Cell Cycle Flashcards
Describe the cell cycle
Only way to make a new cell is to duplicate one that already exists
Orderly sequence of events in which it duplicates its contents and then divides in two
Describe the cell cycle at a minimum
At a minimum, the cell must accomplish its most fundamental task: the passing on of its genetic information to the next generation of cells.
To produce two genetically identical daughter cells, the DNA in each chromosome must first be faithfully replicated to produce two complete copies.
The replicated chromosomes must then be accurately distributed (segregated) to the two daughter cells, so that each receives a copy of the entire genome.
In addition to duplicating their genome, most cells also duplicate their other organelles and macromolecules; otherwise, daughter cells would get smaller with each division.
What are the two phases of the cell cycle?
S Phase: Chromosome duplicaiton occurs. Requires 10-12 hours and occupies about half of the cell-cycle time
M phase: chromosome segregation and cell division. Less time (less than an hour)
More phases
M phase - cell division, mitosis and cytokinesis (10% of cells)
G1 - commitment to cell division
S - amount of DNA in nucleus doubled
G2 - Cell mass doubled
Describe flow cytometry
Measuring DN content in a synchronised cell population as it progresses through the cell cycle. DNA content doubles during S phase - determine the lengths of G1, S and G2 and M
Describe microscopy
- Staining cells with DNA-binding fluorescent dyes, which reveal the condensation of chromosomes in mitosis
- Staining cells with antibodies that recognise specific cell components, such as the microtubules (revealing the mitotic spindle)
- S-phase cells can be identified by supplying them with visualisable molecules that are incorporated into newly synthesised DNA, such as the artificial thymidine analog bromodeoxyuridine (Brd); cell nuclei that have incorporated BrdU are then revealed by staining with anti-BrdU antibodies
- Looking at replicating yeast cells
Discovery of cell cycle regulators through yeast forward genetics
Genetic analysis of cell cycle. Paul Nurse. Elongated cells. Division occurs at a fixed cell size
How to understand essential cell functions
Requires conditional lethal point mutants.
E.g. temperature sensitibe
Mutant protein is functional at the permissive temperatue. Non-functional at restrictive.
Describe forward genetics with S pombe
Cdc mutants
Looked for mutants with two traits:
1. Temp sensitive
2. Cells elongate (cell cycle blocked)
From these can begin to determine the key genes involved in eukaryote cell cycle/cell division. Isolated lots of mutant strains
Cell cycle in all eukaryotes?
Control is similar in all. Progressio is coordinated with growth.
Cells growing in steady state tend to undergo cell division at a characteristic size
Driven and controlled by well conserved control mechanism, many of which are identified in the yeast experiments
Describe the general cell-cycle control system
Based on a connected series of biochemical switches, each which initiates a specific cell-cycle event.
1. Switches are generally binary (on/off) and launch events in a complete, irreversible fashion.
2. Cell-cycle control system is remarkably robust and reliable, partly because backup mechanisms and other features allow the system to operate effectively under a variety of conditions and even if some components fail
3. The control system is highly adaptable and can be modified to suit specific cell types or to respond to specific intracellular or extracellular signals
Entry into te cell cycle
Must receive stimulatory extracellular signals in the form of mitogens.
Mitogens overcome intracellular braking mechanisms that block progress through the cell cycle
Give some examples of mitogens
Platelet-derived growth factor (PDGF) - helps stimulate cell division during wound healing in a broad range of cell types (fibroblasts, smooth muscle cells, and neuroglial cells)
Epidermal growth factor (EGF ) - acts not only on epidermal cells but also on many other cell types, including both epithelial and nonepithelial cells.
Erythropoietin (EPO) - only induces the proliferation of red blood cell precursors.
Many mitogens, including PDGF, also have actions other than the stimulation of cell division - cell growth, survival, differentiation, or migration, depending on the circumstances and the cell type.
Also the proto-oncogenes ras and myc
Describe growth factors
Growth factors activate their receptors and other tyrosine kinases, such as Ras, and mitogen-activated pathways culminating in transcriptional induction of numerous genes, including protooncogenes such as myc.
Cyclin D and E act as growth factor sensors, and activation promotes transition from G1 to S phase.
What does cell cycle control depend on?
Cyclically activated cyclin-dependent protein kinases - Cdks
Describe cdks
Activities of Cdks rise and fall as the cell progresses through the cycle, leading to cyclical changes in the phosphorylation of intracellular proteins that initiate or regulate the major events of the cell cycle.
Cyclical changes in Cdk activity are controlled by a complex array of enzymes and other proteins.
The most important of these Cdk regulators are proteins known as cyclins. Cdks are dependent on cyclins for their activity: unless they are bound tightly to a cyclin, they have no protein kinase activity.
Cyclins were originally named because they undergo a cycle of synthesis and degradation in each cell cycle.
The levels of the Cdk proteins, by contrast, are constant.
How are cdks activated?
Cyclical changes in cyclin protein levels result in the cyclic assembly and activation of cyclin-cdk complexes at specific stages of the cell cycle
An increase in Cdk activity at the G2/M transition increases the phosphorylation of proteins that control chromosome condensation, nuclear-envelope breakdown, spindle assembly, and other events that occur in early mitosis.
Inhibition of cdk activity
The rise and fall of cyclin levels is the primary determinant of cdk activity during the cell cycle. Several additional mechanisms help control its activity.
Phosphorylation at a pair of amino acids in the roof of the kinase active site inhibits the activity of a cyclin-cdk complex
Phosphorylation of these sites by a protein kinase known as Wee1 inhibits Cdk activity while dephosphorylation by cdc25 increases cdk activity
Describe Wee1 kinase
Wee1 phosphorylation on Tyr15 inhibits cdk1
Wee1 inhibits mitosis until the cell has attained a certain size
Wee1 mutant yeast cells initiate mitosis at half the size of wild type cells
What are checkpoints?
A stage in the eukaryotic cell cycle at which the cell examines internal and external cues and “decides” whether or not to move forward with division
Number of checkpoints
What does each checkpoint ensure?
Spindle - Are all chromosomes properly attached to the mitotic spindle?
G1 - before Sphase. Is environment favourable?
G2 - before Mphase. Is all DNA replicated? Is all DNA damage repaired?
Describe the DNA damage response
DNA damage can occur as a result of spontaneous chemical reactions in DNA, errors in DNA replication or exposure to chemicals. Damaged chromosomes must be repaired before replacing them.
- Cell cycle control system detects DNA damage and arrests cell cycle at G1 or G2 checkpoint.
- DNA damage initates a signalling pathway by activating one of a pair of related protein kinases called ATM and ATR, which associate with the site of damage and phosphorylate various target proteins, including two other protein kinases called Chk1 and Chk2.
- Various kinases phosphorylate other target proteins that lead to cell cycle arrest
- Major target is p53, which stimulates transcription of gene encoding p21, binds to G1/S-cdk and inhibits their activites
Describe p53 and G1
DNA damage activates p53 by indirect mechanism
Unstable and present in low concentrations in undamaged cells.
Largely because it interacts with Mdm2. Phosphorylation of p53 reduces binding to Mdm2. Decreases p53 degredation which results in a marked increase in p53 concentration in the cell
Describe the G2 checkpoint
- CyclinB-CDK1 activity is specific to the G2/M checkpoint
- Accumulation of cyclin B increases the activity of CDK1 as cells prepare to enter mitosis
- CDK1 activity regulated by phosphorylation/dephosphorylation of corresponding activators and inhibitors. Activation of cdc25 which deactivates Wee1 (an inhibitor)
- Cdc25 activates the complex through the removal of phosphatases from the active site.
Describe Cdc25
Induces mitosis in a dose-dependent manner
Was discovered by a mutant analysis in fission yeast
cdc25 ts mutants become blocked in G2 on shift to the restrictive temperature
Describe Chk1 and Chk2
Protein kinases that also block cell-cycle progression by phosphorylating members of the cdc25 family of protein phosphatases, inhibiting function
Particularly important in activation of M-Cdk at the beginning of mitosis
Chk1 and Chk2 phosphorylate Cdc25 at inhibitory sites that are distinct from the phosphorylation sites that stimulate Cdc25 activity
Helps block entry into mitosis
Purpose of spindle assembly checkpoint
To make sure that all chromosomes are bound to spindle microtubules at their kinetochores
Important to make sure the precise partitioning of the chromatids to the daughter cells during mitosis
Mistakes in the checkpoint lead to aneuploidy (one or more extra or missing chromosomes)
Describe the spindle assembly checkpoint
Nuclear envelope disassembled, te kinotochores bind to spindle microtubules
Unbound (free) kinetochores lead to the binding of a protein complex (mitotic checkpoint complex)
Cdc20 is part of MCC
Without Cdc20 the APC (anaphase promoting complex) is inactive
Anaphase and spindle assembly
If all kinetochores are bound, Cdc20 becomes free and can activate the APC
Securin and Cyclin B are ubiquitinated and degraded
Separase can cleave Cohesin
Sister chromatids can separate
Describe cytokinesis
Begins in anaphase, ends in telophase
In animal cells, cleavage furrow on cell surface
Uses actin and MTs
Give an overview of the cell cycle
When conditions for cell proliferation are right, various external and internal signals stimulate the activation of G1-Cdk, which in turn stimulates the expression of genes encoding G1/S- and S-cyclins.
G1/S-Cdks unleash a wave of S-Cdk activity, which initiates chromosome duplication in S phase and also contributes to some early events of mitosis.
M-Cdk activation then triggers progression through the G2/M transition and the events of early mitosis, leading to the alignment of sister-chromatid pairs at the equator of the mitotic spindle.
Finally, the APC/C, together with its activator Cdc20, triggers the destruction of securin and cyclins, thereby unleashing sister-chromatid separation and segregation and the completion of mitosis.
When mitosis is complete, multiple mechanisms collaborate to suppress Cdk activity, resulting in a stable G1 period.
