17 + 18 - The Cell cycle and Apoptosis Flashcards
Name the three main checkpoints of the cell cycle, what comes after and what conditions must apply
- Start. End of G1 phase, G1/S-Cdk checks that the environment is favorable and pushes the cell into the S phase. To enter cell cycle and begin synthesis of DNA
- G2/M trasition, the control system triggers early mitotic events that lead to chromosome alignment on the mitotic spindle in metaphase
- Metaphase-to-anaphase transition. The control system stimulates sister-chromatid separation, leaading to the completion of mitosis and cytokinesis
What are the pahses of the cell cycle?
Interphase and mitotic phase. The interphase is divided into a G1 state, an S state, and a G2 state. S for synthesis. there is also a G0 state, in which the cell “hibernates” and does not progress through the cell cycle, usually because of poor external conditions.
The mitotic phase takes 1h-ish (out of 24 h cell cycle)
How to study the cell cycle? (duration of different phases)
by allowing cells to grow with access to a thymine analogue like BrdU or EdU, which can be detected using flow cytometry. These are analogues and will therefore be incorporated into the newly synthetized strands in S-phase, allowing us to see how many cells are in S phase.
To study the duration of the different phases one can measure the amount of DNA in cells, as it will double during S phase and be halved during M.
The cell cycle phase a cell is in can be determined by looking at it in a microscope - how many cells are in interphase/mitotic phase? Budding yeast cells have different sizes in different stages of cell cycle. Staining cells with DNA-binding fluorescent dyes (can reveal the condensation of chromosomes in mitosis) or with binding sites that rec specific cell components (like microtubules, f.ex., revealing mitotic spindle). S-phase cells can be identified by supplying them with visualizable molecules that are incorporated into newly synthetized DNA, such as bromodeoxyuridine (BrdU, artificial thymine analog); cell nuclei that have incorporated BrdU are revealed by staining with anti-BrdU Abs.
What happens in mitosis?
Interphase: not part of mitosis, but there is synthesis of DNA and other important cellular structures that will be divided between the two daughter cells, including mitochondria which cannot be made without an existing one. Also duplication of CENTROSOMES, which are organelles that serve as the main microtubulic organizing center
Prophase: nuclear membrane is still intact, spindle poles separate (to opposite sides of the nucleus), and the chromosomes begin to condense
Prometaphase: the nuclear membrane is opened/dissolved/breaks, and the spindle is attached to the chromosomes
Metaphase: the chromosomes align at the center plane of the cell, sorted so that the sister chromatids will be dragged to different sides of the cell
Anaphase: The chromosomes move to spindle poles (anaphase A) and the poles themselves separate to different sides of the cell (anaphase B)
Telophase: chromosomes decondense, a new nuclear membrane is formed, the spindle releases the chromosomes and dissassembles, and finally cytokinesis happens
talk about the cell-cycle control system
Cdks, cyclins, classes of cyclins, how Cdk-cyclin complexes are regulated, APC/C, Wee1, Cdc25, polyubiquitylation
cyclin-dependent kinases!!! Their activation fluctuates throughout the cell cycle and decides the phosphorylation of intracellular proteins that regulate major events in the cell cycle.
The most important regulator of these Cdks is cyclin (the thing they are dependent on). The [Cdk] is constant, the [cyclin] varies based on what activities the cell needs.
Four classes of cyclins:
1. G1/S cyclins, which activate Cdks in late G1 and help trigger progression thorugh Start. Commitment to cell cycle entry.
- S-cyclins, bind Cdks after progression through Start and help stimulate chromosome duplication. Also contribute to control of some early mitosic events
- M-cyclins, activate Cdks that stimulate entro into mitosis at the G2/M transition.
- G1 cyclins help govern the activities of G1/S-cyclins
Wee1 is an inhibitor of Cdks, inactivating them by phosphorylation. Cdc25 can dephosphorylate them again, activating them.
Progression through metaphase-to-anaphase checkpoint happens due to the anaphase-promoting complex / cyclosome (APC/C) polyubiquitylate target proteins, makring them for destruction by proteasomes. The main targets of APC/C are securin (protects protein linkages keeping sister chromatids together) and S- and M-cyclins (destruction will inactivate most Cdk in cell, triggering anaphase). Cdc20 (mid-mitosis) and Cdh1 (late M to early G1) associate with SPC/C and SCF and regulate them
SCF ubiquitylates CKIs (Cdk inhibitor proteins), helping control the activation of S-Cdks and DNA replication.
What does M-Cdk regulate?
- Chromatin condensation
- Spindle assembly
- Nuclear envelope breakdown
- Reorganisation of the golgi
How does M-Cdk regulate Chromatin condensation?
phosphorylation of condensin subunits by Cdk1 promotes DNA coiling activity. Condensin compacts and structures the chromosomes
How does M-Cdk regulate spindle assembly?
M-Cdk initiates bipolar spindle assembly in prophase: regulation of centrosome separation and maturation, and microtubules dynamics.
XMAP215 delivers tubulin subunits to the plus end to increase the growth rate and suppress catastrophes. Phosphorylation of XMAP215 inihibits its activity
How does M-Cdk regulate nuclear envelope breakdown?
Major structural components of the NE, lamin A, B & C, and
lamina-associated protein (LAP) 2 are phosphorylated by MCdk leading to depolymerization of the nuclear lamina &
retraction of inner nuclear membrane proteins into the ER
How does M-Cdk regulate reorganization of the Golgi apparatus?
M-Cdk initiates fragmentation of golgi
What does a cell need to divide (signal-wise)?
regulation by external growth factors
rebulation by intracellular signals to make sure important processes are complete (like DNA replication, if the chromosomes are attached to spindle, etc.)
Is the environment favorable?
How are G1/S-Cdks and S-Cdks regulated?
mitogens (pro-mitosis) bind to receptors on the extracellular membrane side and stimulate G1/S-Cdk and S-Cdk activity:
- mitogen binds to a mitogen receptor
- Ras complex is activated
- MAP kinase is activated
- increased expression of immediate early genes, including the gene encoding the protein Myc
- Myc increases expression of many delayed-response genes, including some that increase G1-Cdk activity
- Increased G1-Cdk activity triggers phosphorylation of Rb family members, inactivating them
- Rb members are often inhibitors of E2Fs, and when they are inactivated the E2Fs are activated.
- E2F increase trc of G1/S genes, including G1/S cyclin and S-cyclin.
- The resulting G1/S- and S- Cdk activities lead to positive feedback and more activation of the same molecules, and E2F also self-stimulates
How does DNA damage arrest the cell in G1?
Various protein kinases are recruited to the site of damage and initiate a signaling pathway that causes cell cycle arrest
- first kinase is usually ATM or ATR
- other protein kinases like Chk1 and Chk2 are recruited and activated
- these lead to phosphorylation of p53 (normally bound by Mdm2, which marks p53 for degradation by proteasomes bc it is polyubiquitylated)
- phosphorylation of p53 blocks Mdm2 from binding to it, thus stopping degradation of p53.
- high levels of p53 stimulates transcription of many genes, including the CKI protein p21
- p21 binds and inactivates G1/S-Cdk and S-Cdk complexes, inhibiting progression through Start and arresting the cell in G1.
What happens if there is excessive mitotic stimuli?
cell-cycle arrest or apoptosis.
Abnormally high Myc cause activation of Arf, which binds to and inhibits Mdm2, which will increase p53 levels. p53 will then either inhibit G1/S-Cdks and S-Cdks (arresting the cell cycle), or will trigger apoptosis.
How does p53 trigger apoptosis?
p53 stimulates transcription of the p21 gene, which will lead to cell-cycle arrest, and other genes, including NOXA and PUMA. They activate BCL-2, which in turn activates BAK and BAX. A phosphorylated p53 bound to BAX will create holes in the mitochondria, and cytochrome c will be released into the cytosol.
Cytochrome c in cytosol activates Apaf1. Several activated Apaf1s will go together in an apoptosome (each Apaf1 has a CARD, which bind caspase 9). Activation of capsase 9 by these complexes makes capspase 9 cleave and activate downstream executioner caspases like caspase 3
