Nuclear Architecture and Trafficking

Question 1

cell nucleus

Answer 1

mothership of the human genome

Question 2

structures in the eukaryotic cell

Answer 2

-nucleus
-cytoplasm
-mitochondria
-nuclear envelope
-nucleoplasm, which is continuous with cytoplasm
-lipid bilayer

Question 3

mitochondrial membranes

Answer 3

-2 membranes: outer and inner
-outer membrane compartment –> space in between that is the destination for proteins –> inner membrane –> matrix space where proteins can and do need to localize to make the mitochondrion work

Question 4

nuclear membranes

Answer 4

-2 membranes: nuclear envelope with inner and outer membranes
-includes space that is continuous with ER luminal space –> ER emanates from the nuclear envelope

Question 5

where did the nucleus come from?

Answer 5

-when cells were simple cells and had nucleic acid DNA, there were reasons it was advantageous to attach the DNA to a surface
-when DNA has to divide, duplicate, and spread apart, easier to do that when you’re tethered with left or right directions instead of separating in 3D space
-anchoring proteins can hold it all in –> before there was a nucleus (no enclosure), it was a continuation of the plasma membrane that got pinched off and this could be the way the nuclear envelope was originated along with the ER membrane system

Question 6

3D view of the nuclear envelope and ER

Answer 6

-holes are the pores of the nuclear pore complexes
-pore complexes occupy that hole and control what goes in and out
-ER emanates from the nuclear envelope
-outer and inner nuclear membranes are connected at the pores

Question 7

major structures of the nucleus

Answer 7

-outer membrane –> luminal, aqueous space –> inner membrane
-nuclear pores
-heterochromatin- densely compacted chromatin (silent genes)
-euchromatin- actively expressed genes
-ER
-nucleolus- easiest, earliest object recognized with no border –> creates factories to make ribosomes

Question 8

nuclear envelope and nuclear ‘lamina’ networks

Answer 8

-main protein responsible for circularity shape and size –> lamin filaments
-lamina- lamin filaments that are close to the NE since that’s where they’re most concentrated
-they protect the genome mechanically and adaptively from any insult and they anchor the NPCs –> without lamina, the pore complexes drift together and stick
-responsible for rebuilding nucleus after each mitosis- nuclear structure entirely disassembles reversibly
-customize 3D chromosome architecture of each chromosome, which is mediated by which parts are silent –> silent parts of chromatin somehow get associated with lamina near the nuclear envelope

Question 9

retinal cells + heterochromatin

Answer 9

heterochromatin is balled up in the middle and lets light move through efficiently

Question 10

nuclear pore complex

Answer 10

-2 major ring structures with 8 fold symmetry
-total number of proteins- type of proteins that go making up a NPC is ~30 distinct proteins encoded by different genes
-center of NPC has disordered FG repeat-containing nups –> very greasy, swivelly proteins with Serines that can be modified by a sugar (not rigid at all) and together they form a hydrophobic region
-100 nm diameter in yeast and 120 nm in vertebrates

Question 11

nucleoporins (“nups”)

Answer 11

proteins that make up an NPC and all of the NPCs have at least 2 copies of every nup

Question 12

NPCs ‘occupy’ pores and are anchored in the NE membrane

Answer 12

assemble in the pores and few of the nups are integral membrane proteins that are anchored and hold onto themselves on the backside (creating gromit structure to anchor pore complex and controls pore) –> keeps pore from expanding and destroying the NE

Question 13

NPCs are joined by LINC complexes

Answer 13

-SUN-domain proteins and KASH-domain proteins (nesprins)
-SUN-domain proteins- integral membrane proteins with nucleoplasmic domain that binds lamins and KASH domain claws in
-a lot of SUN domains and nesprin genes with a lot of diversity
-nesprins (outer membrane) have ‘talons’ that are disulfide-bonded to SUN domains (inner membrane)

Question 14

how does diversity of nespirin genes come about?

Answer 14

-nespirin genes can create little proteins, big proteins, medium proteins from alternative splicing, transcription, and translation
-nespirins can bind actin filaments, connects plectin to intermediate filaments in the cytoplasm, directly to motor proteins, and drag entire nucleus on microtubules
-nespirins can bind to both directions of motors

Question 15

actual LINC complexes have 3 SUN domains and 3 KASH domain proteins

Answer 15

-assembly and disassembly of these complexes can be regulated –> not gluing the envelope the same way all the time
-KASH domain has some prolines that let it kink and disulfide bonds to one SUN domain and kinky talons sticks into the next SUN domain

Question 16

what is the role of LINC complexes?

Answer 16

take mechanical force that was applied to the outside of the cell and pushes it directly into nucleus –> lamina networks respond by being flexible and springing back

Question 17

distance between nuclear envelope membranes is controlled by LINC complexes (SUN-proteins)

Answer 17

different proteins or genes code for different lengths of this triple alpha helix region

Question 18

lamins are nuclear intermediate filament proteins

Answer 18

-don’t have directionality like an actin or microtubule does –> no motors pulling things along
-ancient, oldest members of the intermediate filament protein family- all animal cells have at least one lamin gene they’re expressing

Question 19

lamin filaments are major components of the nucleoskeleton

Answer 19

-3 genes that code for 4 distinct types of lamins- LMNA, LMNB1, LMNB2 with LMNA encoding lamins A and C by alternative splicing
-all cells express at least one B-type lamin, A and C come in as cells start to not be stem cells anymore and help with cell type specificity
-lamin filaments and their networks are flexible, interconnected by elastic and springy types of proteins
-high tensile strength with breaking force

Question 20

each lamin ‘self-assembles’ (polymerizes) to form strong, distinct rope-like filaments

Answer 20

-each of these newly synthesized lamin proteins make coiled-coiled structures
-regions of lamins coil together and give them stiff rod domains
-a little bit at the N terminus and lots at the C terminus with binding partners
-yields lamin molecules (subunits) –> subunits polymerize ‘head to tail’ –> two ‘head-to-tail’ polymers align side-by-side, staggered, in opposite directions
-lamins assemble –> polymerize to make head-to-tail polymers that are at least 2 of them in opposite directions and polymerization is reversible by phosphorylation during mitosis –> when mitosis is over, de-phosphorylate these sites and it can reassemble

Question 21

lamin filaments concentrate near the NE

Answer 21

-nucleoli exclude lamins
-lamina network can spring back and attached proteins are part of dynamic response (major protective role)
-lamin A and lamin C also localize in the nucleoplasm

Question 22

nuclei evolved to flexibly handle mechanical force INTERNALLY and EXTERNALLY

Answer 22

-forces come from all directions like within the nucleus with large and dense chromosomes (dealt with as masses) plus forces of replication and translation
-from the cytoplasm with microtubule polymerization with microtubules pushing straight into the nucleus, motors that drag nucleus, and actin dynamics
-external forces can squeeze through tiny spaces
-cells can experience contractile forces in their cytoplasm plus gravity

Question 23

3D architecture of chromosomes is tissue-specifically ‘customized’ by association with lamin filaments and NE membrane proteins

Answer 23

-heterochromatin, the more compacted forms of nucleosomes with silencing-specific histone modifications
-euchromatin is more loose and open –> expressible genes being actively transcribed
-chromosomes individually have left and right ends –> heterochromatinized and loops in –> heterochromatized and loops in

Question 24

what controls what is tethered vs loose?

Answer 24

cohesin complexes

Question 25

what are characteristics of heterochromatin and euchromatin?

Answer 25

-liquid domain that self organizes and helps itself blob together -liquid compartment may be true for transcriptionally active regions --> stay away from each other because of intrinsic properties that are conferred by proteins that bind to them -heterochromatin protein one- liquid phase compartmentalizing protein- binds to a nuclear membrane protein and to lamins

Question 26

what is one way to ID lamin-associated DNA (LAD)?

Answer 26

-mark them with a DAM ID mechanism- put an enzyme that methylates certain sequences in DNA --> put it on lamins --> let it mark up all the DNA that's close to the lamins and figure out which DNA that was --> you get LADs in one color and non-LADs in another -LADs is typically associated with heterochromatin, which are methylated

Question 27

what are characteristics of LADs/non-LADs?

Answer 27

-cell-type specific --> whatever is heterochromatin is constitutive heterochromatin but parts interested in for cell specificity are genes that can be turned on if it were that developmental fate allowed -variable LADs are most interesting in terms of how cell fate is controlled and maintained in given cell types

Question 28

what is another way to label LADs and non-LADs?

Answer 28

you can label them different colors Ex. chromosome 12 shows you that the chromosome is organized with LADs near the nuclear envelope and non-LADs further inside being actively expressed

Question 29

nuclear envelope

Answer 29

-lamin filaments anchor all of the nuclear membrane proteins with a single exception -every known characterized nuclear membrane protein can bind laminae C and B -bind lamins to stay at the inner nuclear membrane --> perfectly capable of going to outer membrane

Question 30

Emerin is retained at the inner nuclear membrane by binding to lamin A filaments...what happens in cells that lack lamin A?

Answer 30

-Emerin would localize equally amongst ER and NE membranes so it's free to randomly diffuse (in lipid bilayer) throughout the connected NE/ER membranes -you'll see emission at the inner membrane, equal [] at the outer membrane, and equal [] all over the ER membranes

Question 31

mutations in LMNA cause many tissue-specific disorders

Answer 31

-priscilla lopes-schleip has an autosomal dominant missense LMNA mutation that causes familial partial lipodystrophy (FPLD2) -jill has autosomal dominant LMNA mutation that causes emery-dreifuss muscular dystrophy (EDMD) -other 20 clinical named phenotypes that are caused by single amino acid changes in lamin A and/or lamin C --> difference in how long tail is -range of conditions that are dominant with some exceptions

Question 32

laminopathy (disease) mechanisms may differ in each affected tissue

Answer 32

-hard to pinpoint what's being affected by single amino acid change -could be nucleus is now mechanically weak or not responsive, it might be perturbed signalling in response to something in certain cell types, it could be perturbed functioning of any kind of protein

Question 33

nuclear pore complexes (NPCs) mediate traffic into and out of the nucleus

Answer 33

imported from cytoplasm: -proteins are translated in the cytoplasm by ribosomes --> if the correct location is inside the nucleus, they need to be imported through pore complexes Ex. transcription factors, signalling proteins, replication factors exported to cytoplasm: -ribosomes (small and large subunits) get assembled in the nucleolus -mRNAs, tRNAs, and snRNAs

Question 34

NPCs control the entry and exit ONLY for large molecules

Answer 34

-small stuff (<40 kD) can freely diffuse through the NPC -large stuff (>40 kD) can pass if they have a nuclear localization signal and cannot go through without it, requires a soluble receptor for signal, and abundant Ran-GTP inside (Ran-GTP 'gradient')- this has to be abundant inside the nucleus in the GTP bound state (Ran-GTP gradient)

Question 35

how was NLS discovered?

Answer 35

-a virus that made a protein that normally went into the nucleus and stayed there -mutation in virus was blocking the virus replication pathway and single Lysine to Threonine mutation --> protein localized in cytoplasm -realized that it was 5 positive charges in a row interrupted by Threonine and sufficient to disrupt signal -if you move this signal into a different protein, that other protein will localize to the cytoplasm

Question 36

what are the different types of NLS?

Answer 36

-classic NLS: PKKKRKV -'bipartite' NLS: KRxxxxxKxKK -'proline-tyrosine' NLS: looser consensus and only ~80 human proteins use this

Question 37

export requires a nuclear export signal (NES) on the 'cargo' protein

Answer 37

-equilibrium distribution in cytoplasm -proteins that have nuclear import as well as export signal -hide NLS by phosphorylating it, cutting it, etc. to control when used -with an NLS, things equilibrate to the cytoplasm -if NES is removed, proteins stay in the nucleus -NESes are hydrophobic and some proteins have both NLS and NES

Question 38

proteins DO NOT UNFOLD during nuclear import/export

Answer 38

anything up to 25 nm in diameter can get through NPC if they're coated with nuclear import signals

Question 39

NLS and NES signals are recognized and bound by soluble receptors

Answer 39

cargo protein has accessible NLS (on the surface) --> NLS is recognized by importins and once they bind they can cross the NPC -->favored to release cargo inside due to Ran-GTP gradient -export signal recognized by exportin and bind Ran-GTP --> must have both to leave

Question 40

importins and exportins

Answer 40

-22 genes in humans -if you had mutation in importin that only recognized these 80 random proteins then you will have some disease -redundancy in recognition of common signals

Question 41

NLS or NES must be ACCESSIBLE on the cargo surface

Answer 41

-degrade the protein that covers the signals by phosphorylation or dephosphoryation and also protein can get in with no signal if it's tightly binding to a complex or partner protein with signal (piggyback entry) -large proteins can go in without a signal if they're piggybacking

Question 42

Protein don't know where they are --> system set up based on what is uniquely inside the nucleus (DNA)

Answer 42

-chromatin is inside the nucleus and directionality is interpreted by a protein called Ran, small GTP-binding proteins, which do not burn GTP- they use GTP to switch themselves on -Ran is loaded with GTP inside the nucleus (protein that is needed to empty out its GDP site only happens where chromatin is) -as soon as it's cobound with something being exported and sent out, Ran gets hydrolyzed so it only has GDP to go out in the cytoplasm

Question 43

what does Ran-GEF do?

Answer 43

-associated with chromatin (=inside) -helps Ran release GDP, allowing GTP to bind and switch Ran "on", creating a HIGH [] of RanGTP in the cytoplasm

Question 44

RanGTP 'cycle' or 'gradient'

Answer 44

-GEF-GTP exchange factor- protein that sees GDP and scoops it out to let GTP re-bind -GAP- GTPase activating protein- Ran-GAP is the other protein that helps Ran hydrolyze the GTP to become GDP and turn itself off --> only happens on the cytoplasmic side since RanGAP tends to associate with filaments that are unique to one side of the pore complex

Question 45

RanGTP has opposite effects on importin and exportin

Answer 45

-chromatin is needed for the GEF to make Ran loaded with GTP again --> RanGTP is high in the nucleoplasm and displaces cargo of anything that just came in -if it's an importin, Ran muscles out the cargo -when exportin wants to go out, Ran needs to be there to co-bind with cargo and exportin is allowed to pass -receptors shield the cargo from hydrophobic environment

Question 46

mRNAs are covered with heterogeneous nuclear RNP

Answer 46

-class of RNPs are complicated and coat mRNA in nucleus that have specific jobs -mRNA is coated as it's being spliced -export-read RNA is a big thing with a lot of proteins associated -goes out 5' end first and rest gets squeezed out --> translation can start as soon as first end is out

Question 47

what is another way mRNA can get out of the nucleus?

Answer 47

-by not going through the NPCs at all -proteins in the inner membrane fuse to form vesicle that carry mRNA that fuses with the outer membrane to export mRNA

Question 48

Nucleoli are factories that surround the 100s of copies of genes encoding rRNA

Answer 48

-newly-transcribed rRNAs are processed and co-assemble with freshly-imported ribosomal proteins -large and small ribosome subunits are then EXPORTED to cytoplasm

Question 49

nucleolus and other 'membrane-less' compartments are created by 'organizer proteins' that promote liquid-liquid phase partitioning

Answer 49

-liquid 'condensates' are DYNAMIC- controlled by PTMs -organizer proteins have three key properties: intrinsic disorder, multivalency: mulitple binding sites for self-association and binding sites for proteins/RNA to be []ed, and when purified, form 'liquid droplets' -Ex. heterochromatin protein 1

Question 50

what happens if there is a loss of a nup?

Answer 50

the NPC will be able to expand and cause a huge pore in the nucleus