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
LINC in the DNA damage repair mechanisms
if DNA break takes long to repair
can recruit break to NE
NPC and LINC involved in this
hold them there
facilitates the broken ends finding each other
can stimulate repair
via NHEJ in G1
-ectopic repair in G2/S
-telomere fusions
KO LINC proteins - see issues in DDR
Lamins and DDR response
Lamin A KO
shortened telomeres seen
increase in chromosome nreaks and gamma-H2AX foci
LINC and lamins may work together in DDR
STING protein shown to be involved in DDR via interaction w lamins
(is also an adapter for cytoplasmic sensing of nucleic acids)
so involved in innate immunity and interacts w lamins
LINC nuclear positioning
nuclear positioning important in myotubes
multinucleate cells
need to be clustered beneath neuromuscular junctions to efficiently
SUN/Nesprins involved in positioning nuclei by mediating their interaction with the cytoskeleton
(id assume here that it is to MTs via motor proteins in order to move them)
KO these - see nuclear positioning problems
chromosome territories early experiment
irradiate location in interphase nucleus w focused beam
radiation damage is distributed to only a couple chromosomes but not all
consistent w idea that uncondensed chromosomes in interphase exist in territories and not all mixed together
chromosome positioning basics
gene poor chromosomes
=more compact at nuclear periphery
more active chromosomes
=more internal and spread out
simple trend - bit more complex
different TM nuclear envelope proteins tether chromatin to the periphery
eg Emerin
binds BAF
which binds chromatin
eg inner membrane LBR
binds HP1 (a amrker of heterochromatin)
cell type differential chromosome localisation
concentrations of genes on some chromosomes more important in specific tissue
but less in another
so more internal and peripheral respectively
in cancer:
there tends to be translocations between adjacent chromosomes
since different chromosomes are adjacent in different tissues
some translocations will be more common in some tissues than other
changes in gene expression in development and chromosome positioning
changes in epigenetic markers and activation of factors in differentiation
>get movement of chromatin from cell periphery to internal area
activatory localisation to nuclear envelope
some chromatin localisation to envelope can be activatory rather than repressive (like usual)
if have mechanism to localise certain regions of genome to periphery
and also a mechanism that localises transcriptional regulators to NE
can enrich certain regulators near specific genes
these regulators can be activatory or repressive
Global mapping of genome-NE contacts
looking for LADs (lamin associated domains)
DamID
fuse Dam methylase to lamins
DNA near envelope gets methylated (lamins in envelope)
purify genomic DNA
selectively amplify adenine methylated DNA (ie DNA that was near envelope
-this marks the regions of the genomes that associate at the periphery
different LADs seen in different cells
LADs can change in differentiation
some LADs are constitutive - can see in all cells
LADS and genes
look at relationship between active genes and LADs
gene density lower at LADs
gene expression lower too
epigenetic markers for active chromatin lower
silencing chromatin markers higher
NE localised genome tends to be silenced
heterochromatin and NE protein mutations
most cells have heterochromatin near the NE
in disease
eg AD-EDMD/ x-linked EDMD
heterochromatin broken away from NE forming islands
MAD and Hutchison gilford progeria
see distribution of heterochromatin basically spread out throughout nucleus
NET39 gene targeting
repositions to the NE during myogenesis
repositions genes there - coinciding with their repression
if can retarget it to nucleolus
its targets will go there too
Muscle NETs affect many myogenic genes
affect many of the genes that change gene expression in myogenesis
loss of NETs (NE targeting proteins) causes reduction in myotube organisation
likely due to much reduced differentiation signals
point mutations ;inked to disease (die to blocked gene repositioning activity)
NE recruitment and steric access
NE recruitment could have silencing affect from the affecting of steric access by transcriptional regulators to the genes
Hi-C genome interaction
can look at the 3D genome organisation
cross-link DNA
cut w restriction enzyme
fill ends and mark them w biotin
ligate the ends of these fragments - genes that are close to each other in 3D space will be ligated
creating new sequence from them combining
pull down via biotin
shear DNA
sequence the fragments
can find these new sequences and see what sequences are close to each other in the genome
when plot frequency of interaction of different regions of chromosomes
get strong signal along diagonal (close in sequence)
signal outside this diagonal represents regions distal in sequence but close in space
hypothesis of constrained diffusion
enhancers and genes kept away from each other by envelope association
then when released from NE periphery they can interact with each other
nuclear envelope proteome
eg the types of NETs present
majority of these proteins are tissue specific
makes sense as they regulate what genes are peripherally localised
Nuclear envelope and disease basic
since the NE proteome is very tissue specific
mjutations in NE proteins can cause tissue specific pathology
eg many differetn types of Lamin A present in different tissue types
so different disease phenotypes arise depending on which tissue subtype is affected by the mutation
