Lecture #2 - Nuclear Structure Flashcards

Question

Genes for SUN domain proteins

Answer 1

Humans have 5 gens encoding SUN-domain proteins Genes have different lengths of the alpha region - SUN1 and SUN2 have longer neck - SUN 3 = has even shorter neck - SUN4/5 = smallest neck – only expressed in certain cell types Type of SUN determines how wide apart the inner and outer memebrane is

Answer 2

Intermediate filaments = doing many things in cytoplasm - Intermediate filaments don't have directionality = no motors pulling things along Cytoplasmic IF = Keratine + Vimentin/Vimentin related filaemnts + Neurofilaments

Answer 3

Nuclear IF = nuclear lamins - Nuclear lamins = oldest protein in Intermediate filaments - Found in all aminal cells Function – form major component of nuceloskelaton Lamin Filaments are major components of nucleoskelaton

Answer 4

3 humans genes code for 3 distict lamin filaments: 1. LMNA – codes for lamin A and lamin C - Get lamin A and lamin C by alternative splicing - A and C come in when the cells are starting to know their fate so they can start differentiating to be the right cell type 2. LMNB1 – lamain B1 3. LMNB2 – lamin B2 ALL cells express at least 1 type of B lamin

Answer 5

1. Flexible 2. Interconnected by elastic springing porteins (ex. Spectrins + titins) 3. Extremely strong when extended (have high tensile strength) - They will bend and then snap one structure to get bigger BUT Then they will break = Can take a lot of force but ultimtley they are breakable 4. Filaments have no directionality = no motors

Answer 6

Each lamin self-assmbles (polymerizes) to form thin strong distict rope-like filaments Two proteins are made in the cytoplasm (organge and yellow)) --> Newly syntehszied lamins first dimerize via special alpha-helix to form stiff coil-cloil domain (rod) After rod --> get lamin molecules (Subunits) --> Sununits polymerize head to tail (if stayed like this there would be directionality) --> two head-to tail polymers align side by side + staggered in opposite directions Polymerization is reversible (ex. Mitosis) - Can phosphorylate and the head to tor polymer falls apart the after mitosis you can dephosphorlate and the network will reassemble

Answer 7

Two proteins are made in the cytoplasm (organge and yellow)) --> Newly syntehszied lamins first dimerize via special alpha-helix to form stiff coil-cloil domain (rod) --> Forms backbond of future filament (stiff rod domain is the backbone of the filament - Single SU of lamina = single molecule Have extra protein at the N and lots at the C terminal that is not in the rod domain (back parts stick out) - Things can bind to these C and N temrinus regions (I THNK THAT THE N terinus is more for the head to tail polymerization and C temrinus is for binding partners) Image – see the lamina molecule have rod structure and then lumps at N and C terminus

Answer 8

No directionality because the two filaments will get together in opposite staggard directions (laterally) = had to tail polymerase (considered ‘polymerization) - Because staggered = have parts that stick out more frequentley when 2 have associated side by side - Opposite side staggaring likely happens when in smaller SU

Answer 9

Microtubes (24nm) ; Actin (8nm) ; Vimentin (10nm) VS. Lamins = MUCH thinner (3.5 nm) Even though Lamins are thin they are string enough to anchor the Nuclear Pore complex Image – red dots are the tails that stick out

Answer 10

Lamins = make filaments near nuclear envelope Lamins know what the other filament types are doing even though they are not connected - If get rid of lamin A --> the rest of the filaments freak out (ex. Get herneations of the nuclear envelope)

Answer 11

Lamins were first purified from rat liver cell nuclei - In liver – lamins form an unsually thick layer (‘lamina’) - Image – lamina layers = thick layer in grey --> Lamina proteins – found in grey layer + heterochromatin + place heterochromatin is placed away from pore complex + where pore complexes are located Have 1 million of each type of lamin (1 million lamin A ; 1 million lamin C ; 1 million lamin B1 ; 1 million lamin B2) = have 4 million binding sites - 1 million binding site smight be for proteins that need to bind to lamin A or C ; might have some bidning sites for petins that will ONLY bind to A and not C

Answer 12

Lamina filaments concetrate near the Nuclear envelope (seen in flourescence confocal) Image – shows concentration of the lamina filaments (confocal through center of round object) - Brighter at the edges because have more signal in floruensce BUT you do still have thiner lamina A and C throughout the center of the nucleus (localize to the nucleoplasm) - Lamnina A and Lmanina C = NOT going into the nucleolus

Answer 13

Demonstartion with the ball that extends out Overall - lamina stretch to a certain place - Intermediate filaments take force (flexible) until you stretch them and then they stay extended - Nuclei don’t start maximum extended because then they will break too easily --> Lamina will stay in the crouched smaller position so they can extend more or can go down more and spring back

Answer 14

When sitting in nuelcei and genome is being squished but nuceli are OK because the lamina are doing they job Proteins that connect to laminas are part of dynamic responses to mechanical force (major protective role) Nuceli evoloved to flexibly handle mechanical force

Answer 15

Within the nucleus: 1. Chromosomes are large and dense --> chromosomes have gravitational pull that needs to be dealt with 2. Replication and transcription machinery generate force --> need anchor points for force generation From cytoplasm: 1. Microtubule polymerization --> microtubules might try and push into nucleus 2. Actin Dynamics 3. Motors can DRAG the entire nucleus to distant locations in cell - Nucleus needs to be able to hang on and come

Answer 16

1. Squeezing through tiny spaces (ECM + capillaries + exit blood vessels) --> Cells squeze through tight spaces 2. Contractile forces on the cytoplasm (skelatal + smooth and cardiac muscle) - Lamins = important for muscles 3. Expansion and Shear forces (endothelial cells + airway epithelium) - Cells in ariway pressore or blood flow pressures 4. Gravity and Mass (Ex. Sitting on nuclei)

Answer 17

3D architecture of chromsomes in tissue specially customized by association with lamin filaments and Nuclear envelope membrane proteins Heterochromatin = compact form of nucleosomes + has silencing histone modifications Eurochromatin = expressed (balls on Eurchromatin = Transcrtion Factors) - Eurochromatin = father from the NE Chromsomes heterochromatin is near the Nuclear envelope and then will loop in and be eurochromatin THEN will loop back and be hetrochromatin near nuclear envelope - Who is tethered and loose is controled by cohesin complexes

Answer 18

Lamines would be in same area as heterochromatin (Heterochromatin = associates with lamins) Heterochromatin = itself is in part a liquid domain that self organizes AND the same kind of liquid compartment may be true for euchromatin Euchromatin and heterochromatin stay away from each other because of the intrinsic properties due to proteins that are bound - Example – Heterochromatin HP1 (liquid phase compartimilization protein) binds to nuclear memebrane protein and to lamins

Answer 19

Marks LAD with a DAM ID mechanism --> fuse enzymes that DNA methyalates certain sequences in DNA --> put enzyme on lamins --> enzyme will mark up the DNA that is close to the lamin --> find DNA that was close to the lamin = get LAD - Has been done for whole chromosome (know ehere the LAD is) - LAD defined the heterochromatin (Heterochromatin = associates with lamins) Example – LADs found on chromosome 12 (LAD = red vs. Non-LAD eurochromatin = blue ) Image - Can stain for LAD vs. Non-LAD

Answer 20

LAD = cell type specific (Ex. Different in muscle cells) Have things that are always heterochromatin (never expressed) BUT the cell type specific genes are turned on in certain developmental states - Variable LAD (tissue specific) = more interesting for cell fate control and cell fate maintance)

Answer 21

Each chromosome occupies a territory BUT can intermingle with close neighbors - NOT a harsh boarder --> more that the chromosomes are generally in that area BUT they can still interact with one another Image - Chromosomes can be painted different florescent colors and visualized by microscopy

Answer 22

NE has outer and inner membrane + pore complex + nuclear membrane proteins - Know we have proteins that bind to BAF NE = unique compartment with hundreds of resident membrane proteins - NE membrane protein localize by binding to lamin filaments Image – shows lamin filaments anchor nuclear membrane proteins (have 1 exception) - All known nuclear membtane proteins bind lamin A/B/C

Answer 23

Proteins bind to lamnins to stay at the inner nuclear membrane - To bind and stay at the inner membrane the proteins need to bind to something that is only in the nucleus = bind to lamins or chromatin Proteins can bind to the outer membrane

Answer 24

ANSWR – D --> If have mutations in lamin A and lamin B --> proteins can move freely/diffuse = will do random diffusion – proteins won’t be concentrated in any 1 place (get equal concentrations in ER and equal in the inner membrane and equal in the outer membrane) - If proteins can diffuse = Proteins are not functionally localized where they needed to be (could get LOF of all the proteins that might be depending on binding to a specific site on the lamin)

Answer 25

Mutations in LMNA (or other lamins) cause many tissue specific disorders Images – shows people with laminopathy phenotypes - Both have the same lamina A mutations --> one has a muscular body types and prominnet veis (sisters) - Priscilla = has autosomal dominant LMNA missense mutation that causes FPLD2 - Jill = has autosomal dominant LMNA mutaions that casues EDMD (Has LMNA mutation and supressore in somad 7 = more severe phenotypes )

Answer 26

Have disease models in Drosophilla (can model complex disease in flies) Have Jill and Prisiclla flies - Flies show why Jill’s pheotye is so severe ; Pricilla flies are fine - Jill flies = stay on the bottom because their muscles are weak + see exacerbation of phenotypes when have supressor mutations

Answer 27

Difference of Lamin A and Lamin C = lamin A has a longer tail Have 20 phenotypes that are caused by a single amino acid change in lamin A or lamin C - The 20 single Amino Acid chnages make no sense (The mutations map all over the molecule (not one place always being chnaged)) - Have ranges of conditions (Ex. Heart disease or nueropathy) - All single chnages that are dominant (only need 1 copy)

Answer 28

Hutchinson-Gilford Progeria Syndrome = Defects in protolysis for cutting precursors to lamin A = Failure to cut off back end of lamin A= leads to accelerated aging (now can be treated) Missence/deletions cause segmental progeroud syndromes (accelerated aging)

Answer 29

Changing 1 amino acid in lamin A can disrcupt any aspect of function leaidng to: 1. Mechanical weakness or rupture of nucleus or the nucleus is not responsive 2. Perterbed signaling (ex. In repsonse to mechanical force) 3. Perturbed function of proteins or multi-protien complexes that must bind lamina 4. Perterbed 3D organization of one or more chromosomes - Essential for differentiated cells to know who they are an which genes to turn on or off in response to signaling

Answer 30

Affect of perterbed function of proteins or multi-protin complexes that must bind lamina A: 1. To localize correctly (Ex. At the inner nuclear memebrane) - Protein might have a place where it needs to bind to something else to do its job --> if the binding site is not available then it can’t do its job 2. Function correctly 3. Co-assemble with partners 4. Regulate signaling or chromatin modifying proteins or transcription factors 5. Remain inactive

Answer 31

Nuclear pore complexes = mediate traffic into and out of the nucleus - Proteins are translated in the cytoplasm and need to get to the correct location (If location is in nucleus THEN they need to be imported through NPCs) Nuclear pore complex control the enter and exit of larger molecules

Answer 32

1. Signaling proteins 2. Regulatory rpoteins 3. Transcription Factors 4. Histones 5. Replication Factors 6. Ribosomal proteins (assembley of ribosomes is in the nucleus) 7. Nucleolar proteins 8. SnRNPs

Answer 33

1. Ribosomal SU --> Ribosomes are asmbled in the nucleolus and then need to get out of the nucleus 2. mRNA complexes 3. tRNA 4. snRNA

Answer 34

Nuclear pore complex control the enter and exit of larger molecules - Only control for large things (lager can't diffuse freely) - Larger = can go to the nucleus if that have a nuclear localization signal Small round/globular things (<40 kD) - can freely diffuse through NPC (enter/exit freely) - Histones = smaller than 40 kD BUT histones still have a nuclear localization signal to make the movement even more efficient

Answer 35

Larger things (>40 kD) requires: 1. A peptide signal on the protein or its parter (NLS) 2. A soluble receptor that binds the signal 3. Enough Ran-GTP inside the nucleus (uses the Ran-GTP gradient) - RAN = RNA binding protein ; Ran = abundent in the nucelus (needs to be in GTP state)

Answer 36

Import requires a nuclear Localization signal (NLS) on the cargo protein - If proteins has NLS = it goes to the nucleus BUT only goes to the nucleus if the sequence is on the surfce of the protein (needs to be accesible to the receptor) Types of sequneces: 1. Classic NLS = PKKKRKV (60% of nuclear proteins) 2. Bipartide NLS – KRxxxxxxxxKxKK --> Xxx needs to be able to loop out to bring the two positive amino acids together 3. Proline-Tyrosine NLS – 30 disorder hydrophobic or basic residues followed by (R/K/H)-X(2-5)-PY - Weird NLS – has looser concenseus seq - Only 80 human proteins use this NLS

Answer 37

NLS - Found because virus made protein that normally goes into the nucleus and says in the nucleus IF have mutations in the virus that blocks viral replication pathway --> NOW the virus localizes in the cytoplasm and can’t enter the nucleus Found +++++ Amino acids --> if interrupt the sequence then the virus does not go to the nucleus ; IF add the NLS signal to a protein then the protein will go to the nucleus

Answer 38

Nuclear export requires a Nuclear export signal (NES) on the Cargo protein Protein with NES = Have equilibrium distribution in the cytoplasm - With active NLS – things will equibrilate (mainly to the cytoplasm) ; IF remove NES = protein stays in the nucleus Proteins with NES usually also have NLS because they had to go into the nucleus first in order to be sent out NES – harder to predict because hydrophobic

Answer 39

Protein goes in = NLS doesn't matter anymore ; the NES can be used and the protein would be sent back out --> creates a futile cycle To break the cycle = phosphorylate the NLS = hide the NLS --> NLS can’t be seen by the receptor - Alternatively can cut off NLS - Can hide NES or NLS to control when the signal is used on the protein

Answer 40

Males are male because they have post translational control by modification of import and export signals that make sure that the genetilia developes properly (need right import export signaling control)

Answer 41

Proteins do NOT unfold to go in and out of the nucleus --> because anything can go through the pore complex if coated by nuclear pore signals - Protein are pre assembled and then go through the pore Anything up to 25 nm with an accessible NLS or NES can go through the nuclear pore complex

Answer 42

NLS and NES signals are recognized and bound by soluble receptors (receptors = importins and exportins) IF proteins have accesible NLS --> NLS is recognized by importins and importins bind to NLS --> complex diffuses across the complex disordered region in NPC --> THEN importins will release the cargo into the nucleus - Importins = abundent in the cytoplasm - Release happens because of Ran Exportin recognizes proteins with NES --> exportin binds to NES and then the complex will bind to Ran-GTP --> complex will exit the nucleus - Need exportin + Ran-GTP to take things out

Answer 43

Encoded by 22 genes in humans Some recognize common NLS or NES - Have redundancy in importins that recognize common signals BUT not specialized signals Some are specialized for rare NLS or NES Mutations can cause strange phenotypes because certain proteins fail to enter or exit the nucleus - No importin = not getting proteins into nucleus

Answer 44

The signal (NLS or NES) must be accessible on the cargo surface - Proteins COULD get in with no signal IF the protein is bound to a complex that does have a signal ('piggy back entery') Transport is often controlled by partners or modifications that hide or expose the signal --> Exposure or covering of signals can be controled in many ways - Can degrade protein that covers a signal = NOW access the signal - Can phosphorylate or dephosphylate a signal

Answer 45

Answer – E

Answer 46

Directionality = interpreted by Ran (used for import/export) - System = based on DNA (uniquely inside of the nucelus) Ran = small GTP-binding protein - Uses GTP to switch on/off (GTP = on ; No GTP = off) - OVERALL - Switched off in the cytoplasm THEN reloaded and switched on in the nucleus

Answer 47

Inside nucleus – Ran is reloaded with GTP (Ran-GTP) = switches Ran on - Ran-GTP = swicthed on Happens inside the nucleus because the protein used to make site empty for GTP (remove the GDP) to be able to fill in only happens where chromatin is because the protein that helps Ran remove GDP to add GTP is associated with chromatin - MY NOTES- JUST saying that only add GTP in teh nculeus because the protein that removes teh gDP needs chromatin

Answer 48

Outside the nucleus – Ran is forced to hydrolyze GTP (now have Ran-GDP) When Ran is bound to something being exported out --> Ran gets hydrolyzed = has GDP the minute it gets to the cytoplasm - Ran-GDP = swicthed off

Answer 49

Ran binds to GTP BUT it needs GAP to hydrolyze GTP and turn Ran off (get Ran-GDP) THEN Ran-GDP can re-enter the nucelus BUT can’t release GDP by itself = Ran needs Gef Ran is a bad GTPase itself = can’t hydrolyze or remove GDP itself = needs Gef and Gap

Answer 50

Ran binds to GTP BUT it needs GAP to hydrolyze GTP and turn Ran off (get Ran-GDP) - Gap = GTPase activating protein --> Gap helps Ran hydrolyze GTP to GDP - Gap = associates with NPC filaments on the cytoplasmic side Turn off = happens in the cytoplasm because Ran-Gap tends to associate with filaments that are on that side of the pore complex - Filaments on basket side of the pore complex are different proteins

Answer 51

Ran-GDp can re-enter the nucelus BUT can’t release GDP by itself = Ran needs Gef Gef = GDP exchange factor --> when Gef sees GDP it will remove the GDP --> NOW GTP can bind = switches Ran on - Creates a high concentration of Ran GTP in the nucleoplasm Gef = assciates with chrimatin (only on the inside of the nucleus)

Answer 52

Ran-GTP has opposite effects on importin Vs. Exportin to make sure cargo is released inside and exported cargo gets outside For importin - RanGTP = low in cytoplasm and RanGTP is high in nucleoplasm - For importin – RanGTP competes/dipalsaces cargo stimulating cargo release (release occurs because Ran pushes cargo off) For exportin – Exportin wants to take somthing out = Ran needs to be there to co-bind with cargo--> NOW the exportin is loaded = allowed to pass (exportin can exit when Ran-GTP co-binds)

Answer 53

Ran-GTP are high in the nucleus (defines the inside of the nucleus) Chromatin is needed for Gef to make Ran loaded with GTP = Ran GTP is high in nucleoplasm - RanGTP - Job in nucleoplasm = displace the cargo of anything that just came in

Answer 54

Importins and exportins = shaped like shrimp --> bind cargo on bendy side and backside is greasy - greasniess = how they slither through the hydrophobic disordered compartment in a pore)

Answer 55

High Ran-GTP in the nucleus has known mechanisms impacts on what happens to cargo Exportins get back in by themselves

Answer 56

mRNA use different mechanisms compared to porteins mRNAs are covered with hnRNP proteins and exit either via NPCs or by entering Nuclear envelope vesciles - RNPs = RNA binding proteins that coats the mRNA (proteiins coat the mRNA and have specific jobs) - mRNA is coated with proteins as it is being spliced - Export ready mRNA has many proteins on it (has been visualized in slik worms)

Answer 57

Export ready mRNA has proteins (C shape) - blue image - Image - see the mRNA is leaving with the 5’ end first and the rest is squeezed as it goes through the pore - Because 5’ end goes first – translation can start once the first end is in the cytoplasm

Answer 58

1. Pore complexes 2. Really large mRNA/mRNA that needs to travel get out without the Nuclear pore complexes - Proteins bud on inner membrane (makes a vesicle from the inner membrane = get vesicle with RNA inside --> vesicle fuses to the outer membrane --> release the RNA into the cytoplasm - Happens in neurons - Exploited by viruses

Answer 59

Answer – A THIS IS not about using energy it is about switching Ran on/off

Answer 60

The nucleus also has conserved membarne-less compartments where essential RNA-protein machines are being built or recycled/stored - Ribosomes assemble (tranlation machinery) - Storage/recycling of mRNA splicing machinery - Assmeble of snRNPs that remove introns during mRNA splicing

Answer 61

Nucleoli are factories that surround the hundreds of copies of genes encoding rRNA - Newly transcribed rRNAs are processed and co-assemble with freshly imported ribosomal proteins --> Large and small ribosome SU are then exported to the cytoplasm - 1 rRNA = 1 ribsome (not expanded) - RNA gets made where genes are --> factories assemble around them Ribosome proteins get made in the cytoplasm --> proteins will come back in will bind to RNAs to make ribosome SU --> SU will be exported

Answer 62

Can have 1 or 10 nucelolus - Each nucleus have 10 chromosomes (5 chr with 2copies each) - 10 chromosomes can have repeated RNA genes in 100s of copies --> repeated genes get transcribed to rRNA Have large nuclei if making lots of ribosomes OR could have 2 or 3 nucleoli because they couldn’t get the chromosomes together - 1 big nucleus if have al 10 chromosomes together to do in 1 place - 10 chromosomes each contribute a loop containing rRNA genes to nucleus

Answer 63

Nucleolus and other ‘membrane-less’ compartments are created by ‘organizer proteins’ that promote liquid-liquid phase partitioning Liquid ‘condensates’ are DYNAMIC --> controlled by posttranslational modifications Organizer protein(s) have three key properties: 1. Intrinsic disorder (~50% or more) 2. ‘Multivalency’: Multiple binding sites for self-association and binding sites for proteins/RNAs to be concentrated 3. When purified, form ’liquid droplets’ Examples: Fibrillarin & nucleophosmin (nucleolus) - Heterochromatin protein 1 (Hp1a)

Answer 64

Youtube video (minutes 17-46) - https://www.youtube.com/watch?v=AP47mIkd-h0 what organizer proteins are - how they interact with one anotehr + binding sites + what proteins or RNA they receruit = create plates that cotrate the right place for proteins to do the job