Nuclear Domains Flashcards

Question

molecular players in mechanotransduction

Answer 1

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

Answer 2

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)

Answer 3

interact w nesprins embedded in INM domains located in the periplasmic space then interact w proteins lying just underneath the nuclear envelope (Emerin, Lamins)

Answer 4

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)

Answer 5

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

Answer 6

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

Answer 7

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)

Answer 8

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

Answer 9

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

Answer 10

-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

Answer 11

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)

Answer 12

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

Answer 13

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

Answer 14

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

Answer 15

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

Answer 16

Linked to laminopathies hutchison-gilford progeria - advanced aging in children also -in coiled coil a-helical regions: cardiomyopathies -in globular domain - Associated with HGP

Answer 17

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

Answer 18

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

Answer 19

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

Answer 20

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

Answer 21

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

Answer 22

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

Answer 23

loops around a scaffold proteins form and organise loops anchored to scaffold 70x compaction loops DNA into Topologically Associated Domains TADs

Answer 24

highest compaction driven by SMC, motor proteins, nucleosomes, histones gives rise to mitotic chromosome shape

Answer 25

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

Answer 26

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

Answer 27

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

Answer 28

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

Answer 29

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

Answer 30

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

Answer 31

localises to chromatin forming large loops then Condensin I comes in and extrudes nested loops within these larger Condensin II formed loops

Answer 32

separate regions of dense heterochromatin and less dense euchromatin

Answer 33

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

Answer 34

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

Answer 35

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

Answer 36

peripheral: gene poor less transcription more hetrochromatin internal gene rich more transcription more euchromatin

Answer 37

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

Answer 38

fluorescence in nucleus photobleach part position of bleaching pattern in mother cell reappears in daughter patterning is inherited and is not randomly established

Answer 39

diff cells use diff sets of genes position relates to transcriptional activity chromosome positioning changes in differentiation

Answer 40

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

Answer 41

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

Answer 42

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

Answer 43

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

Answer 44

instead of primers to amplify biotinylate idk how this works though

Answer 45

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

Answer 46

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

Answer 47

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

Answer 48

stain for RNA pol II see transcription structures

Answer 49

stain for pATM then gamma irradiate cell to induce double strand breaks stain shows where activity happens

Answer 50

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

Answer 51

are depots for splicing factors splicing factors move in and out all the time

Answer 52

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

Answer 53

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

Answer 54

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

Answer 55

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

Answer 56

concentration of splicing factors not clear if just storage or provide extra activity

Answer 57

mysterious may have function or could also be storage depot for several types of proteins also act to sequester different factors to affects expression

Answer 58

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

Answer 59

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

Answer 60

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

Answer 61

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

Answer 62

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

Answer 63

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

Answer 64

go into nucleus to cajal body some level of assembly here

Answer 65

contain PML proteins and SUMO -Promyelocytic leukemia protein -Small ubiquitin-like modifier

Answer 66

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

Answer 67

proteins in PML bodies are SUMOylated SUMO motif added on Lysine (K65, K160, K490)

Answer 68

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

Answer 69

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

Answer 70

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

Answer 71

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

Answer 72

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

Answer 73

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

Answer 74

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

Answer 75

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

Nuclear Domains Flashcards

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