Lecture #2 - Nuclear Structure Flashcards
Nuclear Pore Complex (image)
NPC = whole thing
- Ball in center = Liquid compartment (determines the size and nature signal needed and receptor to go from the cytoplasm to the nucleus )
Nuclear envelope = boarder zone
Nuceloskelaton = actin + spectrin (both supportive)
Heterochrmatin Vs. Eurochromatin
Heterochromatin = condended DNA (silcenced)
Eukrochromatin = Less dense (expressed ; active genes)
Nucleolus
Nucleolus = liquid phase organelle inside of the nucleus
Nucleolus = denser area in nucelus
Has no membrane boarder (spontenously self organziing structure)
Function - Makes factories to make ribosomes
Function of the nucleus
Nuclear structure = protects and serves the genome
Mutations in lamins or other Nuclear envelope structural proteins cause a spectrum of tissue specific diseases (called ‘laminopathies’)
Diagram of Eukaryotic Cell
Has nucleus
Line around = lipid bilayer (often shown as a single line)
Each organelle = bounded by bilayer
Nucleus memebranes
Nucleus = has 2 membranes (nuclear envelope has inner and outer membrane)
- The luminal space between the membranes (green) is continuous with the ER luminal space (ER emanates from the nuclear envelope )
Center (brown) = nucleoplasm –> aqueous space that is continuous with the cytoplasm
- Have free diffusion all over the inside of the nucleus (water + solute can diffuse fast)
- Little things will free diffuse and will reach an equilibrium between the cytoplasm and the nucleoplasm
Where did the nuclease come from
Before nucleus = fold was continuous with plasma membrane that was pinched off
Think that when cells had DNA there were regions where it was advantageous to attach DNA to a surface
- Easier for DNA to replicate/divide when it is tethered instead of separately in 3D space
- Anchroing protein can fold eveyrthing in
END - Nuclear envelope and Endoplasmic reticulum likely evolved by in-folding of the plasma membrane
- NOW once have things in different compartments need vesicle to bring things in/out
3D view of Nuclear envelope and ER
Outer and inner nuclear membranes are connect at pores ; ER extends out from the nuclear envelope
Image:
- Green = inside of nucleus
- Holes = Pores of the Nuclear pore complex (Holes reach to the inside of the nucleus)
- ER emanates from the nuclear envelope
- In cross section – the outer membrane folds down and becomes the inner membrane inclosing the white luminal space (Luminal space has proteins + water) –> Luminal space has proteins + water
- Pore = where the folds bend
NOTE - YOU SHOUDL BE ABLE TO DRAW IMAGE (of nuclear envleop cross section)
What is in the pores of the Nuclear Pore complex (overall)
Pore complex occupy the holes and control what goes in/out
Major Structures of nucleus
To note:
- Eurochmatin active gene = loop in
- In image – can’t see individual chromosomes (just know they are organized there)
- White space between the membranes = luminal aqeous space
- See nucleolus = denser area in nucelus
- Can see outer membrane –> luminal space –> inner membrane
- Arrows pointing to pores
What proteins organize the shape and size of the nucleus
Lamin filamnets = main protein for organizing the shape and size of the nucleus
Lamin filaments form networls (called ‘Lamina’)
- Lamin filaments close to nuclear envelope = lamina
- Lamina filmanents = most concetrated at the nuclear envelope
What do the lamin filaments do:
- Protect the genome (mechanically and adaptivley)
- Anchor nuclear pore complexes (If lamins are gone – pore complex drift and stick together = issue)
- Mechanically anchor the cytoskeleton
- Rebuild the nucleus after mitosis
- Customize 3D chromosome architecture by tethering silent chromatin (heterochromatin)
- Regulate tissue speficic signaling
What do the lamin filaments do - Rebuild the nucleus after mitosis AND Customize 3D chromosome architecture by tetherting silent chromatin (heterochromatin)
Nuclear structure disassembles - NOT breaking INSTEAD it is reversibly disassembling by disassembling the lamin filament networks and and reuilding the entire struture every time the cell divides
Lamins ALSO Rebuild 3D arcitechture of each chromosomes
- Mediated by the heterochromatin –> heterochromatin is associated with the lamina near the nuclear envelope
Nuclear envelope Proteins
Nuclear envelope includes hundreds of Nuclear envelope membrane proteins
Proteins = involved in nuclear lamina structures + roles in tissue specific signaling + chromatin silencing
- Most are proteins uncharacterized
- We know things about the tissue specific signaling but we don’t know all
- There might be some proteins expressed in specialized cell types (need special nuclear membrane proteins in specialized cel types)
Nuclear pore complex - Structure
Includes 2 major ring structures with 8 fold symmetry (see top ring and the bottom ting)
- 8 proteins that make up each ring (16 total)
Have ~30 types of distict proteins make up NPC (nuceloporins - ‘Nups’)
- All proteins have 2,8,16 or more copies per NPC (16 copies if its in the ring structures)
- High molecular weight per pore complex
Center filled with disordered Phe-Gly (FG) rich domains
NPC = 120nm in diameter in vertabertes
Center of NPC
Center (speghetti looking thing) filled with disordered Phe-Gly (FG) rich domains (proteins have repeats of Phe-Gly throughout the length)
- Proteins can have Ser that is modified by a sugar
Function - Acts as hydrphibic barrier – makes hydrophobic region where need the right things to go through to enter and exit
- Greasy swiveling porteins (NOT rigid)
Where are NPCs
Nuclear pore complexes = occupy pores and are anchored in the Nuclear envelope membrane (assemble in pore zone)
Some of the Nucleoporins ate intergral membrane proteins that hold onto the membrane
- Intergal proteins = make a gromet structure that anchores the core complex and controls the pore so the pore does not get too big (Anchored and hold onto the backside)
- Function - Integral porteins = control the size of the hole (size control is part of the assmebly of the pore complex)
What links the nucleoskepaton and the cytoskelaton
LINC complexes mechanically link the nuceloskelaton and cytoskeloton (LINC complex = binding to the cytoskalton)
- Nuclear pore complex = joined by LINC complexes
LINC complex = SUN-domain proteins (orange) and Nesprins (KASH domain protein)
- Each LINC complex structure has 3 SUNs domain and 3 Nesprin proteins
Proteins in LINC
LINC complex = SUN-domain proteins (orange) and Nesprins (KASH domain protein)
SUN doman = integral memebrane protein –> has a nucleoplasmic domain that binds to lamins which anchors them
KASH domain of nesprin binds to SUN domain
Assembly and disassembly of complexes can be regulated from the luminal side
Diversity of SUN and KASH proteins
Have many SUN proteins and Nesprin genes = there is a lot of diversity in proteins (Have alternative transcrtion initiation + alternative splcining + alternative termination = nesprin genes can make many types of proteins (ex. Can make big or small proteins or medium sized)
- Each protein has different combination of who they bind to in the cytoplasm (which cytoskeletal component do they anchor to)
Example – nepsrins can bind to actin or can plectin to intermediate filements in cytoplasm or can bind to motor proteins and drag the nucleus on microtubules
Moving Nucleus on microtubules
Moving of nucleus on microtubules happens during development with nuerons (ex. Perkinjie nuerons in brain) + occurs when cells crawl so the nucleus stays in the center
- Nesprin can bind to both direction of motors (depends on if the nucleus needs to go away from towards the center)
Movement of nucleus = requires LINC complexes to grab the microtubule motors and be moved to the correct location (need LINC complex connections to the cytoskelaton)
KASH proteins binding to SUN domain proteins
Overall - Nesprins (outter membrane) have ‘talons’ that are disulfide bind to SUN domains (inner membrane)
Image:
- Left = NOT fully developed SUN domain protein (not straightened some alpha helical regions to expose to SUN domain) –> Think have assembly and unfolding to get SUN domains proteins to the right configuration
- Red lines hanging down = KASH domain –> KASH domain has prolines to let it kink –> disulfide bonds to 1 SUN domain then ‘talons’ sticks into the next SUN domain = forms string (KASH = claw into the SUN domain to hold on)
Function of LINC
SUN domain and KASH = have a mechanical connection
LINC complexes take mechanical force on outside of cell (felt by the cytoskalton) –> pushes the mechanical forces into the nucleus
- When push on cell = push on nucleus via LINC complexes directly to the lamina network –> lamina network responds by being flexible and springing back
- System = set for LINC to be mechanical force transmitters to be attached to lamina filaments which will then respond
Second function of LINC
Distance between nuclear envelope membranes (distance between the inner and outer membranes) is controlled by LINC complexes (SUN-proteins)
Genes for SUN domain proteins
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
Intermediate filaments
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
Nuclear Intermediate Filaments
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
Genes for Lamins
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
Mechanical properties of lamin filament networks
- Flexible
- Interconnected by elastic springing porteins (ex. Spectrins + titins)
- 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 - Filaments have no directionality = no motors
Assmebly of lamins
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
Assembly of lamins - forming coil-coil interaction
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
Why don’t lamins have directionality
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
Thickness of Lamins
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
Location and communication between lamins
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)
Purification of Lamins
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
Where do lamina filaments concentrate
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
What do lamina networks look like
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
Sitting on nuclei
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
Forces that the nucleus experience
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
Forces that the nucleus experience - External forces (lots of mechanical forces)
- Squeezing through tiny spaces (ECM + capillaries + exit blood vessels) –> Cells squeze through tight spaces
- Contractile forces on the cytoplasm (skelatal + smooth and cardiac muscle)
- Lamins = important for muscles - Expansion and Shear forces (endothelial cells + airway epithelium)
- Cells in ariway pressore or blood flow pressures
- Gravity and Mass (Ex. Sitting on nuclei)
3D architecture of chromosomes
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
Heterochromatin + Lamins
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
Lamin Associated DNA - How to find them
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
LAD in cell types
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)
Chromosome territories
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
Nuclear envelope
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
Why do proteins bind to lamins
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
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)
Mutations in LMNA
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 )
Disease modeals of laminopathies
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
Mutations in Lamin A and Lamin C
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)
Hutchinson-Gilford Progeria Syndrome
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)
Ways for Laminopathies to arise
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
Ways for Laminopathies to arise - Perturbed function of proteins or multi-protien complexes that must bind lamina
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
Function of NPC
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
What gets imported into the nucleus
- Signaling proteins
- Regulatory rpoteins
- Transcription Factors
- Histones
- Replication Factors
- Ribosomal proteins (assembley of ribosomes is in the nucleus)
- Nucleolar proteins
- SnRNPs
What gets exported out of the nucleus to the cytoplasm
- Ribosomal SU –> Ribosomes are asmbled in the nucleolus and then need to get out of the nucleus
- mRNA complexes
- tRNA
- snRNA
What does NPC control
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
What do proteins larger than 40kD need to enter/exit the nucleus
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)
Nuclear Localization Sequence
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
How did they find NLS
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
What does nuclear export need
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
Potential Issue with NES and NLS
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
Why are males male
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)
Protein folding during nuclear import and export
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
What recognizes NLS and NES
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
Importins and exportins
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
Where is NLS or NES on protein
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 – E
Why do importins take Cargo in and why do exportins take cargo out?
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
Ran Inside the nucleus
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
Ran outside the nucleus
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
RanGTP cycle/gradient
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
GAP
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
GEF
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)
Effects of Ran-GTP on importin Vs. Exportin
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)
Concetration of Ran-GTP
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
Visual summary are how Ran-GTP controls the directionality of nuclear export
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)
Summary of nucelocytoplasmic transport
High Ran-GTP in the nucleus has known mechanisms impacts on what happens to cargo
Exportins get back in by themselves
How do mRNAes leave the nucleus
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)
Export ready mRNA
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
Ways RNA can get out of the nucleus
- Pore complexes
- 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
- 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
Answer – A
THIS IS not about using energy it is about switching Ran on/off
Check your understanding
Membrane-less compartments in Nucleus
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
Nucleoli
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
Number and size of nucleoli
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
Organizer proteins
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)
Youtube video
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
