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Dynamic Cells > Dr. Pool's lectures L18-L22 > Flashcards

Dr. Pool's lectures L18-L22 Flashcards

1
Q

How is nucleocytoplasmic transport different to othe rprotein translocation systems eg er and mitchondria?

A

Substrates often large and complex
Folded cargo
Bidirectional transport

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2
Q

Nuclear Pore complex

A

Large structure approx 125MDa. To allow large proteins through, e.g. ribosomal subunits. * fold -symmetry, looks like flowers through microscope.
Composed of 50+ nuceloporins

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3
Q

Nucleoporins

A

Make up Nuclear pore complex- Contain FG repeats- short clusters of phenylalanine and glycine. Hydrophobic. These clusters are separated by hyrdophilic linkers.

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4
Q

ron laskey 1982 experiment

A

Worked on protein called nucleoplasmin (pentamer). Injected into cell and found all of it in nucleus even though much bigger than 60KDa.

Wanted to find which part of protein let the nucleoplasmin into nucleus. Treated with protease (papain) which chops off tails. Tails were part that let the protein into nucleus.
When injected into nucleus also got stuck without tails. So showed needed for import and export, kpet in nucelus by selective retention.

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5
Q

Nuclear Localisation Signals (NLS)

A

1-2 stretches of basic residues.
Only thing you need to go to nucleus.
Added to pyruvate kinase- usually cytosolic, goes to nucleus.

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6
Q

Saturable import process

A

It is saturable. If you attach NLS to albumin (v. abundant) blocks import of most proteins.
Implies existence of NLS-receptors.

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7
Q

Cytoplasmic factors in import

A

Took culture cells. Treated with low conc of detergent. Punctured PM but not \ER and nucleus.
Wash out cytoplasm and then detergent do PM reseals in buffer or old cytoplasm if you want. In control withold cytoplas, gel normal nuclear import. In buffer no nuclear import- so—-> cytoplasmic factors needed

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8
Q

Gel filtration of cytoplasm

A

Get fractions. Test each fraction with semi-permeabilised cell import assay. See from each fraction how much enters nucleus.

Fraction a-
comes out first. Importin alpha which binds NLS
Importin beta- dimerises with importin alpha and interacts with NPC fg repeats.

Fraction b-
Ran gtpase
NTF2- interacts with ran

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9
Q

2 stages of import

A
  1. Docking- energy independent
    Just fraction A, importin alpha and beta you see “rim staining” with fluorescent-BSA-NLS. Shows the proteins get to nuclear pore comples but cant get in.
  2. Translocation- energy, Ran and NTF2- dependent
    Add fraction B- form of energy in GTP, get complete translocation of proteins into nucelus. So “nuclear staining”.
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10
Q

RCC1- in ranGTPase cycle

A

Is a GEF- Activates RanGDP to Ran GTP

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11
Q

RanGAP1- in RanGTPase cycle

A

deactivates RanGTP to RanGDP

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12
Q

Ran gradient

A

RanGAP1 and RCC! are asymmetrically distributes. Important. RCC1 in nucleus. A lot of RanGTP in nucleus. RanGDP in cytoplasm.

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13
Q

Evidence for needing ran gradient

A
  1. If you delete RCC1 by using restrictive temp. (usually 39.5) then there’s no Ran gradient. With a GFP-NLS so can see there’s no nuclear accumulation. Shows you need ran gradient for nuclear transport.

IN permissive temp, GFP-NLS can see it accumulate in nucleus

  1. Use a Ran-gtp sensitive probe. See high RanGTP conc in nucleus. Then use mutant of Rsn- RanT24N. which is unable to exchange its nucleotide. So once it’s hydrolysed GTP it can;t be reloaded again (from GDP bound form)
    Low RanGTP in nucleus. Don’t get nuclear import.
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14
Q

ran gtp gradient is providing what?

A

Directionality. Tells nuclear importin receptors where they are. Importins bind cargo in absence of rangtp.
Binding of rangtp releases cargo.
Rangtp binds to importin-beta so separated from alpha.
When beta and alpha have dissociated, alpha can;t bind to NLS on cargo anymore.

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15
Q

Nuclear import cycle

A

In cytoplasm intergin alpha and beta bind cargo. Beta can interact with FG repeats on nuclear por complex.
Goes into nucleus.
High RanGTP in nucleus. RanGTP binds to beta, so beta dissociates from alpha. Then alpha cant bind to cargo anymore, releases it. Beta can still bind to FG repeats on nuclear pore complex. leaves nucleus again.
incytoplasm lots of ranGAP, hydrolyses RanGTP immediately. starts again.

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16
Q

Ecidence that export needs ran gradient

A

INject RanGAP into xenopus oocyte nucleus. Usually only in cytoplasm. GAP will immediately hydrolyse GTP. So high RAN GDP everywhere. Then inject radiolabelled RNAs (that would usually be exported). Then seperate nucleus and cytoplasm fractions gently.

At time zero- should just have signal in nucleus.

Control- injected with buffer- rnas are found in cytoplasm, control protein still in nucleus shows nucleus isn’t leaking.
RanGAP1- No RNA in cytoplasm, has stopped export.

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17
Q

What else does export need?

A

Also requires receptors of the importin-beta family.

Work like import receptors but in reverse. All family can bind GTP and FG repeats on nuclear pore complex.
Respond conversely to Ran gradients! IN nucleus with high RanGTP- will binds to cargo. In cytoplasm with low Ran GTP- will release cargo

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18
Q

CAS

A

Importin alpha has its own export machinery called CAS> So we dont run out of alpha in cytoplasm.
IN high gtp cas binds to importin alpha.
Whn gets to cytoplasm, ranGTP hydrolysed so CAS dissociated from RANGDP. CAS then goes back round to enter nucleus.

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19
Q

How are proteins exported?

A

They possess an NES- nuclear export signal.
Rich in leucine residues. Short leucine-rich sequence.
Specific export receptor fot he proteins with NES. Again part of importin Beta family called CRM1.

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20
Q

CRM1

A

export receptor for proteins with NES. Can also export RNA, eg in HIV.

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21
Q

HIV rna export

A

HIV encodes a protein called REV. Rev has a NLS which gets into nucelus. Also has NES which allows it to interactw ith CRM1. So can leave nucleus. Also has rna binding stie and can bind into unscpliced viral RNA. USually a quality control mechanism stopping unscpliced rna being exported. HIV can overcome this and export unspliced RNA.
Rev binds RNA and CRM1 and leaves nucleus.

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22
Q

Bulk export of mRNA

A

Does not require Importin B like receptor and not dependent on ran gradient.
If you collapse ran gradient, wont affect mRNA.

Found mex67-5 important for export. In nonpermissive temp, export blocked in mutant. Get strong fluorescence from nucleus.

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23
Q

Splicing and export of mRNA link

A

Unspliced mRNA. Splicing machinery deposits a footprint on the mRNA after splicing where the intron was, like a signal to say it has been spliced. This proteins is recognised by mex67.
So mex67 can only see mrna that has been splices.

Mex67 homologue in mammals=TAP. Both interact with spliced mRNA and nuclear pore complex.

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24
Q

NTF2

A

Binds to NPC and RanGDP. NTF2 found in fraction B in filtration. Can bind NPC, can only bind gdp form of Ran in cytoplasm so lets it go into nucleus, otherwise would end up with all of ran in cytoplasm.

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25
Q

Ran in cell division

A

Ran is required for assembly od mitotic spindle.
Need RanGTP for spindle formation.
RCC1 is not fre, it is tightly bound to chromatin! So even though ran is free, only get RanGTP by chromatin, so forms spindles there.

26
Q

TPX2-

A

involved in nucleation of MT spindles. Has NLS so binds importin alpha. when it isbound its inactive. So doesnt stimulate MT formation, when near chromatin, rcc1 so RanGTP. So importin alpha binds rangtp and tpx2 is free. So chromosomal spindle formation.

27
Q

mRNA localisation examples

A

Beta actin mRNA only at leading edge where actin needs to be made.
OSkar mrna in drosophila oocyte..
Gurken mrna in drosophila oocyte.

28
Q

Neurone mrna localisation

A

MAP2 mRNA and myelin basic protein (MBP) mRNA are found in the dendrites of neurones.

29
Q

Budding yeast mrna localisation

A

ASH1 only in daughter cell - at bud tip.

OXA1 mrna is localised to mitochondria, better than delivering each protein there after made.

30
Q

mRNA localization mechanisms

A
  1. Directed transport on cytoskeleton. Use tracks
  2. Random diffusion and transport. Cytoplasm movign gently protein then captures mrna at target.
  3. Generalized degradation in combination with local protection. so capture the mrna at target, then switch on RNAase that degrades everything not at target.
31
Q

main mechanism- active transport steps-

A
  1. Assembly of transport complex
  2. Transport along cytoskeleton needs motor proteins to move mrnas along mTs or actin.
  3. Anchoring at destination.
32
Q

ASH1 localisation

A

Uses active transport.
ASH1 protein represses transcription. Represses HO endonuclease-? mating-type switching.
Cell is either A or alpha. Mother cell changes mating type. Ash1 keeps daughter so it cant change.

33
Q

ASH1 genes found by mutants

A 
ash1- transcriptional repressor
she1- type V myosin motor
she2- rna binding protein
she3 adaprot protein, can interact with other proteins
BNI1- actin cytoskeleton organisation
34
Q

Ash1 complex

A

Ash1 has zip codes in 3 utr and orf. Stem loops.
she2 protein binds to these zip codes in cytoplasm
she2p can then bind to adaptor protein0 she3p.
she3p binds to Myo4p to binds and allow complex along the actin.
Usually the plus end in daughter end to transport mRNA to daughter cell.

35
Q

Myo4 test in ash1

A

Use GFP-RNA to see ash1 mRNA movement. Can see RNA move into daughter cell in wild type. IN Myo4 mutant- rna just bobs arounf. so need myo4 for ash1.

36
Q

ASH1 anchoring

A

requires bn1p and translation of ash1 mrna.

ash1 translation anchors it at bud tip. bni1p isnt doing the anchoring. its just organising the actin cytoskeleton.
indirectly anchoring.

37
Q

mrna in drosophila oocyte

A

Oskar at posterior. Bicoid near muddle.
Contain targetting signals in their 3’UTR. when these are swapped, they swapped places. Added osk 3@utr to lac z and that went to posterior. ven with just stem loop structure.

38
Q

Oskar transport complex

A

Staufen protein binds to stem loop.
Staufen will only bind to oskar mrna loop strcutre.
Recruites proteins including bruno- bruno represses translation until its at the target.

It’s MT dependent, delivered to plus end of MTs.

39
Q

motor proteins of oskar and bicoid

A

oskar- kinesin. to plus end of mts

bicoid- dynein- minus end of mts.

40
Q

Oskar anchoring.

A

Similar to ASH!, linked to translation. Bruno represses until plus end. Then bruno released. Oskar is made this anchors on. Cortical actin needed too.

41
Q

Nanos (nos

A

Random diffusion/trapping model.
Also localised to posterior of oocyte like oskar.
Not linked to MTS, Mt mutants dont matter.
Dependent on 3’UTR for localisation.
trapping proteins at posterior are dependent on prior localisation of oskar.

42
Q

Neuronal migration growth cone

A

The midline of spinal cor produces signal that is chemoattractant, nectrin from floorplate. cells go towards it. Then when it gets to high point, doesnt need to move toward sit anymore so switched off receptor (dcc). Then repellents, slit and semaphorin are made at floor plate and wall of neural tube. Moves towards brain to get away from repellent.

43
Q

B actin in neuronal growth cone

A

localised to growth cone by 3’utr. binds to zip-cod binding protein. binds to stem loop. more b actin where nectrin is highest so more actin so growth cone moves towards nectrin.

44
Q

Spiinal muscular dystrophy

A

Degenerative disease - motor neurone death. Cayse by mutation in SMN. SMN is an mrna binding protein. Binds specific mrnas in axons including B actin.
Normally b actin is taken to growth cone. Here they lack SMN so stunts axon growth and cone sixe. poor neuromuscular junctions.

45
Q

NGF

A

Target tissue secret Nerve Grwth Factor.
Imprortan for timulationg neruones to grow outwards like a firework when they get to location. also controls cell survival.
Growth cones that arrive first to NGF (target tissue) survive. others die.

46
Q

TrKA

A

NGF binds TrKA cell surface receptor. NGF stimulation of migration occurs locally at growth cone rhoguht receptor- mediated activation of ras/rac/rho.

when trka binds to ngf:
activated gtpases, drives actin formation
survival signals.
Trka slef phosphorylates and phosphorylates CREB which goes into nucleus and cells dont apoptose.

47
Q

LTP and LTD

A

early phase of both- done need nucleus, or protein synthesis. short term.

late phase- communication between synapse and nucleus. long term memory. proteins made. activation of NMDA receptor.

48
Q

LTP

A

NMDA receptors make complex with importins and TFs eg CREB2. TF cant go into nucelus because in complex with receptor. LTP triggers nuclear translocation of dendritic importins. when stimulat ltp, localisation of importin receptor into nucelus . not holding onto receptors anymore.

49
Q

ltd nulcear accumulation of creb2

A

Use photoswitchable fluorescent protein, linked to creb2. shine laser on part of dendrite. so tf turns red. after stimulation can follow protein. can see signal at dendrtite move and accumulate in nucleus when receptors are stimulated.

50
Q

Fragile X syndrome

A

leading cause of autism.
mutation of FMR1 gene.
encodes FMRP protein which regulated translation like BRUNO.
Loss of FMRP leads to disregulation of translation at the dendrite.
FMRP critical for LTD

in autism too much ltd. stimulated mGlR, triggers dephosphorylation of FMRp, leads to too much ltd.

51
Q

ER function

A

protein secretion and lipid synthesis
entry to secretory pathway
disulphide bond formation and N-glycosylation to fold proteins.

(hb70s stop aggregating proteins)

52
Q

DTT and tunicamycin treatment

A

Mimicks when there’s too much protein load in ER.
Dtt stops disulphide bonds (s-s bonds)
tunicamycin stops n-glycosylation.

However cells still grow normall, can cope with unfolded protein response. Transcribe more folding machinery eg chaperones.

53
Q

yeast mutants upr genes

A

Searched for yeast mutant which cant cope with dtt or tunicamycin- found genes for upr-

Ire1- er membrane protein, kinase and endonuclease

Rtg1- tRNA ligase

Hac1- transcription factor

54
Q

1st steps of upr

A

misfolded proteins bind and activate ire1 in the membrane

  1. triggers dimerization
  2. activates kinase via trans-autophosphorylation
  3. this activates ribonuclease domain on Ire1
55
Q

Ire1 in upr steps

A

Hac1 unusal i that it’s exported from the nucleus with intron still intact.
Ribonucleare domain of Ire1, cuts out intron of Hac1
the tRNA ligase sticks these exons back together again. get hac1 protein.

56
Q

HAc1 function

A

A TF and has an NLS so interacts with importiins. enters nucleus.
It binds to target genes and induces transcription of ER chaperones.
Chaperones taken to er to help out with folding.

57
Q

UPR components in mammals

A

Evolutionary consevred
ire1= ire1
hac1= xbp1 (already knew it was needed for plasma be cells because more er, differentiation
rtg1= human tRNA ligase complex

but not the only pathway in eukaryotes for upr

58
Q

upr mechanisms in eukaryotes

A
  1. ire1—xbp1—activate genes that increase upr
  2. PERL–phosphorylates translation machinery to reduce translation, leads to selective translation of regulatory proteins—-activates genes that increase upr
  3. ATF 6— regulated proteolysis so releases TF— activate genes that increase upr
59
Q

upr in secretory cells

A

e.g. pancreas. upr is always switched on because lots of secretion needed. PERK knockout in islets of langerhans leads to diabetes. shows upr always needed.

60
Q

mitochondria fusion and fission

A

promoted by distinct gtpases.
needed for mitochondrial inheritance
mitochondrial distribution for energy distribution

61
Q

mitochondria dynamics disease

A

usually neurodegenerative eg parkinsons because neruones need the mitchondria to move so much. e.g. parkinsons

Dynamic Cells (5 decks)

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