Nuclear Domains Flashcards
Nuclear envelope basic
double membrane
outer membrane joined w ER
nuclear lamina supporting inside, located just beneath envelope
numerous perforations - nuclear pore complexes
made from curving of membrane
act as gatekeepers
allows nuclear proteins to be concentrated inside
nuclear traffic basic
import:
proteins required for nuclear structures
-histones
-DNA/RNA pol
-TFs
Export:
transcription and splicing occur outside of nucleus at external ribosomes
gives extra level of transcriptional control
-mRNA
-tRNA
-rRNA
-Ribosomal particles
Nuclear import/export selectivity - Ribosome Biogenesis
mRNA encoding ribosomal proteins transcribed in nucleus
the mRNA is shuttled out of the nucleus
translation of ribosomal proteins outside of nucleus
ribosome assembly occurs inside nucleus
ribosomal proteins imported back in
find rRNA in nucleus - assemble
final ribosomes then exported again
so entails export -> import -> export
Nuclear pore complex structure basic
large 125MDa
50 distinct proteins
multimerise into 8 fold rotational symmetrical structure
form:
-nuclear basket: involved in interactions inside the nucleus (eg chromatin regulation)
-central core
-cytoplasmic fibrils on cytoplasmic side
have diff layers of proteins forming rings to generate the hole
diff rings diff purposes
have cytoplasmic filaments projecting out the spoke proteins
cytoplasmic filament purposes
important in determining traffic
NPC division into structurally similar modules
Halves:
-cytoplasmic
-nuclear
Spokes:
-8 radial spokes
columns:
-16 radial columns
Rings:
-Outer
-inner
-outer
outer and inner rings make up core scaffold
NPC membrane ring
interacts with nuclear membrane
Nuclear pore flexibility
mRNA unwinds itself to pass through
but there is decent flexibility in what NPC can let through
pore can flexibly change in size?
nuclear import speed in S phase
nuclear import generally rapid
during S phase need to import many histone molecules to wrap newly replicated DNA
works out to 2-3 histones imported per second over 8hr period of S-phase
nuclear membrane transport types
Passive:
diffusion
no energy
-metabolites and other small molecules/proteins
Active:
requires energy and transport proteins
-large proteins
-protein complexes
-mRNA
-tRNA
-ribosomal particles
experiemnt for passive transport size limitations
coat dextran sugar w fluorescent probe
inject it into xenopus oocyte
watch how long it takws for diff sized dextrans to enter nucleus
~60-80kDa limit
larger proteins require nuclear localisation signal for import
Nuclear localisation signal
NLS:
Peptide - Stretch of basic Amino Acids
-Lysines/Arginines (K/R)
can tag on to other proteins - cause them to be imported
mutation of it prevents import
can be at beginning, middle, end of protein as long as it is exposed
so can be attached to any protein of interest
E.g. GFP (normally ends up in cytoplasm)
NLS directs their import from cytoplasm
coating with this signal is sufficient for import of a large gold particle
Nuclear export signals
NES
many are a leucine (L) rich sequence
can function autonomously like NLS
certain RNA sequences/structures also work as NES
cytosolic proteins necessary for nuclear import
Nuclear import receptors - Karyopherins
soluble cytosolic components
Experiment for testing cytosolic protein necessity for import
treat cell w Digitonin
permeabilises cell
removes cytoplasmic medium - leaves just some cytoskeleton
take NLS fused fluorescent protein tags
add ATP + cytosolic lysate
or just ATP
when Just ATP no import happened
need cytosolic components
Karyopherin binding
directly bind cargo
or via an adapter protein which binds cargo
binds the NLS
also bind the Nucleoporins (NUPS) in the nuclear pore complex
interact with the Phenylalanine-Glycine (FG) repeats in the NUPs
FG repeat importance in nucleoporins
FG/Phe-Gly repeat containing nucleoporins constitute the PERMEABILITY BARRIER of nuclear transport
form a Phe-Gly meshwork in the NPC core
once you bind and enter the meshwork can then pass through pore
Karyopherin/Import receptor interaction with FG repeats
Cargo binds Karyopherin
Karyopherin has FG binding sites
Binds the FG repeats on the NUPs
Cargo/Karyopherin complex can pass through
Importin-Beta
founding member of karyopherin import receptor family
has ~19 HEAT repeats
one part binds NPC FG repeats
another binds cargo
another binds Importin-Alpha - an adapter that binds cargo
Ran-GTP mediated nuclear IMPORT
High Ran-GTP in nucleus
high Ran-GDP outside
Protein binds import receptor
imported thru pore
Ran-GTP competes w receptor for cargo - kicks it off inside nucleus
Ran-GTP/Karyopherin exits nucleus
Ran-GTP is hydrolysed outside of nucleus
loses affinity fot import receptor
receptor now free for more cargo to bind
Ran-GTP mediated nuclear EXPORT
Exportins
Binds Ran-GTP inside nucleus
this allows it to bind cargo
leaves nucleus
Ran-GTP hydrolysed by Ran-GAP
kicks off Ran-GDP and cargo outside nucleus
Exportins can also bind importin alpha
helps the adaters to get out of nucleus
Establishing the Ran-GTP concentration gradient inside/outside
Ran-GEFs inside nucleus (RCC1)
Guanine nucleotide Exchange Factor
Ran-GDP + Pi => RanGTP
Ran-GAPs in cytoplasm
GTPase activating protein
Ran is slow GTPase
GAPs promote GTP hydrolysis
Keeps GTP form high inside nucleus
and GDP form high in cytoplasm
RanGDP and Pi freely diffuse i guess
Nucleus Mechanosensing
Nucleus and rest of cell are mechanocoupled via the cytoskeleton
pulling the actin filaments changes nucleus shape
Cells respond to mechanical forces in their environment (eg in the ECM)
Cells of most soft connective tissues need to do this to maintain the ECM during development, remodelling, repair
Mechanotransduction
Transduction of extracellular mechanical stimuli into signals
triggering a cellular response through gene expression eg
controls:
-cell motility
-nuclear motility
-gene expression
communication btwn cells and environment
issues can lead to disease
molecular players in mechanotransduction
Nuclear envelope TM proteins
Cytoplasmic players:
-cytoskeleton: Actin, IF, MT
connect to Nesprin family of nuclear envelope outer membrane proteins
Nesprins interact with inner membrane SUN domain proteins (SUN1/2)
then have other proteins embedded in inner membrane:
-Lamin B receptor
-LAP2Beta
These connect directly with IF lamins and with chromatin
Nesprins
Outer membrane TM proteins
connect nuclear and cytoplasmic cytoskeletal networks together
-Nesprin 1/2 - to actin
-Nesprin 3 to Plectin (an IF)
-Nesprin 4 - to MTs, motors, and centrosome
all contain KASH domains - embedded in outer nuclear membrane
Their C-terminus Interacts with SUN domains embedded in the INM (stick out in space between the ONM/INM)
SUN proteins
interact w nesprins
embedded in INM
domains located in the periplasmic space
then interact w proteins lying just underneath the nuclear envelope (Emerin, Lamins)
LINC complex
Nesprins
+SUN proteins
+Emerin
called the LINC complex
linker of Nucleoskeleton and cytoskeleton
-facilitates mechanitransduction
-positions nucleus within cell
-tehter centrosomes to nuclear envelope
-have other domains eg Spectrin repeats and Calpolin Homology (CH) domain (which in Nesprin 1/2 bind actin CS)
Nucleus mobility
nucleus can take different locations in cell depending on context
eg Epithelial cells
-in culture: is in middle of cell
-in tissue: locate towards the basal membrane
eg Myotubes(polynucleate)
-in culture: many nuclei randomly distributed in cell
-in tissue: nuclei found exactly where the synapses contact the cell
Nesprin mediation of nuclear migration -Klarsicht
In drosophila:
Klarsicht (a nesprin) can connect to motor proteins (Dyenin) on the MTs and allow nuclear migration on the MTs
relevant in photoreception
MT and dyenin align the nucleus towards the apical side
in mutants
migration perturbed
many stay towards basal side
this migration failure results in eye morphology/function defects
Human brain neural celll migration dependent on nuclear migration
scaffold of radial Glial cell bodies
other cells migrate on this from ventricular zone to primitive cortex (brain surface)
2 types of movement in these neurons
1. rapid movement by leading neurite
2. followed by second phase which is the cell body following along and also the nucleus (NUCLEOKINESIS)
general neural cell migration dependent on ability to perform nucleokinesis
mutation in eg Dyenin compromises nucleokinesis, can lead to human classical lissencephaly (smooth brain)
neuromuscular junction nuclear localisation
enrichment of myotube nuclei at neuromuscular junction is dependent on Nesprins
1 WT allele sufficient for WT phenotype
need -/- to disrupt
nuclei scatteres not directly below surface junction
SUN domain protein/Nesprin centrosome tethering
centrosome= cytoplasmic organelle
but is v close to nuclear envelope
Mediated by Nesprin 4 (ONM)
connected to SUN domain
connected to Emerin
Centrosome connects to Nesprin 4 tethering it to the nucleus
emerin mutation breaks this connection
INM proteins
-Lamin B receptor!
-Lap1!
-LAP2!
-Man-1
-Emerin!
can make contact to proteins on
outside
and connect to important nuclear structures:
-Lamins (IF)
-Chromatin
many INM proteins that interact w these can affect gene expression
allows mechanical stimulus to transduce signal all the way to chromatin
Nuclear lamina
intermediate filaments
just underneath envelope
meshwork of filaments
nuclear lamins regulate size, shape, mechanical integrity of nucelus
breaks down at onset of mitosis (phosphorylated lamins)
Lamins
Type V intermediate filaments
mutations linked to many diseases
genomic locus:
V large
many exons
typical structure:
-Domain at N-terminal half - Alpha helical rod domain - stiff part of filament
-followed by flexible part - globular domain - the Ig fold, important for contacts inside nucleus (eg w chromatin)
Have 2-6 separate genes
get Lamin A and C from alt splicing
and B1 and B2 isoforms
arrangement of monomers in lamin filaments
alpha helical stiff rod domains connected in filament
globular Ig domains stick out of side
periodicity of Ig domains (connected by flexible linker to rod shaped N terminus domain) are about 20nm apart
relatively short persistence length - measure of stiffness
allows it to be bent in diff directions
Assembly of lamin filaments
Lamin dimer
coiled coil from rod domains
(cross section = 2 a-helices in coiled coil)
both Ig domains toward C-terminus - both at same side of dimer
dimers assemble in staggered head to tail longitudinal assembly
come together overlapping in small region -giving the staggered tetramer
head to tail polymer
can be staggered or half staggered lateral assembly (parallel or antiparallel)
tetrameric cross section at overlap
dimeric at non-overlap
then form protofilament
hexameric overlap region
tetrameric region where not overlapped just 2
Processing steps for mature functional lamins
CAAX region at end is modified
-farnesylation
-endopeptidase
-carboxyl methyltransfer
then important step:
Have Pre-Lamin A:
protease cuts this region away producing mature lamin A
Likewise get mature Lamin B1 and B2 in diff pathways which dont undergo proteolysis - instead undergo farnesylation that remains there
if this important step goes wrong (mutations, protease cleavage region absence)
end up with progerin molecule
Lamin A mutations
Linked to laminopathies
hutchison-gilford progeria - advanced aging in children
also
-in coiled coil a-helical regions: cardiomyopathies
-in globular domain - Associated with HGP
Processing of Progerin protein
eg in HGP
15AA deletion
all the other modifications take place
EXCEPT
protease cannot recognise the proteolysis site
end up with just Prelamin A (Progerins)
has modifications on the c-terminus that is usually cleaved
in HGP imparis nuclear mechanics leading to altered gene expression
leading to premature aging
Genome interaction with nuclear lamina
there are dense heterochromatin regions next to the nuclear envelope
silenced regions
regions of heterochromatin away from lamina are often around the nucleolus
Progeria patient -reorganisation of heterochromatin
all the HC normally under membrane is interior - leading to massive change in gene expression
Mapping Nuclear lamina - chromatin interactions via DamID
DNA methyltransferase from bacteria (Dam)
methylates the Adenosine in GATC sequence
fuse Dam enzyme to Lamin A protein
methylates and hence gives way to ID interacting with the Lamins
this methylation not normally present in Eukaryotes i guess
have one with Lamin A/Dam fusion
and one with just general Dam methylation for control
digest GATC using Dpn1 enzyme
size fractionate cut regions
label Experimental and control diff lables (Cy3, Cy5)
then hybridise them to microarray with wells containing probes for known regions covering the entire genome
colour assay tells us which genomic regions interact with lamins
saw large regions mapped to genome that were enriched for GATC methylation
LAMIN ASSOCIATED DOMAINS
LADs
LADs in diff cell types
look in cells where differentiation was induced and cells in more polypotents state
regions were LADs were not currently in the more polypotent
would then form in later differentiated cells
association w nuclear envelope way to regulate gene expression
solidifying silenced state
TFs are more important so may be extra way to solidify silenced state ON TOP of other regulatory elements
LADs dynamicity
LADs can change in differentiation
Breakdown of Nuclear envelope in mitosis provides opportunity to rearrange envelope-chromatin interactions
lamins phosphorylated by kinases
NE breakdown
after mitosis
membranes assemble around chromosomes in daughter
lamins start connecting to those chormosomes again
Nuclear envelope fragments surround them and then eventually fuse to make new NE
this is stage where breakup can disrupt former LADs and make new ones
-also some heterochromatin regions associated to envelope become associated to nucleolus
Chromatin fibre hierarchical organisation
DNA double helix
7-11x compaction: nucleosome beads on string
30nm fibre - 35x compaction: nucleosomes packed into 30nm fibre (not in humans but in urchins)
higher order organisation
Higher order organisation in interphase
loops around a scaffold
proteins form and organise loops anchored to scaffold
70x compaction
loops DNA into Topologically Associated Domains TADs
Higher order organisation in mitosis
highest compaction
driven by SMC, motor proteins, nucleosomes, histones
gives rise to mitotic chromosome shape
Chromosome scaffold
purify chromosomes
add salt to spread out DNA
2 things seen:
1. loops of DNA
2. Separated scaffold proteins
isoltate this scaffold structure and see if anything important for chormosome formation:
-Topoisomerase II
-condensin
Cohesin in mitotic chormosomes
required for their organisation
SMC = structural maintenance of chromosomes
2 coiled coil proteins
Smc1
Smc3
(are motor proteins)
held together by Scc1
Scc3 bound to Scc1
cohesin complex
forms ring complex that holds the 2 sister chromatids together in mitosis
(can promote loops in interphase?)
-Smc1 and 3 connext with Scc1 to form ring
-Scc3 bound to Scc1
Sisters line up - each to one pole
once properly connected to poles Scc complex begins to degrade Securin (inhibits Separase)
then Separase can cleave Scc1
frees up sisters
Condensin in chromosomes
v similar to cohesin
Smc2 and Smc4 motor proteins and CAP-H form ring
CAP-G/G2 and Cap-D1/2 bound to CAP-H
this ring complex maintains chromosome folding and loop formation
condensin in maintaining Metaphase chromosomes
maintains the folding and loop formation of metaphase chromosomes
add EDTA
chelates Mg
gives amorphous structure of chromosomes, removes their normal structure
SMC2 WT: remove magnesium - chromosomes disappear, add Mg back-chromosomes restore and reappear
can cycle this many times
SMC2 OFF: gets rid of condensin, chromosomes start off fuzzy to begin with
and when remove magnesium chromosome structures completely disappear
adding Mg back gives some compaction but chromo shape is completely absent
meaning:
condensin is important for Establishing AND maintaining the shape of the chromosome
Structural memory of chromosome
helps chromosomes recover their morphology after repeated disruption/packaging
dependent on condensin complex
and non-histone proteins
anchors keep memory intact
get rid of condensin - no way of knowing whate sites of scaffold to go back to
memory lost so chromosomes not restored in restoring Mg buffer
Mitotic chromosome formation Pathway
used Chromosome conformation capture
and highly synced cells
prophase: condensins mediate formation of arrays and consecutive loops
Prometaphase: chromosomes - now a spiral staircase (helically arranged axial scaffold of condensin II at base of loop)
condensin I helps further compaction into clusters of smaller nested loops
gives final 10,000x compaction
Condensin II and I action
localises to chromatin forming large loops
then Condensin I comes in and extrudes nested loops within these larger Condensin II formed loops
arrangement of interphase eu-/heterochromatin
separate regions of dense heterochromatin and less dense euchromatin
Rabl orientation
centromeres located at one side of nucleus
telomeres to other
this configuration seen in embryogenesis during cleavage divisions - no G phases in cell cycle
chromosome territories
Interphase chromosome organisation
chromosome Territories experiment
experiment:
local UV radiation damage part of nucleus
if chromosomes in territories then fewer would be damaged than if randomly intermingled
only 1 or 2 were so supports territories
3d organisation of interphase chromosomes
use multicolour FISH
target diff chromos with diff colours based on sequence
visualises 3d organisation
each chromosome occupies distinct territory within nucleus
proportional to chromosome size
some chromosomes have extensive contact with the nuclear envelope
-located externally
-more heterochromatin - more repressed regions/inactive genes in this cell type
some chromosomes are positioned internally
-will have more neighbours
can interact with more
may explain why certain chromosomal translocations occur with more frequency than others in cancer cells
eg btwn 4, 5, 10 - close vicinity
position of interphase chromosome and gene expression
peripheral:
gene poor
less transcription
more hetrochromatin
internal
gene rich
more transcription
more euchromatin
measuring dynamicity of chromatin in interphase nucleus
photoactivated GFP tags
activate in stripes
in mouse ES cells - lines disappeared completely - has dynamicity in interphase nucleus
in fibroblast no movement of lines - so more stable
Global chromosome position transmitted in mitosis
fluorescence in nucleus
photobleach part
position of bleaching pattern in mother cell reappears in daughter
patterning is inherited and is not randomly established
Chromosome positioning in diff tissues
diff cells use diff sets of genes
position relates to transcriptional activity
chromosome positioning changes in differentiation
Gene migration out of territories
inactive genes located by periphery
if want to activate expression of gene in periphery
can loop it out
can probe certain genes and watch as they migrate out of territory during differentiation
Transcription factories and territories
RNA pol II distrubution is not homogeneous in nucleus
localises together in transcription factories
can cluster these together and loop out certain genes
ensure all components for transcription are localised together
another layer of gene expression regulation from internal chromosome organisation
long range interactions and chromatin loops
enhancer can be distal from promoter of gene (several kb)
but still affect expression
cohesins bind DNA and extrude loop
can bring the distal enhancer near to the promoter
useful for regulating expression and isolating DNA elements away from each other too
Chromosome Coformation Capture (3C)
Can show Distal enhancer and promoter in the sequence are actually together in cell
gives idea of what sequences are interacting
add cross linker
cross links DNA in close proximity (ONLY these close sites)
then digest with restriction enzyme
can ligate these two cross linked pieces together
use known primers of candidate regions to amplify
then sequence the new sequence this ligation created from these interacting sequences
can show promoter regiosn and enhancers that are together and apart in diff cell types
High-C (3C method)
instead of primers to amplify
biotinylate
idk how this works though
diff sequence detection methods for 3C
PCR amplification
-one vs one
-known primers to check if candidate region is interacting
-if not interacting then cannot PCR between bound primers
High throughput sequencing:
-one vs all
Microarray:
-linker mediated amplification
-compare many vs many
biotin fill in/blunt end ligation
-compare all vs all
Activity dependent subnuclear compartments
4 nuclear processes
-replcation
-transcription
-splicing
-DNA repair
can detect subdomains when stain for these particular activities
clusters of certain protein activity
are Membrane-less organelles
and can self organise
DNA replication staining
PCNA stain
clamp protein that loads replication factors
stain it: shows clusters of it
no. of replication forks needed to replicate DNA is more than the no. of PCNA clusters
so the replication factories’ existence depends on replication activity
Transcription cluster staining
stain for RNA pol II
see transcription structures
DNA repair staining
stain for pATM
then gamma irradiate cell to induce double strand breaks
stain shows where activity happens
interchromosomal compartments
site of transcription and splicing
occupied by splicing speckles - storage compartments for splicing factors
couples transcription of new mRNA that needs splicing and the splicing process
so splicing occurs co-transcriptionally
splicing speckles dynamics
are depots for splicing factors
splicing factors move in and out all the time
visualising interchromosomal space
interchromatin compartment
GFP-H2B fusion protein expressing cells
expose to hyper-osmolar medium
undergo chromatin condensation
and expansion of interchromosomal space
amplifies these regions making them easier to see
these are the regions in between DNA occupied by diff subnuclear compartments
nuclear components and compartment dynamicity
unlike cytoplasmic ones where protein enters and stays
either in or out
in nucleus
no membranes
so more accessible
lots of fluidity
compartments are self organised and have their own integrity
but allow for this fluidity of components
Proteinaceous Nuclear Bodies (NBs)
Nucleolus
Speckles
Cajal bodies
PML bodies
distinguished by unique subset of proteins in each
can be sites of activity
OR just storage structures - no meanignful activity within
Nucleolus
site of ribosome biogenesis
-transcriptional processing of rDNA
-rDNA transcribed by RNA pol I in nucleus
-~80% of RNA in growing cells is rRNA
nucleolus also acts as storage compartment for proteins
eg Cdc14 phosphatase - needed for regulation of cell cycle exit of mitosis into interphase
during this it is in the nucleolus
Speckles
concentration of splicing factors
not clear if just storage or provide extra activity
PML bodies
mysterious
may have function or could also be storage depot for several types of proteins
also act to sequester different factors to affects expression
finding out if certain protein localises to a certain nuclear body?
stain that protein
stain protein that is known to be in that NB
see if they co-localise in nucleus
use Ab (paul Mcloughlin shaking in his boots)
or GFP
or can use immunoprecipitation + mass spec to see if protein of interest interacts with proteins in that NB
layers of the Nucleolus
3 distinct layers
inside - lots of rRNA genes - Fibrilar Centre
surrounding nucleolus - Dense Fibrilar componenrs - where pre-rRNA transcripts mature
several proteins break them into pieces so can be targeted to diff ribosome subunits
outside - granular components - site of assembly of pre-ribosome particles into Ribosomes
appears granular during this process
mammal cells have 1-5 nucleoli per cell
Nucleolus Dynamicity
fuse Fibrallarin to GFP
use FRAP (fluorescence recovery after photobleaching)
assesses mobility of molecule
photobleach area of fibrillarin on nucleolus
look for recovery speed of GFP fluorescence
fibrallarin highly diffulible
nucleoplasm has low affinity Fibrillarin sites
nucleolus has high affinity sites
establishes an equilibrium of Fibrillarin in the nucleolus
similar mechanism in many nuclear compartments
-core affinity holds it together
-but many components move in and out dynamically
Nucleolar Organiser regions (NORs)
regions of multiple DNA encoding rRNA genes
found clustered together on few chromosomes
always on edge of Acrocentric chromosome (on P arm i think)
need many rDNA genes as necessary for production of lots of ribosomes in stages where cells are growing and translationally active
NORs where rRNA genes are clustered is site of nucleolus formation
Nucleolus formation on NORs
fusion of multiple NORs
nucleolus disassemble at start of mitosis
at end of Mitosis (G1 onset) - NORs organise together into one or a few Foci
these Foci fuse togetehr in G1 - can end up with 1 or multiple
not all NORs engage in forming nucleolus
some are inactive transcriptionally
-stain all rRNA
-some will be found outside these fused NORs
Cajal Body purpose
processing of snRNAs and maturation of snRNPs (sn = small nuclear)
these are required for SPLICING
contain mature splicing factors stored here
supply them to ready to go splicing machinery during transcription
keep these in diff subcompartments
because in stress conditions
need to generate certain types of machinery
better to have them clustered in close vicinity for higher efficiency
maturation of snRNAs
go into nucleus
to cajal body
some level of assembly here
PML bodies
contain PML proteins and SUMO
-Promyelocytic leukemia protein
-Small ubiquitin-like modifier
PML in acute myeloid leukemia
affected by a chromosomal translocation
15:17 translocations between PML and retinoic acid receptor (RAR)
RAR recruits proteins that repress transcription
by virtue of fusing to PML protein causes it to repress Tumour suppressor genes
retinoic acid can alleviate this repression
treating the cancer caused by it
Post-translational modification of proteins in PML
proteins in PML bodies are SUMOylated
SUMO motif added on Lysine (K65, K160, K490)
SUMOylation pathway
SUMO is small protein added on
similar to ubiquitin process (hence the U)
– SUMO E3 ligase brings together SUMO E2 conjugating enzyme
– bringing of SUMO E3 and E2 to target/substrate causes SUMO to transfer from E2 to target
SUMOylation changes activity of protein
diff SUMO species
can be mono or poly SUMOylated
on lysine residues
carboxy terminal Glycine residue of SUMO forms an isopeptide bond w the amino group of lysine residue
PML body struture
PML constitues part of outer structure of PML body, mono-SUMOylated there
inside of PML body is the poly(2/3)-SUMOylated proteins
many proteins are transiently recruited there
wehre they become SUMOylated
attachment of SUMO changes their properties
SUMO can act like a glue between SUMOylated proteins
PML body protein diffusion
proteins can diffuse in and out
Nuclear localisation signal fused with GFP
photobleach the GFP specifically in PML body
quickly recovers inside
shows high dynamicity in proteins diffusing in and out
Function of PML bodies
increase in size and number under cellular stress conditions or senescence conditions
many proteins sequestered/recruited there to be SUMOylated
sequesteration of transcriptional repressers/activators affects gene expression
PMLbody sequestration of Daxx
cell stress
activates SUMOylation machenery
increase in PML-SUMO
SUMOylated PML interacts with Daxx and sequesters is to PML body
Daxx sequestration leads to Derepression of Pro-apoptotic genes
Formation of heterochromatin domains
active euchromatin has H3K9 acetylation
at some point can be deacetylated
Suvar3-9 methylation comes and methylates H3K9
methylation interacts with HP1 protein via its chromodomain
HP1 recruits more Suvar3-9
cycle
propagates heterochromatin domains
heterochromatin domain formation
-Liquid phase separation
diff proteins can separate based on characteristics
HP1 does this
associate with each other
form droplets
these HP1 droplets fuse together fomring larger droplets forming these domaisn
the droplet domains exclude other proteins incl. TFs - this is how thees domains are maintained
involved in making heterochromatin nuclear subdomains
