4 - nucleus and the cell cycle Flashcards
1
Q
what is piggyback import?
A
- some proteins are imported into the nucleus without a nuclear localization signal (NLS)
- called piggyback nuclear protein import
- nascent (newly synthesized) protein without NLS binds to a protein with NLS in the cytoplasm
- importin receptors mediate targeting and import of protein/protein complex into nucleus
2
Q
how is nucleocytoplasmic transport achieved?
A
- many proteins shuttle between the nucleus and cytoplasm
- participate in nuclear and cytoplasmic functions
- often contain an NLS and NES
- strength of NLS and NES controls the distribution of the protein in either the cytoplasm or nucleus
- if NLS is stronger than NES then most protein at steady state/at any time is localized in the nucleus
- strength of an NLS or NES can be controlled by post translational modifications or where it is in a cell
- e.g. phosphorylation of amino acid residues that have or are adjacent to the targeting signal
- decreases its ability to be recognized by importin/exportin receptor
- NES: LxxLxxL
- NLS: KKQRKKx
- x: S/T/Y
3
Q
how does ARC1 get transported?
A
- ARC1 shuttles between the cytoplasm and nucleus in plants
- has an NLS and NES
- before pollination, NLS is stronger than NES
- ARC1 localized mostly in nucleus
- during self pollination
- NLS is disrupted due to phosphorylation of adjacent amino acid residues
- phosphorylated NLS is weaker than NES
- ARC1 localized mostly in the cytoplasm
- ARC1 in the cytoplasm functions in the UB/proteasome dependent turnover of other proteins
- leads to pollen rejection
- two experiments with ARC1
- experiment 1: phosphorylation of NLS results in ARC1 being mislocalized to only the cytoplasm
- put phosphomimic residues on amino acids beside the NLS
- aspartic acid is chemically similar to phosphorylated s/t/y
- experiment 2: mutation of NES results in ARC1 being mislocalized to only the nucleus
4
Q
how is coip used in studying nucleocytoplasmic transport?
A
- assess cargo (meant to go to the nucleus) protein to importin binding via in vitro coimmunoprecipitation assay
- two main components
- bait: purified epitope tagged nuclear protein (protein of interest that presumably won’t bind to importin without an NLS)
- e.g. myc-ARC1 or ARC1 missing NLS
- prey: purified importin of alpha and beta subunits
- both subunits are needed to work as the importin receptor
1. mix bait and prey proteins in vitro
2. add agarose beads with anti epitope tag IgGs
3. isolate beads via centrifugation along with all associated proteins
4. SDS-PAGE and coomassie blue staining
questions
- both subunits are needed to work as the importin receptor
5
Q
what is the cell cycle?
A
- nucleocytoplasmic proteins are involved in the control of the cell cycle
- synthesized and degraded during each cell cycle
- e.g. cyclins
- there are two main phases of the cell cycle
- interphase, has three stages
- G1: cell performs normal cellular activities and can respond to its environment
- S: DNA replication and increased synthesis of factors required for chromosome duplication
- G2: cell grows and prepares for mitosis
- there is also G0: non dividing cells, when a cell arrests during G1
- M phase: mitosis
- consists of PMAT
- prophases involves chromosome condensation, mitotic spindle formation, and reversible breakdown of the nuclear envelope (membranes, lamina, NPCs)
- duplicated chromosomes are separated into two nuclei
- also has cytokinesis where the mother cell becomes two daughter cells
6
Q
how is the cell cycle controlled?
A
- understanding cell cycle regulation helps with studying cancer
- cancer is the inability of the cell to regulate its own division
- progression or arrest through the cell cycle is regulated as distinct stages called checkpoints
- surveillance mechanisms ensure that the cell cycle is proceeding properly
- if not, signals lead to cell death, cell cycle arrest, or disease
- involves positive controls
- nucleocytoplasmic factors stimulate phase transitions
- the transition through checkpoints is controlled by mitotic cyclins and CDKs
- CDKs: cell cycle specific kinases in the nucleus that phosphorylate nuclear proteins in order to turn them on or off
- histones and condensins: phosphorylation leads to chromatin packing and chromosome condensation in preparation for mitosis
- lamins: phosphorylation leads to disassembly of the nuclear lamina
- nups: phosphorylation leads to disassembly of the NPCs
7
Q
what are the checkpoints of the cell cycle?
A
- three primary checkpoints in the cell cycle
- mid G1: restriction point or 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
8
Q
how do cyclins work?
A
- cyclins: nucleocytoplasmic proteins that bind to CDKs and regulate their activity during specific stages of the cell cycle
- called cyclins because their concentration is cyclical
- mitotic cyclins vary during the cell cycle
- early interphase: mitotic cyclins are low so CDK activity is low
- end of G2: mitotic cyclins are high so CDK activity is high
- -results in phosphorylation of target proteins and cell enters M phase
- oscillations in cyclin are due to the relative rates of protein synthesis and degradation at different points during the cell cycle
- decrease in mitotic cyclins after the start of M phase is due to the decreased synthesis of new cyclin proteins and degradation of preexisting cyclin proteins via proteasomes
- proteasome: cell protein degradation machinery
- CDKs are inactivated by phosphorylation by CDK kinases
- after start of M phase, preexisting cyclins are also prevented from targeting to the nucleus
- thus they can’t activate CDKs in the nucleus
9
Q
what is open mitosis?
A
- dynamic process in higher eukaryotes
- entire nucleus completely disassembles by metaphase
- starting in prophase, both nuclear membranes fragment and vesiculate
- lamina and NPCs disassemble
- soluble nuclear proteins are released into cytoplasm
- two daughter nuclei reassemble during end of mitosis in telophase after chromosomal division
- mitotic cyclins are low so CDK activity is low
- nups and lamins are dephosphorylated
- nuclear lamina, envelopes, and NPCs reform
- soluble NLS proteins are imported back from the cytoplasm
10
Q
how are cyclins transported?
A
- cyclins normally shuttle between the nucleus and cytoplasm
- have both an NLS and NES
- the relative strength of each targeting signal results in the cyclin protein being localized primarily in the cytoplasm or nucleus
11
Q
what is cyclin B1?
A
- e.g. localization of the mitotic cyclin B1 in mammals
- up to and during G2: B1 shuttles between nucleus and cytoplasm but localizes mostly in cytoplasm so NES is stronger
- after start of M phase: NES in B1 is phosphorylated which results in the NLS being stronger and B1 is mostly in the nucleus now
- cyclin B1 in the nucleus activates CDKs required for transition from G2 to M phase
12
Q
what is closed mitosis?
A
- closed mitosis is seen in lower eukaryotes
- nuclear envelope remains intact during mitosis
