Cell Division Flashcards
Caner
Unregulated Cell division
***cancer = relates to cell division
Cells + nucleus
Some cells do not have a nucleus
Cells without a nuclues
- RBCs
- Human Skin
- Lens of the eye
RBCs
Simple cell that can replicate – bag of hemoglobin
Human RBCs vs. other animals RBCs
Humans = RBCs do not have a nucleus
Aviation + Amphibians (birds + frogs) = RBCs do have a nucleus
Amatopoisis
Shed nucleus
Human Skin Layers
Bottom layer = has nuclei
Strayum Corneum = No nucleus –> has cells but as the cells move up to the top they lose their nuclei –> allows the cells to compress = makes barrier properties
Why does the Starum Corneus lose nuclei
So that as the cells move up they can compress = gives it barrier properties
Lens of the eye Shape
- In lenticular shape
Layers of lens
- Has epithelial Layer
- Has germal layer
Lens of the eye division
Cells divide in the lens at the equator in the GERMAL LAYER –> as they divide = they differentiate into lens fiber cell with crystals
Lens over lifetime
Lens throughout your whole life = keeps growing –> reason old people get glaucoma
Lens fiber
No nucleus (Anuclease)
Enucleated
Verb!!! – like in SCNT when you enucleate (remove the nucleus)
Why shouldn’t lens have nuclei
Relates to phase microscopy –> because you can see differnt patrts of the cell in a phase microscope because of diffreent refractive indicies –> This would happen in the lens if it had a nucelus
- Having a nucelus would ruin your ability to see because it would interfere with refractive indicies
Parts of the Nucleus
- Nuclear envelope
- Nuclear Pores
- Histones
- Non-Histones
Nuclear envelope
Double membrane system around the nucleus
- Comes from the ER
***NE = connected to the ER because the ER generates the Nuclear Envelope
Nuclear Pores
VERY COMPLEX
***Sites where mRNA + Proteins leave
Use - passivley diffuses proteins less than 62,500 da
Diffusion of nuclear proteins
Most proteins in nucleus = can diffuse passively through nuclear pores BECAUSE most of the proteins in the nucleus are Histones
Histones
Small 20,000 da proteins in the nucleus –> they can diffuse passively through nuclear pores
***Most proteins in nucleus = histones
Use of Histones
Involved in Epigenetics – can be methylated + Phosphorylated
Non-Histones
Includes Transcription factors + Other proteins
- Have many different molecular weights
Example – Lamins (Type of intermediate Filament)
Example Active transport
Nucleoplasm (Nuclear protein) – exceed the passive diffusion weight
- Big protein = 165,000 da = can’t diffuse
- It is a Pentomer –> 5 SU each 33,000 da
- First molcular chaparone discovered
Discovery of Nucleoplasm
Found originally in African frog toad – found in Xenous Leavits
10% of the protein in the X. Laeveis egg
Where do xenobots come from
X.levits
Function of Nucleoplasm
Chaperone + nucleosome assumably + Genome stability + Transcriptional regulation
Purpose of Nucleosome
Helps histones arrange
Synthesis of Nucleosome
Question – is it synthesized in the cytoplasm and then shipped through a pore?
Experiment – track synthesis:
***Use ultrastructural radiograhy
- Use gold labeled anti-nucleoplasm tag – use Ultrasturctural radiography
- Idea = that it uses an ATP dependint synetshis to be trasnported through the pore SO you want to remove ATP BUT you can't remove all ATP or the cell will die -- SOLUTION = cool the cell to 4 degrees celcius
Result: Nucelosome gets transported through the nucleo pore
Lamins
Overall – type of intermediate filament
Found – in the inner envelope of nucleus
Types of Lamins
- Lamin A
- Lamin B
- Lamin C
Function of Lamins
Functionally important because they have many functions
- Provide structure for keroskeloton – important for circularization of the nucleus + localizing it in the center of the cell
- Connects chromatin to nucelar envelop – at the nuclear pore
- Phosphorylation of Lamin B triggers nuclear dissolution (MOST IMPORTANT FUNCTION)
Nuclear Dissolution
Nuclear envelope disappears
Inducing nuclear dissolution experiment
Experiment = demonstrates nuclear dissolution occurs during mitosis
Exp:
Fuse a mitotic cell with a cell in G1
Results:
1. Get nucleus disappears = have nuclear dissolution
2. G1 chromatin condenses –> means that something in the mitotic cell that can cause premature formation of chromosomes
***Some entity (MPF) = causes nuclear dissolution via Lamin Phosphorylation
Lamin + Nucleus
Have lamin inside nucleus –> when nucleus goes through mitosis = have nuclear dissolution + lamin B is phosphorylated (triggers dissolution)
Late mitosis – lamin B is dephosphorylated BUT doesnt mean that Lamin B comes back to make new nuclues –> the new nucleus comes from the ER
Defect in Lamin A
Associated with progeria
Progeria
Precoccious aging disease – age faster
Issue = not elliptical nucelus –> mishappen nuclei – not spherical
Normal – the Fornesyl comes off after the lamin is insertes –> Normal = have Lamina around the rim
Affected – The fornesyl stays on – make the Lamin BUT the Fornesyl foes not come off in the end = cause smishapen nucleus
Lamin Assmebley
Known process – know that formesyl inserts lamin into memebrane
***Faciliates lamnin into nuclear envelope
Lonafarnib
Farneyltrasnferace inhibitor used to treat Progeria
***Farneyltrasnferace inhibitor = researched a lot in cancer because Farnesyl links ocoproteins = get cancer but the inhibitors didn’t help
Cell Cycle (overall)
G0 –> G1 –> Restiction/START point –> S (DNA synthesis) –> G2 –> M phase (mitosis) –> daughter cells
What is cell cycle related to
Relates to embryogenesis + Stem cells
Fidelity of cell division
Remarkable –> to not make mistake
Cell division Occurance
Cell divison = occurs all of the time –> theoretically you should get bigger – Need a balance between cell division and cell death
Hematopoeis
Type of cel division – RBC division
G0
First part of cell cycle
***Technically NOT part of G1
- Quecet period –> part of the cell cycle chrachteristic of differenated cell
***Cells in G0 have no interest in dividing unless they are triggered to do so
What does G stand for?
Gap – gap between mitosis
G1
G1 length
For a cell that has a cell cycle of 24 hours –> 9 hours
G1 = 9/24 hours
Cancer cell cycle
Cancer cells can’t go through the cell division any faster
Example – some cancer cell cycle = 38 hours
Aurthur Pardee
Looked at G1 cycle – looked at the cell cycle in general BUT he mostly looked at G1
Asked – what does it take to get through G1 – what do you need to get from G1 –> DNA replication
Results: Found that you need Growth factors
Cells authur used
Used 3T3 cells – easy to convert from normal to cancer cells
**He introduced 3T3 cells – made them famous
**Commonly used for cell cylce and oncogene studes because they are easy to convert from normal to cancer cells
***They are isolated from mouse embryo tissue
What did authur show?
Showed that you need Growth factors to get from G1 –> S phase
Need PDGF –> EGF –> Insulin – need the growth factors in THAT order
PDGF –> EGF –> Insulin (ALL before S)
Genes in G1
Can identrify early and late response genes – All activated by Growth factors
To Study – look at mRNA over time
Results:
1. Get early response genes (see increase in mRNA early)
2. See late response genes (increase in mRNA later)
Early + late response genes
If you prevent the degradation of early response genes = then you don’t get the late response genes = SHOWS that they are connected
***Don’t get late response without the early
Studying Cell cycle
- Most of the work done = done by fusing cells
- Need to synchronize the cells so that they are all going through the cell cycle at the same time – to study you need cell synchrony (doesn’t occur naturally – need to induce it)
Inducing Cell synchrony
It won’t occur natural – you need to induce it
***Most wats are based on G1
- Amino Acid deprivation – stall cells in G1 –> because no AA = the cells can’t make proteins = stalls
- Can add back later
- Serum deprivation – stalls in GA (Prefered way)
- remove growth factors - Protein synthesis inhibtors – stalls in G1 + can stall in G2 (since it can stall in both = this is not prefered method)
- DNA synthesis inhibition – stalls in S
- Nucleosome inhibitor – stalls in M because spindle us inhibited
Cell synchrony experiment
Take synchronized cells and watch them divide
Result: Get a graph with a very strange profile –> need to figure out what happened
- Found that the only way to get that data was if the cell cycle was 24 hours +/- 6 hours – means that the cycle is varaible
What part of the cell cycle is variable
G1 phase – most variable in time
Variability = explains why the cells fall out of synchrony over time
Two points in the G1 cycle
- G1 checkpoint
- Resiction point
G1 checkpoint
Checking fidelity of process –> anylzye the fidelity (correctness) of G1 process
***Look at DNA
G1 restiction point
G1/S boarder – Go or no go point
***At this point – goes to S phase
IF it doesn’t go to S = it goes backwards in G1 = spends more time there
***aka “pardee point”
Where is restiion point found
Found in mammal cells + non-mammal cells BUT not in yeast cells
***Start in yeast
Do cancer cells differ from normal cells in their repsective cell cycle conpartment time?
Answer – they are not different
Experiment – look at different compartment times (look at each separately)
Take G1 synchronized cells – take synchrionized cells and watch
- In G1 –> Add H3-thimadine –> Look at how much time it is before you see labeled cells – because the labeled cells = tell you they are in S phase
- Once see labeled cells = know they are done in G1 – now in s (Because they are doing DNA replication)
- In S –> look at randomly dividing cells – all go through the cell cycle at different times
- At H3-thiamdine –> look at the number of labeled cells – if 3/10 are labeled = then do 3/10 X 24 hours = gives you the S phase time
- G2 Time –> use randomly dividing cells again
- Use H3 thiamdine to pick only cells in S phase –> once they are in M = you will see lableed chromsomes
- can see m cells with radioactive probe = tells you length of G2
- can use the same system to find length of m Phase
Result: Normal cells + cancer cells = have the same compartment times
Adding H3-Thimadine
Add to cells to probe cells that are going through DNA replication (cells starting to divide)
What controls cell cycle transit
Cyclins + associated Cyclin dependent kinases
Rhudamin + hunt
Did classic experiment about cell cycle movement
Exp:
Took naturally occuring synchronized cells – used sea urchin embryos (used ionmycin or sperm – added at once to the eggs)
THEN they ran SDS gel to see proteins
Results: Discovered Cyclins
Types of cyclins
There are many BUT only two are important:
1. Cyclin B
2. Cyclin A
Mitotic Cyclins
Cyclin A and Cyclin B – govern whether the cell goes through mitosis
What is associated with cyclin
Cyclins have partner cyclin dependent Kinases
***Cyclins won’t do anything unless has partner CDK
NEither alone can do anything –> they are a pair
***They can be influenced by many things – many things inhibit their function
CDKs
CDKs = increase and decrease over the cell cycle
Amounts of Cyclinc over cell cycle
Cyclin e – G1 –> S
Cyclin A –> G1 –> S –> G2 (decrease at m)
Cyclin B – M phase
Cyclin D –> G1
Mitosis
What is required for G1 passage
Cyclin D
***Cyclin D = associated with G1
Cyclin D experiment
Questioon – Us Cyclin D required for G1 cell cycle transit
T
ake G0 cell –> Add Growth factors –> Add BrdU –> Look at BrDU incorporation after time
Control cell – leave alone
- Result: have BrdU positive cells – means they are in S phase
Experimental cell – add anti-cyclin D antibody – blocking cyclin D
- Result: BrdU negative cells
***BrdU = like H3-Thiamine BUT safer
If add AB early in G1 = the control go to S BUT the experimental do not
If add AB late in G1 = the conrtol go to S and the experimental go to S (if add the antobody late in G1 = teh cells still go to S phase)
Use of BrdU
BrdU = like H3-thiamine BUT is safer – floursecnt alternative to H3-Thimadine
S phase
DNA synthesis
How do we study S phase
We can use electron microscope to tell us about S phase
Exp – Take cells in S phase –> Open cells + open the nucleus
Result: See replicons/replication bubbles – bubbles tell you there may be multiple points of replication + DNA synthesis may be directional
Two ways to study S phase
- Elecrton microscope
- Use Fiber autoradiogrphy
Autoradiography in S phase
Use fiber Autoradiography – Use autoradiography with chromosome inside –> Shows that electron microscope were right
Replication origins
Know there are many points where replication generates + H3 thiamine incorporates
Have Point ORC –> ORC makes replication bubble
What controls S phase
Cyclin A-CDK –> move cell cycle to mitosis
Cyclin B
Main mitotic cyclin
G2 phase
Some protein synthesis is required
***We know very little about G2 because non one cares about G2 = no one studies G2
What Happens in G2
- Cell verifies that al of the DNA has been correctly duplicated and all DNA errors have been corrected
- Chromosome condensation is initiated
- Early organization of the cell cytoskeleton
- Mitotic cyclin dependent kinases initiate activity
Mitosis (Overall)
Prophase – Spindle poles
Metaphase – sister chromatids
Anaphase
Telophase + Cytokinesis
M phase
***get new nuclear envelope
Shortest Phase in Cell cycle
M phase – know by looking at cell kinetic studies
Lamins in M phase
Lamin B = gets phosphorylated by MPF
What triggers movement through M Phase
MPFs
E.Leavis – Maturation phase factor
Mammalian cells (Cell fusion) – Mitosis Phase factor
Discovering MPF
2 groups using 2 model organisms:
1. Using X.levais –> perfect organism to study because cycles go through sequence
- take cytoplasm out of cell –> put in new cell –> idnduces mitosis iommediatley = have factors to promote maturing in egg
Found MPF – Matruartion Phase factor
- Used cell fusion experiment –> tells you what tells cell to go through mitosis
Overall – took a test tube + added cytoplasm from mitotic cell + nucleus + ATP –> Nucleus will duplicate
Used mammalian cells –> Did cell fusion –> found MPF
MPF – Mitosis Phase factor
Use of MPF
MPF = phosphorylates Lamin B = causes nuclear dissolution
When is MPF highest
MPF = highest at Metaphase
MPF cycle
Metaphase = high MPF –> In anaohase the Cyclin B is uqiquinated = decrease cyclin B BUT still high CDK–> make more cycle B over the course of the cycke = have more at mitosis again
Low MPF at telophase
What is MPF
MPF = Cyclin B + CDK
MPF function
Phosphorylate Lamin B = have dissociation of nuclear envelope = reason why MPF is high during metaphase
Cyclins in Cell cycle
Cyclin A = S phase + G1
Cyclin B = M phase
Cyclin D = specific to different cell cycle points
Ruth Sager
Discovered Cell cycle check points
***First to suggest that we have tumor supressor genes –> first to suggest that we have genes in system to prevent cancer
P53 (chart)
Cell cycle Checkpoint
Sites of control – we have many cell checkpoints
***Each cell cyle compartment = has checkpoint – check fidelity
Who discovered cell cycle check points
Ruth Sager
Discovering Cell cycle checkpoints Experiment
Take normal cell + cancer cell –> Fuse cells –> Get heterokeryon ( have two nuclei) –> get hybridioma
Look several generation later – Look at the phenotype –> Acts more like a normal cell NOT like a cancer cell
LOOK EVEN LATER – look at phenotypes –> now you have cancer cell
Conclusion: Maybe we have genes in system that prevent camcer cells – maybe we have tumor supressor genes
Heterokeyron
Have cell with two nuclei –> during the fusion process –> have reorganzation of nuclei + chromosome exchange – chromsomes are lost + genes are lost
Where is p53 active
p53 = active in many checkpoints
p53 name
Called “guardian of the genome”
p53 (overall)
Tumor supressor gene
p53 action
Triggered by DNA damage in G1
p53 = very unstable until it is phosphorylated by a ATMR –> activates p53
When p53 is active = it can do two things:
1. Fix probelm – if few DNA errors = fixes the DNA errors –> cell goes to S phase
2. Kill cell –> if there are many DNA errors = cell goes through apoptosis
***p53 = checks for DNA errors
Where were checkpoints first discovered
First discovered in Yeast –> budding yeast + Fission yeast
Budding yeast Cycle
Fission yeast Cycle
