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ANAT 262 > Nuclear lamins, pores and biocondensates 2 > Flashcards

Nuclear lamins, pores and biocondensates 2 Flashcards

1
Q

Name some mutations in nuclear envelop - gen

A

Inner nuclear membrane - lbr = pha, hem/Greenberg
Emerin - lap2 like protein in outer nuclear membrane
Syne-1 = cerebellar ataxia - connects outer nuclear membrane to cytoskeleton

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2
Q

Describe fondue - more tho

A

No starch
As increase starch = becomes with 2 phases and then actual mixture

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3
Q

Describe phase separation

A

Cytoplasm can be one phase
But if change conditions = 2 phases now - NO MEMBRANE around that areas

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4
Q

What do proteins need for liquid liquid phase sep

A

Regulated by proteins containing =
intrinsically disorder regions (highly dynamic stretches of protein sequence that can engage in interactions with other proteins)
Protein concentrations - diff
Post translational modifications of proteins - phosphorylation, methylation and others (acetylation, ub) = can drive or inhibit it

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5
Q

Describe how protein concentration can affect liquid liquid phase separation

A

If proteins prone to interactions = if In high concentration = better chance generating phase separated compartments

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6
Q

Describe what environmental conditions needed for phase sep

A

Temp control
Ph - if cytoplasm acidic = may generated phase separated compartments in cytoplasm
Ionic strength

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7
Q

Describe what else is needed for phase sep

A

Presence of dna binding partners - proteins, rna, dna = many but not all phase separated compartments have this

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8
Q

Describe biomolecular condensates = gen

A

Formed by phase sep = can help inactivate enzyme, bc willl lose enzymatic activity or opposite
Plant temp effect -=sensing
Localization to specific compartments - to change envrionmenr
Filtration - npc

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9
Q

Describe biomolecular condensates = types

A

Many types
Nucleolus - ribosomal synthesis - always there
Some only induced when specific conditions = stress granules = for storage,transcription and regulation
Wide variety of biocondensates - highly dynamic

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10
Q

Describe biomolecular condensates = relevant ex to human health

A

Treatment resistance cancer
Infectious diseases - many viruses rely on phase sep for their porpagtion
Aberrant phase separation - neurodegeneration

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11
Q

What are the advantages of biocondensates compared to organelles

A

Do not have to spend energy and effort making membrane
Can easily move material in and out = bc no memrbame barrier
Can control if compartments there or not
Easily regulated and highly dynamic

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12
Q

Describe npc transport

A

Cytosol to nucleus and vice versa
Occurs through nuclear membrane/envelope
Most at npc - works both ways

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13
Q

Describe fxf nucleoporins

A

Nup358, nup62, nup153
Aka fg-nups = important for nuclear protein import

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14
Q

Describe glfg nucleoporins

A

Nup98
Inside nucleus but can move outside
Important for mRNA export

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15
Q

Where is nup358 found

A

Cytoplasmic filaments

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16
Q

Where is nup62 found

A

Central gated channel

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17
Q

Where is nup153 found

A

Nuclear basket

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18
Q

What is npc made up of

A

120md approx = 30 diff proteins called nucleoporins

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19
Q

Describe modular architecture of npc

A

8 of these modules per npc
Subcomplex = works as module, used during assembly or dissabemly of npc
Single nucleoporins - organized into one module - come together in sub complexes

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20
Q

Describe modular architecture of npc = what happens when mitosis

A

When need to get rid of nuclear evenlop -
Disassemble npc into modules = larger protein complex and can put back together easier

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21
Q

Where are fxf repeats mostly

A

Central gated channel
Hydrophobic

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22
Q

Describe models of npc

A

Oily spaghetti or gummy bear
(Hdyrogel = gummy bear, can be disorganized or highly organized )
True model = mix of all these

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23
Q

Describe exp of fg nuceloporins

A

Can produce fxf repeats - have same sequences as in npc - make them then put into eppendorf tubes and spin them = bench top procedure and see they form sediments = solid hydrogel with npc Like properties
Also if use micro fluid - analyze fg domains = can see liquid droplets with npc like properties - formed by phase sep - can form solid hydrogels - depends on how long they sit

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24
Q

What are functions of fxf repeats

A

Can generate bioconjugates or drop like stcructures that sit in central gated channel = blocks it
Also important for interacting with transport machinery

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25
What is wrong with this picture
Missing another lipid bilateral Only one membrane in pic
26
How can move across npc
Passive diffusion Active diffusion
27
Describe molecular size - passive movement across npc
Molecules have to be smaller than diffusion channel of npc - 8nm Inefficient for proteins of molecular mass >40kda, proteins larger than 70kda = impossible, excluded from nucleus if have no import signal - have to engage active transport one
28
Describe energy - passive movement across npc
No energy required
29
Describe apparatus - passive movement across npc
No transport apparatus needed
30
Describe movement - passive movement across npc
Movement depends on concentration gradient of molecule - high to low concentration till reach equilibrium
31
Describe molecular size - active movement across npc
Molecules that exceed diffusion radius of npc - Particles up to 45nm can be translocated if have proper signal
32
Describe energy- active movement across npc
Requires energy - RAN GTP
33
Describe apparatus - active movement across npc
SPECIALized transport apparatus needed - soluble transport factors and nucleoporins
34
Describe movement - active movement across npc
Nucleus to cytoplasm = ribsomal subunits, mRNA Cytoplasm to Nculeus = tfs Shuttling - in and out - multiple times
35
Can large molecules diffuse across npc
Nawwwrrrrr
36
Name signals that control nucelocytoplasmic distribution of proteins
Signals for transport Signals for retention
37
Name signals for transport
Nls NES Shuttling signals
38
Describe nls
Nuclear localization sequence = cytoplasm to nculeus
39
Describe nes
Nuclear export signal = nucleus to cytoplasm
40
Describe shuttling signals
Protein with sep nls and nes Or One shuttling sequence that combines import and export signals - one sequence can do both
41
Describe nrs
Nuclear retention signals - nucleus
42
Describe crs
Cytoplasmic retention signal - cytoplasm
43
Why are signals - transport/retention needed
If want protein to accumulate in nucleus = have nrs Or if something you don’t want in nucleus = have crs so stays in cytoplasm
44
Are nls permanent
Yes Not cleaved during import
45
WHERE ARE NLS LOCATED
Can be located anywhere within polypeptide chain Not restricted to any certain section But needs to be accessible on surface of proteins so can bind nls receptor
46
WHAT DO NLS MEDIATE
Post translational nuclear import - take folded protein into nucleus
47
NAME TYPES CLASSICAL NLS
SV40 Sv40* SV40inv Nucleoplasmin
48
SV40 NLS
From t antigen virus protein Sequence = tppKKKRKv (k=lysine, r=arginine) Stick short peptide to something Simple or monopartite POS CHARGE aas = important for function nuclear localization YES NUCLEAR targeting= nuclear accumulation of that peptide
49
SV40* NLS
Sequence = tppKtKRKv = threonine instead Nuclear targeting = no/WEAK
50
SV40 INV NLS
Sequence = vKRKKppt (v=valine, t = threonine at c term) Same sequence - flip c and n terminus NO NUCLEAR TARGETTING - Doesn’t work even tho pos charges, bc conformation - structure diff
51
NUCELOPLASMIN NLS
Bipartite - 2 clusters pos charged aas, separated by spacer Sequence = avKRpaatkkagaKKK YES NUCLEAR TARGETTING
52
DESCRIBE NON CLASSICAL NLS
Stretches of aas = contain PY (proline tyrosine) motif SR (serine, arginine) rich regions - often in rna binding proteins RECOGNIZED BY NLS RECEPTORS
53
DESCribe nuclear transport of gfp tagged protein
SHUTTLING protein response to changes in calcium concentration As increase ca with ionophore = protein will into nculeus Shuttling = can response to physiological changes in cell
54
Describe step 1 - model: classical import into nucleus
Nls receptor (2 subunits = alpha and beta (beta = binds ran gtpase in gtp bound form)) Nls containing cargo binds receptor = complex
55
Describe step 2 - model: classical import into nucleus
Docking at npc No energy Occurs at 4 degrees Celsius = low temps, after energy depletion Importin beta binds cytoplasmic filaments
56
Describe step 3 - model: classical import into nucleus
Translocation across npc Requires energy Does not happen at 4 degrees Celsius = active process, cannot do in cold
57
Describe step 4 - model: classical import into nucleus
Terminate import and recycle tfs Do not want nls receptor in nculeus so dissociate complex and then take nls out into cytoplasm = free to do another round trafficking MODEL = only for proteins <45nm, including nls receptor, complex has to fit
58
Describe nuclear import without transport receptors
HIV-1caspid Diameter = 60nm, when bound nls receptor = too big So engages directly with fg contacts - fxf repeats proteins in central gated channel Directly interacts with repeats
59
Describe ran gtpase cycle - in nucleus
More ran gtp in nucelus Rcc1 = stuck in nculeus, associated with chromatin When encounters ran gdp = will exhcnage for gtp RCC1=RAN GEF (guanine nucleotide exchange factor)
60
Describe ran gtpase cycle - in cytoplasm
More ran gdp Ran gap - ran gtpase activating protein = sits at cytoplasmic filaments and mediates gtp hydrolysis on ran
61
Step 1 npc = image
Alpha subunit binds beta Alpha subunit binds nls
62
Step 2 npc = image
Dock at filaments Then get into nculeus - associate with nup153 (fxf repeats) B subunit = steps = fxf repeats at cytoplasmic site, through central gated channel - complex binds nup153 in basket npc
63
Step 3 npc = image
Ran gtp binds beta subunit = import complex unstable and dissociates Takes beta out to cytoplasm first Takes alpha bound to cas and out to cytoplasm
64
Step 4 npc = image
Import complex stable in cytoplasm bc no ran gtp
65
What are fxf repeats - pore odyssey
Stepping stones for nls receptor Also for export receptors
66
Name transporters - factors for nuclear protein import
Nuclear carriers Nls receptor Importin beta Nes recpetor Export in Transportin Karyopherin = all members Importin b fam and Bind ran gtp >20 membrane in mammalian cells
67
Name adaptors - factors for nuclear protein import
Importin alpha Not always required to bind cargo - sometimes beta subunit can do alone
68
Name ran - factors for nuclear protein import
Gtpase - need energy
69
Name ran interacting factors - factors for nuclear protein import
RanGAP1 = gtpase activating protein RCC1= ran gef Ranbp1 Ranbp3 Finish Import process
70
Name nucleoporins - factors for nuclear protein import
Multiple nups Nups with fxf repeats - nup358, 62, 153
71
Name energy - factors for nuclear protein import
Gtp to generate ranGTP
72
Name transport signals- factors for nuclear protein import
Recognized by carriers
73
Describe nes - specifically
Export signal Pki (pka inhibitor) LALKLAGLDI (hydrophobic aas at specific positions, isoleucine and leucine)
74
Describe nuclear export
Exportin, nes receptor, carrier Ran gtp - generated by rcc1 in nculeus = high, to make complex of cargo ran gtp and exportin Then moves through pore to cytoplasm Where gtp hydrolysis = then complex unstable and falls apart
75
Does age matter for nuclear transport
YURRRRR YOUNg = doesn’t let big proteins through But central gated channel can become leaky In old Nuclei = deterioration of permeability barrier So large molecules can get through = bad
76
What could be the consequences when npc permeability barrier breaks down
Tfs that get in shouldn’t be there = transcribe bad genes Lose control of what is in nculeus vs cytoplasm
77
How can we control trafficking = 3 ways = explain all
Control trafficking individual moelcules Can control trafficking of group of proteins - increase conc nls receptor *cancer cells = some have increase importer, so cancer cells more efficient at importing into nculeus At level npc = can change diameter of central gated channel = change distribution of molecules in cytoplasm and nculeus
78
Describe alternative ncuealr export for large cargos
Herpes nuclear egress Capsid diameter - 125nm So capsize interacts with inner nuclear membrane = generate envelop of inner nuclear membrane around capsid = goes into perinuclear space then vesicle fuses With outer nuclear membrane and gets to cytoplasm
79
How to transport something to inner nuclear membrane
Use 2 mechanisms = immobilization trap or importin alpha16 and translocon associated sorting
80
Describe immobilization trap targeting to inner nuclear membrane
Insert proteins into er membrane/one = protein moves by diffusion to nculeus - trap bc binds lamins or chromatins or both Binds to nuclear lamina or chromatin = concentrates here bc gets stuck (lbr = sit in inm, synthesized in er)
81
Describe inserting proteins - targeting to inner nuclear membrane
Insert protein into er / outer nuclear memrbame Use proteins to target protein to inner nuclear membrane In er - bound to importin alpha16 = binds motor proteins and so importin alpha 16 helps translocation, release importin after in inm

ANAT 262 (24 decks)

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