Block E Part 1: Cell Division, Cell Growth and Control Mechanisms Flashcards
What is the basic function of the cell cycle?
To duplicate DNA in the chromosomes and then segregate the copies into 2 identical daughter cells
(Lecture 1, Slide 5)
What phase of the cell cycle does chromosome duplication occur in?
S phase - S for DNA synthesis
(Lecture 1, Slide 5)
How long does the S phase of the cell cycle last and how long is this compared to the full cycle?
~ 10-12 hours and is roughly half the time of the full cycle
(Lecture 1, Slide 5)
What phase of the cell cycle does chromosome segregation and cell division occur in?
M - M for mitosis
(Lecture 1, Slide 5)
How long does the M phase of the cell cycle last?
~ 1 hour
(Lecture 1, Slide 5)
What are the 2 major events that make up the M phase of the cell cycle?
Mitosis (aka nuclear division) and cytokinesis (aka cytoplasmic division)
(Lecture 1, Slide 6)
What helped us understand how the cell cycle worked?
Model organisms
(Lecture 1, Slide 7)
What is the order of the 4 phases of the cell cycle?
G1->S->G2->M
(Lecture 1, Slide 8)
Why does the cell cycle have controls?
To ensure that progression in the cell cycle only occurs in the right situations
(Lecture 1, Slide 8)
What are the 3 main control points of the cell cycle?
Start transition
G2/M transition
Anaphase onset/cytokinesis
(Lecture 1, Slide 8)
What power do the controls points in the cell cycle have?
The power to stop cell division until any problem in the cell cycle is resolved
(Lecture 1, Slide 8)
What is Schizosaccharomyces pombe?
Fission yeast
(Lecture 1, Slide 9)
What was used to isolate mutants blocked at various stages of the cell cycle in Schizosaccharomyces pombe?
Yeast genetics
(Lecture 1, Slide 9)
What type of mutants did Nurse and Hartwell use in Schizosaccharomyces pombe?
Mutants that were temperature sensitive
(Lecture 1, Slide 9)
What are temperature sensitive mutants?
Mutations that produce a protein that functions at one temperature, but not another
(Lecture 1, Slide 9)
What type of mutant is a temperature sensitive mutant?
A conditional mutant
(Lecture 1, Slide 9)
What is Xenopus laevis?
African clawed frog
(Lecture 1, Slide 11)
What is a useful model system to study the cell cycle?
Egg development in the Xenopus laevis frog
(Lecture 1, Slide 11)
Why do Xenopus oocytes (eggs from the African clawed frog) become arrested in the G2 phase of the cell cycle for 8 months?
So they can grow in size to a diameter of 1 mm, stockpiling molecules required post-fertilisation to generate a swimming, feeding tadpole
(Lecture 1, Slide 11)
How do the Xenopus oocytes cells of African clawed frogs arrested in the G2 phase of the cell cycle begin meiosis?
When stimulated by a male, the female ovarian cells secret progesterone, which induces the arrested cells to enter meiosis
(Lecture 1, Slide 11)
What occurs during meiosis of xenopus laevis (African clawed frogs)?
The diploid oocyte (egg) produces mature eggs which are ready to be fertilised by sperm
(Lecture 1, Slide 11)
What does fusion of the sperm and egg generate in Xenopus oocytes and what does it start?
A diploid nucleus, and embryogenesis proceeds with multiple rounds of the cell cycle
(Lecture 1, Slide 11)
Why is it possible to remove the cytoplasm from one cell of xenopus oocytes and mature eggs and introduce it into another?
Due to their large size
(Lecture 1, Slide 11)
How can populations of cells be studied?
By measuring the DNA content of the individual cells in a flow cytometer
(Lecture 1, Slide 15)
How can you tell how much DNA is in a cell and how many cells are present?
Cells are incubated with Hoechst-33342, a membrane-permeant dye that fluoresces when it binds to DNA; the amount of fluorescence is directly proportional to the amount of DNA in each cell
(Lecture 1, Slide 15)
What are the 3 peaks identified in cell population when DNA content is analysed with a flow cylinder?
Those that have an un-replicated complement of DNA and are therefore in G1
Those that have a fully replicated complement of DNA (twice the G1 DNA content) and are in G2 or M phase
Those that have an intermediate amount of DNA and are in S phase
(Lecture 1, Slide 16)
How can we study cell cycle regulation?
Study the localisation of protein (or mutants) in cells in culture using indirect immunofluorescence microscopy
(Lecture 1, Slide 17)
What does the cell-cycle control system depend on?
Cyclically activated Cyclin-dependent protein kinases (Cdks)
(Lecture 1, Slide 19)
What does each Cdk-cyclin complex do?
Phosphorylates a different set of proteins
(Lecture 1, Slide 21)
How can the same cyclin-Cdk complex can induce different effects at different times of the cell cycle?
As accessibility to targets may change in a cell cycle-dependent manner
(Lecture 1, Slide 21)
What can cyclins direct to specific substrates?
The kinase
(Lecture 1, Slide 21)
How is the next phase of the cell cycle started and stopped if needed?
The Cdk without cyclin is inactive - cell cycle is stopped. The cyclin activates the Cdk which phosphorylates its targets, beginning the next phase
(Lecture 1, Slide 22)
How do cdks become partially active?
In the absence of the cell cycle cdks show a loop (T-loop) which blocks access of substrates to the kinase active site , cyclin binding causes this loop to move aside which partially activates the kinase
(Lecture 1, Slide 23)
How do Cdks become fully active?
Full activation requires a Cdk-activating kinase to phosphorylate the T-loop driving a further change in conformation, making the Cdk now fully active
(Lecture 1, Slide 23)
What triggers the metaphase to anaphase transition?
Regulated proteolysis
(Lecture 1, Slide 28)
What drives progression through the start and G2/M transitions?
Specific cyclin-Cdk complexes
(Lecture 1, Slide 28)
What triggers progression through the metaphase-to-anaphase transition?
Protein destruction
(Lecture 1, Slide 28)
What is the key regulator in the metaphase to anaphase transition?
Anaphase promoting complex (sometimes called cyclosome; APC/C)
(Lecture 1, Slide 28)
What is anaphase promoting complex?
A member of the ubiquitin ligase family of enzymes
(Lecture 1, Slide 28)
What is ubiquitin?
A small protein added to proteins by the concerted action of three enzymes; E1, E2 and E3 - this is called uniquitylation
(Lecture 1, Slide 29)
What is polyubiquitylation a trigger for?
Proteins to be degraded in cells
(Lecture 1, Slide 29)
What does polyubiquitin target proteins to?
The proteasome
(Lecture 1, Slide 29)
What does the proteasome do to proteins?
It chews them up and allows the amino acids to be re-used
(Lecture 1, Slide 29)
What happen to proteins in the proteasome after they are chewed up?
Ubiquitin is cleaved off and is recycled by the cell
(Lecture 1, Slide 29)
What are 2 examples of enzymes that can function as the E3 enzyme to help make ubiquitin?
Anaphase promoting complex (APC/C)
SCF
(Lecture 1, Slide 29)
What does anaphase promoting complex (APC/C) catalyse?
The ubiquitylation and destruction of two major types of proteins; Securin and S- and M-cyclins
(Lecture 1, Slide 31)
Why does APC/C catalyse the ubiquitylation and destruction of securin?
Securin holds the sister chromatids together, separating the sister chromatids starts anaphase, which is accomplished by destroying securin
(Lecture 1, Slide 31)
Why does APC/C catalyse the ubiquitylation and destruction of S- and M-cyclins?
To stop the cell cycle progressing
(Lecture 1, Slide 31)
When does APC/C activity significantly increase?
When it associates with an activating subunit such as Cdc20
(Lecture 1, Slide 33)
When is Cdc20 concentration elevated and by what control?
By the action of Cdk-M through feedback control
(Lecture 1, Slide 33)
What does the APC/C-Cdc20 complex (acting as an “E3” recruit and why?
Ubiquitylation enzymes E1 and E2 to target a specific protein
(Lecture 1, Slide 33)
What does a protein being ubiquitylated signal?
The cell to destroy that protein
(Lecture 1, Slide 33)
When in the cell cycle is SCF active?
Constantly
(Lecture 1, Slide 35)
What does SCF do to CKI proteins late in G1 phase?
It poly-ubiquitylates them
(Lecture 1, Slide 35)
What does SCF poly-ubiquitylating CKI proteins in G1 control?
Activation of S-Cdks and DNA replication
(Lecture 1, Slide 35)
What is ubiquitylation by SCF controlled by?
Changes in phosphorylation of its targets, recognised by associated F-box proteins which can only bind phospho-targets
(Lecture 1, Slide 35)
What stimulates the activation of G1-Cdk?
Various external and internal signals
(Lecture 1, Slide 36)
What does the activation of G1-Cdk in turn stimulate?
The expression of genes encoding G1/S and S-cyclins
(Lecture 1, Slide 36)
What does the activation of G1/S-Cdk drive?
Progression through the start transition
(Lecture 1, Slide 36)
How does G1/S-Cdk’s initiate S phase?
They unleash a wave of S-Cdk activity, which initiates chromosome duplication in S phase
(Lecture 1, Slide 36)
What does M-Cdk activation trigger?
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
(Lecture 1, Slide 36)
What 2 things make up the total cell mass?
Total number of cells and their size
(Lecture 1, Slide 37)
What does cell number depend on?
The amount of cell divisions and death
(Lecture 1, Slide 37)
What do mitogens stimulate?
Cell division
(Lecture 1, Slide 38)
What do growth factors stimulate?
Cell growth
(Lecture 1, Slide 38)
What is cell growth?
An increase in cell mass
(Lecture 1, Slide 38)
What do survival factors do?
Support cell survival by suppressing programmed cell death (apoptosis)
(Lecture 1, Slide 38)
What do some human cells that do not divide do instead?
Terminally differentiate
(Lecture 1, Slide 38)
What limitation do many human cells have built into them?
How many times they can divide
(Lecture 1, Slide 38)
How did the path of isolation of platelet-derived growth factor start?
With the observation that fibroblasts in a culture dish proliferate when provided with serum but not when provided with plasma
(Lecture 1, Slide 39)
How is plasma prepared?
Plasma is prepared by removing the cells from blood without allowing clotting to occur
(Lecture 1, Slide 39)
How is serum prepared?
By allowing blood to clot and taking the cell-free liquid that remains
(Lecture 1, Slide 39)
What happens when blood clots?
Platelets incorporated in the clot are stimulated to release the contents of their secretory vesicles
(Lecture 1, Slide 39)
What suggested that platelets contain one or more mitogens?
The superior ability of serum to support cell proliferation
(Lecture 1, Slide 39)
How was the hypothesis that platelets contain one or more mitogens confirmed and what was the crucial factor?
By showing that extracts of platelets could server instead of serum to stimulate fibroblast proliferation, and crucial factor in the extracts was shown to be a protein, which was purified and named platelet-derived growth factor (PDGF)
(Lecture 1, Slide 39)
What does PDGF liberated from blood clots help stimulate?
Cell division during wound healing
(Lecture 1, Slide 39)
How many mitogens does our body have and why?
Around 50, to stimulate a broad range of cells types
(Lecture 1, Slide 39)
How do mitogens control the rate of cell division?
By acting in the G1 phase of the cell cycle
(Lecture 1, Slide 40)
What do mitogens interact with?
Cells surface receptors
(Lecture 1, Slide 40)
What do mitogens interacting with cell-surface receptors trigger?
Multiple intracellular signalling pathways
(Lecture 1, Slide 40)
What does the activation of the small GTPase Ras lead to?
Activation of MAP kinase cascade
(Lecture 1, Slide 40)
What does activation of a MAP kinase cascade lead to?
Increase expression of numerous immediate early genes, including the gene encoding the transcription of the regulatory protein Myc
(Lecture 1, Slide 40)
What is Myc thought to promote and what does it stimulate?
It promotes cell-cycle entry and plays a major role in stimulating the transcription of genes that increase cell growths
(Lecture 1, Slide 41)
What is one of the mechanisms that Myc uses to promote cell entry?
It increases the expression of genes encoding G1 cyclins (D cyclins) thereby increasing G1-Cdk activity
(Lecture 1, Slide 41)
What is the key function of G1-cdk complexes in animal cells?
To activate a group of gene regulatory factors called the E2F proteins
(Lecture 1, Slide 41)
What to E2F proteins do?
They bind to specific DNA sequences in the promoters of a wide variety of genes that encode proteins required for S-phase entry, such as G1/S-cyclins and proteins involved in DNA synthesis and chromosome duplication
(Lecture 1, Slide 41)
What happens to E2F-dependent gene expression in the absence of mitogen stimulation?
It is inhibited by an interaction between E2F and members of the retinoblastoma protein (Rb) family.
(Lecture 1, Slide 41)
When cells are stimulated to divide by mitogens, what does active G1-Cdk do to retinoblastoma (Rb) family members and what does this result in?
G1-Cdk accumulates, and phosphorylates Rb family members which reduces their binding to E2F, which then leads them to activate expression of their target genes
(Lecture 1, Slide 41)
What does abnormally proliferation signals cause and what is the exception?
Cell-cycle arrest or apoptosis, except in cancer cells
(Lecture 1, Slide 42)
Why were many genes that code for components of the mitogenic signalling pathways originally identified as cancer-promoting genes?
As mutations in them contribute to the development of cancer
(Lecture 1, Slide 42)
What does a single amino acid mutation in the GTPase Ras cause?
The protein to become permanently overactive
(Lecture 1, Slide 42)
What does the GTPase Ras becoming permanently overactive lead to?
Constant stimulation of Ras-depending signalling pathways, even in the absence of mitogenic stimulation
(Lecture 1, Slide 42)
What do mutations that cause an overexpression of Myc stimulate and therefore promote?
Excessive cell growth and proliferation, promoting the development of cancer
(Lecture 1, Slide 43)
What are 2 possible results of experimentally overproducing a hyperactive form of Ras or Myc in most normal cells?
Instead of excessive proliferation, cells undergo either permanent cell-cycle arrest or apoptosis
(Lecture 1, Slide 42)
What does most normal cells going into permanent cell-cycle arrest or apoptosis in response to an experimental overproduction of a hyperactive form of Ras or Myc prove?
That the normal cell seems to be able to detect abnormal mitogenic stimulation, and it can respond by preventing further division
(Lecture 1, Slide 42)
What is Mdm2?
It is a E3 ubiquitin ligase
(Lecture 1, Slide 43)
What is the main function of Mdm2?
To ubiquitinate P53, signalling it for destruction by the proteosome
(Lecture 1, Slide 43)
What does abnormally high levels of Myc activate?
Arf
(Lecture 1, Slide 43)
What do Arf do once activated by Myc?
It binds and inhibits Mdm2, increasing p53 levels
(Lecture 1, Slide 43)
What 2 scenarios can P53 cause once activated and what 2 things decide which one it picks?
It can either cause cell-cycle arrest or apoptosis depending on the cell type and extracellular conditions
(Lecture 1, Slide 43)
What is cell proliferation accompanied by?
Cell growth - there is little point in telling a cell to divide if it is not triggered to grow
(Lecture 1, Slide 44)
What does a growth factor binding to a cell surface receptor activate?
PI 3-kinase
(Lecture 1, Slide 44)
What does PI 3-kinase promote?
Protein synthesis
(Lecture 1, Slide 44)
How does Pi 3-kinase promote protein synthesis?
Through a complex signalling pathway that leads to the activation of the protein kinase TOR
(Lecture 1, Slide 44)
On top of the signalling pathway PI 3-kinase uses to activate TOR, what else can help activate it?
Extracellular nutrients such as amino acids
(Lecture 1, Slide 44)
How does TOR stimulate protein synthesis?
By phosphorylating a range of transcription regulatory proteins leading which contribute to an increased production of ribosomes
(Lecture 1, Slide 45)
In addition to binding to cell surface receptors to activate PI 3-kinase what do growth factors stimulate increase production of?
Myc
(Lecture 1, Slide 45)
How does Myc help promote cell metabolism and growth after it’s production is increased by growth factor stimulation?
By activating the transcription of various genes that promote cell metabolism and growth
(Lecture 1, Slide 45)
