Lecture 4 - nucleus part 3

Question 1

What is piggyback nuclear protein import?

Answer 1

some proteins being imported into the nucleus without an NLS
- newly synthesized protein lacking NLS binds to NLS-containing protein in cytoplasm

Question 1

What determines the distribution of protein in either the cytoplasm or the nucleus?

Answer 1

strength of the NLS or NES

Question 2

What is the strength of the NLS or NES controlled by?

Answer 2

post translational modifications

Question 3

What does ARC1 possess?

Answer 3

both NLS and NES

Question 4

What occurs before and during pollination in ARC1 in regard to NLS and NES?

Answer 4

before pollination - NLS>NES results in ARC1 being localized mostly in nucleus
during self pollination - NLS disrupted due to phosphorylation of adjacent AA residues
NLSphos < NES results in ARC1 localized mostly in cytoplasm, ARC1 in cytoplasm functions in proteosome dependent turnover of proteins

Question 5

What are two experiments used to determine the nucleocytoplasmic transport of ARC1?

Answer 5

experiment 1: phosphorylation of NLS results in ARC1 mislocalized to only cytoplasm. Introduced phosphomimic mutations to residues adjacent to NLS - aspartic acid physiochemically similar to phosphorylated S/T/Y

experiment 2: mutation of NES results in ARC1 mislocalized to only nucleus

Question 6

What are the steps to an in vitro coIP assay to assess cargo protein importin binding?

Answer 6

(does a nuclear localized protein interact with importin?)
1. mix bait and or prey proteins in vitro
2. add agarose beads coated with anti-epitope tag IgGs
3. isolate beads via centrifugation along with all associated proteins
4. SDS-PAGE and coomassie blue staining

Question 7

What are the two main components to a CoIP assay?

Answer 7

bait - purified epitope tagged nuclear protein
prey - purified importin (alpha beta subunits)

Question 8

What are the two main stages to the cell cycle?

Answer 8

interphase and m phase (mitosis)

Question 9

What are the three(four) stages in interphase?

Answer 9

G1 - Gap 1 cell performs normal cellular activities and can respond to the environment
S - synthesis DNA replication and increased synthesis of factors required for chromosome duplication
G2 - gap 2 cell grows and prepares for mitosis
G0 - gap 0 non-dividning cells, stage when a cell arrests during G1 rather than proceeding to S

Question 10

What occurs in M phase (mitosis)?

Answer 10

prophase, metaphase, anaphase, telophase, cytokinesis (mother cell divides into two daughter cells)

Question 11

What regulates cell cycle progression or arrest?

Answer 11

by checkpoints, involve surveillance mechanisms to ensure cell cycle proceeds properly, if not cellular signals can lead to death, or cancer

Question 12

Where are the checkpoints in the cell cycle?

Answer 12

mid G1 - START cell commits to DNA replication in S and organelle duplication begins
end of G2 - cell commits to entering M phase
end of metaphase - cell commits to chromosome segregation

Question 13

What are nucleocytoplasmic mobile factors?

Answer 13

responsible for mediating transitions from one phase to the next in the cell cycle

Question 14

What is transition through checkpoints in cell cycle controlled by?

Answer 14

mitotic cycles an cycle-dependent kinases (CDKs)

Question 15

What are CDKs?

Answer 15

cell-specific kinase enzymes located in nucleus; phosphorylate various target nuclear proteins - turn on or off

Question 16

What are cyclins?

Answer 16

nucleocytoplasmic proteins; bind CDKs and regulate their activity during specific stages of the cell cycle

Question 17

How do cyclin and CDK activity vary throughout the cell cycle?

Answer 17

early interphase (G1) - [mitotic cyclins] low -> CDK activity low
end of G2/start of M phase - [mitotic cyclins] high -> CDK activity high

Question 18

What are three examples of CDK target proteins in the nucleus at the end of G2 and start of M phase?

Answer 18

histones/condensins - phosphorylation leads to chromatin packing and chromosome condensation in prep for mitosos

lamins - phosphorylation leads to disassembly of nuclear lamina

nups - phosphorylation leads to disassembly of NPCs

Question 19

What occurs to the nucleus during mitosis?

Answer 19

nucleus completely disassembled by metaphase
prophase - outer and inner nuclear membranes break down, lamina and NPCs disassemble, membrane-bound and soluble nuclear proteins released into ER membrane and cytoplasm respectively
two daughter nuclei reassemble during end of mitosis

Question 20

What are the steps to mitosis in the nucleus and activity of CDK and cyclin?

Answer 20

[mitotic cyclin] low -> CDK activity low -> dephosphorylation of nups and lamins -> reformation of nuclear lamina, nuclear envelope and NPCs -> re import of soluble NLS proteins from the cytoplasm

Question 21

Why are there oscillations in [cyclin]?

Answer 21

relative rates of protein synth and degradation at diff points in cell cycle

Question 22

why is there a decrease in [mitotic cycle] after the start of M phase?

Answer 22

due to decrease synth of new cyclin proteins and degradation pre-exisitng cycle proteins via proteosome

Question 23

What are CDKs inactivated by?

Answer 23

phosphorylation by CDK kinases, CDK kinase kinases etc.

Question 24

What happens to pre-existing cyclins after the start of M phase?

Answer 24

prevented from targeting to nucleus (cannot activate CDKs in nucleus)

Question 25

Do cyclins normally have both NLS and NES?

Answer 25

yes, relative strength of targeting signals results in cyclin protein localized primarily in cytoplasm and or nucleus

Question 26

What are the activities of cyclin B1 in mammals during the cell cycle?

Answer 26

up to and during G2 - cyclin shuttles between nucleus and cytoplasm, but localizes primarily in cytoplasm (NES >NLS)
end of G2/start of M phase - NES in cyclin phosphorylated - NLS > NES phos localizes primarily in nucleus; activate CDKs
after start of M phase - NES cyclin phosphorylated; NES > NLS.. localizes primarily in the cytoplasm

Question 27

What is the difference in nuclear mitosis in higher vs lower euk?

Answer 27

higher - open mitosis
lower - closed mitosis