you may not believe it but by the end of the semester this will make sense! hanahan and weinberg,...
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You may not believe it but by the end of the semesterThis will make sense!
Hanahan and Weinberg, Cell 100:57-70 (2000)
Cells must be able to proliferate
- during development- wound healing- stem cells in blood, small intestine, immune system
For cells to copy themselves they need to:
- Grow; make more stuff; e.g. proteins, lipids
- Copy their genetic material
- Segregate contents to daughter cells, especially…
- Segregate replicated chromosomes to daughter cells
Many of the images in the cell cycle part of the course are taken from The Cell Cycle , by David O Morgan (New Science Press)
Interphasecells duplicate chromosomes Mitosiscells segregate duplicated chromosomes into two daughter cells
The Cell Cycle
Interphase has 3 periods: G1, S, G2
G1: cells decide whether to divide or not:- Have I grown big enough to enter the cell cycle?- Am I OK?
Restriction Point / START
S: chromosomes are duplicated
G2: cell prepare to enter mitosis by asking:- Have I completed DNA synthesis properly?- Am I OK?
Execution of these decisions commits a cell to complete a full division cycle
The main jobs of the cell cycle:
1. To accurately transmit the genetic information!2. To maintain normal ploidy; i.e. diploidy!
Regulatory mechanisms:- Accuracy in the “assembly line” (e.g. DNA
polymerase)- Extrinsic regulatory mechanisms (all processes
follow a correct order)
Early G1 Pre-replicative complex (origin licensing)
Early SActivation of helicase;Assembly of pre-initiation complex
Helicase
DNA does not come naked
It is packed into chromatin
Mainly, histone proteins
Thus, duplicating chromosome = duplicating DNA and duplicating histones
In addition, we need to repack the duplicated DNA
Histone synthesis increases sharply during the S phase
Increase in transcription, in processing, and in stability
Chromatin Inheritance
- TelomeresCis-elements: sequences recruiting proteins that
modify histones
- Centromere Epigenetic mechanisms, not clearly understood
Reproducing chromatin organization during the S phase
Mitosis
During the S phase, the duplicated DNA is rearranged through cohesion to form two sister-chromatids attached to each other by cohesins
Gradually, the cohesins will be removed to allow sister-chromatid separation
- Sister-chromatids condense
- Centrosomes move to opposite poles of the cell, nucleating microtubules (MTs)
- Nuclear envelope breakdown
Prophase
Prometaphase
- Nuclear envelope breakdown is completed
- The centrosomes nucleate MTs towards each other, forming the spindle MTs
- The growing (+) ends of the MTs capture the chromosomes at the site of the centromere through a protein complex called the kinteochore
Prometaphase
- Nuclear envelope breakdown is completed
- The centrosomes nucleate MTs towards each other, forming the spindle MTs
- The growing (+) ends of the MTs capture the chromosomes at the site of the centromere through a protein complex called the kinteochore
Silverman-Gavrila lab
MitosisProphaseChromatid condensationPrometaphaseKinetochore-MTs bindingSpindle assemblyMetaphaseChromosomes align at the midline
Telophase and CytokinesisBirth of two daughter cells
AnaphaseSegregation of sister-chromatids
- Fuse S phase cell with G1 cell: The G1 nucleus enters S phase
Rao and Johnson (1970)Cell fusion experiments
- Fuse M phase cell with interphase cell: Interphase nucleus enters M
For example, anaphase-metaphase transition will take place only if ALL the kinetochores are attached to MTs
If the checkpoint regulators are compromised, unattached chromosome might be lagging behind, resulting in aneuploidy
Experimental Systems Important for Cell Cycle Studies
Arbacia punctulata
Xenopus laevisSchizosaccharomyces pombe
Saccharomyces cerevisiae
Lee Hartwell
Hartwell was interested in the protein synthesis machinery
Budding Yeast: a genetic eukaryotic model organism
Let’s look for mutants that cannot synthesize proteins
Lee Hartwell
Budding Yeast: Saccharomyces cerevisiae
Serendipity, our old friendBrian Reid, an undergrad, needs to look at a microscope to follow a mutant. They realize that bud size stores information about the cell cycle
Brian Reid
Permissive (low) temperature(mixed population of cells in different
stages of the cell cycle)
Restrictive (high) temperature
An assay for isolating cdc mutantscdc: cell division cycle mutants
cdc mutant growingat permissive temp
cdc mutant growth arrested after 6 hrs at restrictive temp
Temperature sensitive cdc mutant
DNA
How to clone cdc genes in yeast?
Let’s say you have a candidate sequence
cdc28 (-)
If the candidate sequence complements (rescues) the mutated phenotype: that’s your gene!
WT
cdc genes encode proteins needed for the G2-M transition: studies in s. pombe
cdc2D = gain of function mutant
Cloning cdc2The same approach used in budding yeasts:
complementation by a library
Only using a budding yeast library
This is all great
Yeast are really cute and interesting
Can we really learn something from that about humans?
Schizosaccharomyces pombe
Sir Paul Nurse
Crazy idea
Let’s try to complement (rescue) the cdc2 (-) mutant of pombe with a human cDNA library
It worked for us with budding yeast genes. Why not try human genes?
Human cdc2 rescues cdc2 mutants
Elongated cdc2 mutants, failing to undergo mitosis
cdc2 mutants, complemented by a
human cdc2 gene
Melanie Lee
Summary
- A genetic approach in fission and budding yeasts reveal genes that are essential in promoting the cells through the cell cycle
- These genes encode kinases proteins and are called CDKs for Cyclin-Dependent Kinases
Cdk1 = the protein encoded by cdc2/CDC28
can be stimulated to lay lots of eggs
Sea urchins
The summer project: to follow protein synthesis upon fertilization by following incorporation of S35 - Met and getting samples every 10’
Proteins X,Y,Z are synthesized only in unfertilized eggs Proteins A,B,C are synthesized upon fertilization
Protein A disappears 10’ before completion of mitosis
mitosis mitosis mitosis
In clams two proteins, A and B, express this cyclic behavior
Cyclins are synthesized and degraded in a cyclic manner and with correlation to the cell cycle
ProteinLevel
Time
cyclin A cyclin B
M M M
Something needs to go away in order for the cell cycle to proceed
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