Sept 24 - Nucleus lecture

Question 1

How many layers does the nucleus have?

Answer 1

It is a double bilayer - it has an inner membrane and an outer membrane

Question 2

How crowded?

Answer 2

2 meters of DNA in a volume of 5 E-16 m^3, mass of histones, lots of RNA, many other macromolecules

Question 3

Lamins

Answer 3

Fibrous proteins that form the nuclear lamina. Note that Lamins are not Laminins.

Question 4

Nuclear lamina

Answer 4

2D network of polymers along the inner surface of the inner nuclear membrane

Question 5

How are lamins assembled?

Answer 5

They are Type V intermediate filaments, 60-70 kd, small amino-terminal head, a-helical rod domain, and globular tail.
Lamins undergo self-assembly.

Question 6

How are lamins disassembled?

Answer 6

Phosphorylation mitosis. Cdk and PKC disassemble (these are kinases). Reassembly requires dephosphorylation.

Question 7

LMNA mutations - LMNA is lamin A.

Answer 7

These cause CMT2 - charcot marie tooth, Progeria…

Question 8

FRAP

Answer 8

Fluorescence recovery after photobleaching. Use this to reveal mobility of nuclear proteins.

1. GFP-labeled protein is irreversibly photobleached in a defined area using a laser
2. The time taken for the area to regain fluorescence gives the rate for protein mobility. Slow = immobile. Fast = very mobile.

Question 9

Pseudodicentric chromosome

Answer 9

Fusion resulting in 2 centrosomes on one chromsome, and then one centromere gets permanently silenced.

Question 10

Neocentromere

Answer 10

Chromosomal fragment without a centromere can result in permanent activation at a new site

Question 11

Are DNA sequences sufficient or necessary for specifying centromere location?

Answer 11

This is the centromere paradox. No - they do not explain centromere location. instead, CENP-A is thought to be the epigenetic centromere mark.

Question 12

CENP-A

Answer 12

Histone H3 variant. Always at active centromeres, including neocentromeres. Always absent from inactive centromeres.
It gets added at early G1

Question 13

How did they show that CENP-A gets added at early G1?

Answer 13

Using a Quench-chase-pulse experiment to label only new protein and then seeing that it localized at the start of G1.

Question 14

NPC

Answer 14

Nuclear pore complex. Big, 120 mDa in vertebrates. 2000-3000 NPCs per mammalian nucleus.
SMALL MOLECULES DIFFUSE <35 kDa but larger proteins are restricted - it is selectively permeable.
Resting state is 5-10 nm but dilates to up to 39 nm.

Question 15

Nucleoporins

Answer 15

Also known as Nups.
NPC is composed of 30 Nups.
Vertebrate Nups are O-glycosylated with N-acetyl-glucosamine (GlcNAc)
A few Nups have transmembrane domains which anchors NPC in the membrane.
Many Nups have phenylalanine-glycine (FG) repeat motifs

Question 16

FG repeats

Answer 16

This is something that nuclear pore proteins (Nups) have. and if you mutate it you get a very poor formation of a network.

Question 17

Stationary phase of nuclear transport

Answer 17

NPC

Question 18

Mobile phase of nuclear transport

Answer 18

Cargoes and soluble factors

Question 19

Cargoes

Answer 19

Many protein cargoes and RNPs are imported into the nucleus. All mRNAs, tRNAs, ribosomes, and many proteins are exported.

Question 20

Import signal

Answer 20

Nuclear localization signal (NLS)

Question 21

Export signal

Answer 21

Nuclear export signal (NES)

Question 22

Can cargoes contain both import and export signals?

Answer 22

Yes - these cargos have the ability to shuttle.

Question 23

KR(X10)KKKK

Answer 23

Consensus bipartate nuclear localization sequence.

Question 24

What were the results of the heterokaryon experiment?

Answer 24

One of the hnRNPs that they were studying ended up in the other nucleus, suggesting that there was continuous shuttling from nucleus to cytoplasm and back

Question 25

Properties of digitonin

Answer 25

Binds to and precipitates cholesterol so it could attack the outer membrane but not the nuclear membrane.

Question 26

Importin alpha

Answer 26

Binds to the nuclear localization signal. NLS–Imp alpha – Imp beta – NUPS

Question 27

RanGTP

Answer 27

Binding terminates NPC gate / permeability barrier

Question 28

Importin alpha’s importin beta binding domain does what?

Answer 28

The IBB binds importin beta when that’s around but when it’s not, IBB binds imp alpha on the same site that recognizes cargo (specifically the NLS of cargo)

Question 29

Importin beta super-family of transport receptors

Answer 29

All bind directly to either NLS or NES of their cargo. Some also use adapter proteins. All can bind directly to the FG domain in Nups, allowing docking at the NPC. all bind to small GTP-ase RAN which regulates nuclear transport receptor cargo binding.
They are composed of HEAT repeats.
All continually shuttle between nucleus and cytoplasm.

Question 30

Exportins

Answer 30

Imp-beta superfamily members that mediate nuclear export.

Question 31

Crm1

Answer 31

Best characterized Imp-beta superfamily member exportin.
Exports many proteins bearing leucine rich NESs.
Target of leptomycin B

Question 32

CAS/Cse1

Answer 32

Exports Imp-alpha from the nucleus

Question 33

Exportin t/Los1

Answer 33

exports tRNA - binds RNA directly

Question 34

Msn5p

Answer 34

Exports several transcriptional regulators. Cargoes must be phosphorylated in the nucleus. Also functions in import

Question 35

Pse1

Answer 35

Required for import of pho4 into the nucleus in low phosphate settings

Question 36

Msn5

Answer 36

Required for export of Pho4 in high phosphate settings

Question 37

RanGTP

Answer 37

Small GTPase essential for nuclear transport. RanGTP concentrations are high in the nucleus and low in the cytoplasm.
Promotes export cargo/receptor interaction
Promotes import cargo/receptor dissociation.
The only known energy consuming protein involved in nucleocytoplasmic transport.

Question 38

RCC1

Answer 38

Converts RanGDP to RanGTP

Question 39

RanGAP1

Answer 39

Converts RanGTP to RanGDP