus as no intact NPCs to enrich them there - so diffue throughout cell
vps4 ESCRT protein
delete
get lots of broken off pieces of envelope that never reconnect w NE
NPC channels
NPC has 8 fold symmetry
complex structure
has central channel
39nm max cargo
cytoplasmic proteins through here
peripheral channels that can handle 10nm
-ER is continuous w nuclear envelope, proteins from ER can pass through these peripheral 10nm channels
NPC v labile
components can dynamically connect and break apart
allows removal of clogs
NPC structure investigation
immuno-gold EM
Ab to GFP tag w gold on it for EM
can also IP/MS to see interactors of each protein
can use this localisation and IPMS interaction data to build model of each protein relative to each other
NPC transport directionality
Ran-GTP/GDP gradient
higher GTP inside (GEFs inside nucleus)
higher GDP outside (Ran-GAPs outside nucleus)
importins:
can bind the FG repeats in the NPCs via their own FG motifs (hydrophobic - prefer to be with other FGs within the NPC)
bind cargo in cytopasm
go through NPC
bind Ran-GTP inside and release cargo
exit
Ran-GTP->GDP - releases of importin
Exportins:
bind Ran-GTP in nucleus
can bind cargo in nucleus now
exit via NPC
GTP-GDP
cargo and Ran released
NPC components and genome function
NPC components can facilitate disassembly of the NPC
can retain diff regions of chromatin association with NPC
mammalian:
involved in silencing in
and also in activation (genes right next to the pore complex can have their mRNA shuttled out quickly
yeast:
act as boundary elements
transcriptional activation on one side
repression on the other
soluble NPC components:
-associate more with genes involved in tissue development/differentiation
-associate w origins of replication
-mitosis: NPC proteins associate w spindle
-Ran-GTP associates chromatin w MT spindle
NPC proteins and cancer
many specific cancers associated w particular NPC proteins
Nup - Nucleoporins
many cancer related fusion proteins w NPC proteins and other genes
NPC proteins and viruses
many viruses shown having interactions w NPC proteins to facilitate entry to nucleus
Nup98 targeted by HIV
Nup63 by herpesvirus
NPCs can help in viral disassembly so while thing can get through
-things can go wrong at this step
-eg if oriented wrong, components can go into cytoplasm and not through NPC and be detected by cytoplasmic sensors
-if virus stresses NPC too much it gets blocked and virus captured there
HIV capsid and NPC
wider than central channel
dynamic interaction of components allow stretching of this central channel to be big enough to accomodate HIV without it dissasembling
if HIV disassembles too soon then viral genome will be detected in cytoplasm
need to protect genome until docked on NPC
>specific interactions between viral capsid and NPC proteins (cytoplasmic filaments, basket, etc…)
HIV and Nup153
Nup153 interacts with the integrase inside the virus (so virus needs to be close to pore)
though Nup153 overexpression inhibits virus activity though
maybe because it is strengthening the capture to the NPC too mich - prevents virus from going on to do other stuff in interaction - lowering replication
LINC basics
LIncker of Nucleoskeleton and Cytoskeleton
50nm spacing between ONM and INM
NPC numbers alone not enough to maintain this spacing across the double memrane
have SUN domain and Nesprin proteins
>Nesprin: binding motif that binds actin directly and IFs (via plectin), and to MTs through motor proteins
> Sun1: dimer with coiled coil domain that goes through intermembrane space
binds to domains of nesprins that stick through the membrane
Sun 1 and 2 act redundantly - KO both = spacing between ONM and INM becomes less regular
some TM proteins bind lamins and some chromatin directly
LINC complex trimer
trimer of SUN and other KASH proteins interacting in the intermembrane space
usually drawn as coiled coil but this is impossible for this structure
less than 300AAs are present in the trimeric “coiled-coil” region
lamins need 350AAs to make 40-50nm (distace btwn O/INM)
would need even more to make a trimeric coiled coil of this length - so the proteins here are too short
would need some random unstructured coiling in there to make it long enough instead of an organised coiled coil
this unstructured region would allow the KASH/SUN complexes to form a mesh network with many more KASH/SUN complexes
this would be better at distributing force from cytoskeleton and chromatin from each side of NE
parts of the protein sequence of Sun and KASH domain proteins that would exist in the lumen space:
only very short regions of this sequence predict coiled coils
SUN-KASH complexes also form diverse 6:6 assemblies
SUN2 and meiosis
SUN2 localises at meiotic telomere attachment sites on the NE
connections btwn telomeres and NE help align chromosomes when homologues are recombining
millions of bases in condensed chromosome
lots of force
this concentration of SUN proteins at the telomere-connected regiosn of the NE allows cytoskeleton to link with the telomeres and help support them
SUN1-SPDYA at meiotic telomere tether interface
point mutations in SPDY protein can affect SUN binding
get phenotype with small testes - reproduction affected
i guess failed meioses leading to apoptosis
in this mutant -loss of connectivity of telomeres lined up on the nuclear envelope
