2_3 Flashcards
cell cycle
- Multicellular eukaryotes depend on cell division for Development from a fertilized cell, Growth, Repair
- Cell division is an integral part of the cell cycle, the life of a cell from formation to its own division.
- Most cell divisions result in daughter cells with identical genetic information.
- The exception is meiosis
genome
- All the DNA in a cell constitutes the cell’s genome.
- A genome can consist of a single DNA molecule (common in prokaryotic cells) or a number of DNA molecules (common in eukaryotic cells).
- DNA molecules in a cell are packaged into chromosomes.
binary fission
- Cell Division in a prokaryote
1. DNA contained in a single circular chromosome with an origin of replication. First step is DNA replication.
2. Cell gets larger as chromosomes move to opposite ends of the cell.
3. After replication, cell pinches in two, resulting in two identical daughter cells.
Eukaryotic chromosomes
- Eukaryotic chromosomes consist of chromatin, a complex of DNA and protein that condenses during cell division.
- Every eukaryotic species has a characteristic number of chromosomes in each cell nucleus.
Cell division in eukaryotic cells more complicated because
because they have…
More than one chromosome
Cell organelles
Cytoskeleton
cell cycle phases
interphase
- G1 (40 percent) - separates M phase from S phase. Cell grows and accumulates substrates needed for DNA replication. Centrioles in centrosome separate.
- S (39 percent) - DNA synthesis
- G2 (19 percent) - separates S from M. Cell produces molecules needed for mitosis.
M phase
(2 percent); Mitosis (nuclear division) and cytokinesis (division of the cytoplasm)
G0
(variable) The non-dividing (resting) phase in which most cells exist
cell cycle differences
- The frequency of cell division varies with the type of cell
- These cell cycle differences result from regulation at the molecular level
cell cycle experiments
- The cell cycle appears to be driven by specific chemical signals present in the cytoplasm
- Some evidence for this hypothesis comes from experiments in which cultured mammalian cells at different phases of the cell cycle were fused to form a single cell with two nuclei
- one cell in S; one in G1; G1 nucleus immediately entered S phase
checkpoints
- In normal cells that divide, the cell cycle does not proceed unchecked. Cells maintain control over the stages of the cell cycle through what are called checkpoints—steps along the cycle that must be completed before the next step is started.
- Many of these signals are kinases (cyclin-dependent kinases - CDKs) that activate other proteins and allow the next phase to proceed.
G1 checkpt
regulates the entry of the cell into the S-phase of DNA replication.
One of the conditions that must be met is:
Is DNA undamaged?
G2 checkpt
regulates the entry of the cell into the M-phase of mitosis
One of the conditions that must be met is:
Has all of the DNA been replicated?
M checkpt
regulates the entry of the cell from metaphase to anaphase
One of the conditions that must be met is:
Are the chromosomes aligned at the metaphase plate?
Cdk/cyclin concentrations
- Cdks are proteins that activate other proteins and allow the next phase to proceed. Their concentrations do not vary throughout the cell cycle. However, they must be bound to cyclins to be active.
- Cyclin concentrations do vary during the cell cycle.
Cdk/cyclin compelx and example
- Cyclins and CDK bind together to form complexes that control the progression of the cell cycle.
- ex: Maturation (or M-phase)-promoting factor (MPF)
- This protein allows the cell to progress past the G2 checkpoint and enter mitosis. It is formed when a cyclin binds to a CDK.
- Activated MPF is a kinase and controls some of the processes associated with mitosis, including the breakdown of the nuclear membrane.
- MPF is only a transiently active molecule - only when it is bound to cyclin.
MPF concentration
- MPF also initiates a sequence that leads to its separation back into a cyclin and the inactive cyclin-dependent kinase. The cyclin is then broken down, and levels remain low until the correct signal occurs to increase cyclin concentrations.
- MPF activity tracks (and lags behind) changes in the cyclin concentration
internal signal at checkpt
An example of an internal signal is that kinetochores not attached to spindle microtubules send a molecular signal that delays anaphase
external signal at checkpt
- Some external signals are growth factors, proteins released by certain cells that stimulate other cells to divide
- Another example of external signals is density-dependent inhibition, in which crowded cells stop dividing
PDGF
- example of growth factor (external signal)
- platelet-derived growth factor (PDGF) stimulates the division of human fibroblast cells in culture
experiment:
1. sample of human connective tissue is cut up into small pieces w/ scalpel, pieces placed in petri dish
2. enzymes digest the ECM, resulting ins uspension of free fibrobasts
3. cells transferred to culture vessels; PDGF added to half of the vessels
4. vessels w/ PDGF: many more cells than vessels w/o
anchorage dependence
- example of external signal
- Most animal cells, in addition to density-dependent inhibition, exhibit anchorage dependence, in which they must be attached to a substratum in order to divide
- Cancer cells exhibit neither density-dependent inhibition nor anchorage dependence
density-dependent inhibition
- crowded cells stop dividing
- cells form a single layer; cells divide to fill a gap and then stop
S-Cdk
S-Cdk controls G1 checkpoint (entry into S-phase)
- if DNA is damaged, S-Cdk is inhibited so cell won’t
enter S-phase and replicate damaged DNA.
Ubiquitylation
is the targeting of molecules to be destroyed
when p53 gene is normal
- Mdm2 binds to the unphosphorylated p53 and directs it to be destroyed. (Ubiquitylation)
- In response to the presence of damaged DNA, kinases are activated that phosphorylate and thus increase the concentration of p53 (it’s no longer ubiquitylated).
- Active p53 binds to a regulatory site on the DNA just upstream from a gene that codes for a protein called p21.
- This stimulates the transcription of the p21 gene and through translation increases the production of the p21 protein.
- p21 protein is a CDK inhibitor and it binds to and inhibits the activity of the S-cyclin CDK complex.
when there’s something wrong w/ p53
- Cells that lack the p53 gene or gene product fail to make p21 and thus do not inhibit the S- cyclin CDK complex when their DNA is damaged.
- As a result, the entry into the S-phase is uninhibited and the damaged DNA is replicated. This can result in the production of cells that have damaged DNA and can become cancerous. In fact many human cancers have been traced to mutants in the p53 gene.
kinetochores
attachment points of microtubules
M-Cdk
allows entry into anaphase (M checkpoint)
by activating APC (anaphase promoting complex)
- Initiates the separation of sister chromatids at the metaphase-anaphase boundary.
APC
- Cohesin proteins hold sister chromatids together.
APC (anaphase promoting complex) is activated by phosphorylation by M-Cdk. This activated APC then can bind cdc20. - Active APC then ubiquitylates and destroys securin, which had been bound to separase, thus inactivating securin.
- Freed separase then cleaves the cohesin molecules.
- Tension on the chromatids exerted by the mitotic spindles (microtubules) pulls chromatids to opposite poles.
microtubules
- essential for cell division
- tubulin dimer from alpha and beta monomers
- 25 nm diameter
centrosomes
- Centrosomes are the point at which microtubules are formed in some but not all cells. (microtubule-organizing region)
- In animal cells they contain a pair of centrioles, may contribute to but are not required for microtubule formation.
Nucleolus
Consists of ribosomes and RNA. Is well developed in cells that have a lot of active protein synthesis. The nucleolus is the site of synthesis of ribosomal proteins.
Chromatin
Consists of DNA and its associated proteins (histones and non-histone proteins) , in particular a group of proteins called histones that are structural proteins are closely associated with DNA. Also many regulatory proteins that have many functions in controlling gene expression.
- in non-dividing cell.
packaging of DNA in non-dividing phase
- During the non-dividing phase, DNA is not organized into an easily visible structure within the cell nucleus. Chromosomes, which are the characteristic feature of dividing cells, are not present. Instead, DNA is found in chromatin.
- Chromatin is distributed throughout the
nucleus, and in this form would be difficult
to distribute accurately between the
daughter cells.
packaging of DNA in dividing phase
- In dividing cells chromatin is organized into more readily recognizable structures called chromosomes.
- During cell division a complex of proteins called kinetochores attaches to the centromere. Specialized microtubules attach and pull the chromosome apart.
chromosomes
Each chromosome is comprised of a single strand of DNA. In a somatic cell, the chromosome has two sister chromatids which contain identical strands of DNA. The chromatids are joined at a centromere, which is the point at which the chromosomes are pulled apart during mitosis.
How is DNA organized within the nucleus?
- The human genome has 6 x 10^9 base pairs, each of which occupies 0.34nm. This means that the DNA in every cell is 2m long. How is this packaged into a nucleus that is often only 10µm in diameter?
- it’s accomplished by a complex folding process that involves a close association of the DNA with a series of proteins.
nucleosome
a structural unit of a eukaryotic chromosome, consisting of a length of DNA coiled around a core of histones.
mitosis
The cell begins mitosis from G2 of interphase. Mitosis consists of five phases: 1) Prophase 2) Prometaphase 3) Metaphase 4) Anaphase 5) Telophase
Mitosis is just the nuclear division.
It is followed by cytokinesis, which is the separation of the cytoplasm and the formation of the two daughter cells.
Two key events occur after the cell enters S phase:
1) DNA is replicated
2) The centrosome replicates.
Centrosome replication
replication is controlled by a cyclin-cdk complex whose concentration peaks at the G1 checkpoint and is the key event in directing the cell towards mitosis (cell enters S phase).
centrosome movement
During G2-to-M transition, the two new centrosomes separate from each other and move to opposite ends of the nuclear envelope.
The orientation of the centrosomes within the cell determines the cell’s plane of division.
structures involved in prophase
1) Chromosomes
2) Centrosomes and microtubules
chromosomes in prophase
- During prophase, chromosomes become visible as distinct structures, but are fairly elongated.
- Prophase chromosomes consist of two sister chromatids held together over much of their length by cohesins.
- The kinetochore is a structure associated with the centromere and is the site where microtubules attach to the chromatids. They develop in late prophase.
microtubules in prophase
- Polar microtubules and astral microtubules make up the non-kinetochore microtubules and begin to extend from each centrosome complex in a formation called an aster (‘star’).
- These tubules will develop into mitotic spindles. Microtubules are formed by addition of tubulin dimers to the end of each microtubule.
polar microtubule
Each polar microtubule runs from a centrosome to a point where it overlaps and interacts with a microtubule from the other side.
astral microtubuel
Astral microtubules extend between the centrosome and the cell membrane.
prometaphase structures involved
- chromosomes
- nuclear lamina
- microtubules
- kinetochores
chromosomes in prometaphse
- The chromosomes become more tightly coiled and appear shorter and fatter. They remain held together by cohesins.
- Chromosomes move toward the center of the cell—the metaphase plate. At this point, chromosomes often demonstrate a jerky back and forth motion as they line up in the middle of the cell.
nuclear structures in prometaphase
The nuclear lamina disintegrates (remember MPF?) and the nuclear envelope fragments to permit spindle microtubules to infiltrate the nuclear region.
kinetochore vs centromere
centromere: The ‘waist’ of a chromosome. The structure where mitotic spindles attach.
kinetochores: Proteins that attach microtubules to centromeres.
non-kinetochore microtubules
The spindle microtubules ones not attached to kinetochores are called non-kinetochore microtubules: polar microtubules and astral microtubules.
microtubules in prometaphase
- Some of the spindle microtubules begin to associate with kinetochores. These are called kinetochore microtubules.
- The microtubules from one pole associate with the kinetochore of one of the sister chromatids of each chromosome. Microtubules from the other pole associate with the kinetochore of the other chromatid.
nuclear structures in metaphase
The nuclear membrane and laminar have completely disintegrated.
metaphase: ____ begins ____
The cohesins begin to be broken down (remember m-Cdk and separase?).
microtubules in metaphase
Chromosomes held in center by tension of kinetochore microtubules—each is being pulled toward one or the other end, but chromosomes are still attached together so they stay in the center.
chromosomes in metaphase
- Chromosomes aligned at metaphase plate in the center of cell, midway between each spindle pole.
- Chromosomes held in center by tension of kinetochore microtubules.
chromosomes in anaphase
- The sister chromatids of each chromosome (now referred to as daughter chromosomes) are pulled apart during anaphase.
- Cleavage of cohesins is completed.
- Daughter chromosomes pulled to opposite poles.
microtubules in anaphase
- Kinetochore microtubules shorten—pull chromosomes to poles.
- Polar microtubules lengthen—push spindle poles apart.
- Astral microtubules shorten—pull spindles apart.
At the end of anaphase,
each pole of the cell has an identical complement of genetic material.
microtubule experiment
- Microtubules labeled with yellow fluorescent dye. - Then, a portion of the dye was bleached with a laser. - Later, when the chromosomes were pulled to the poles, the bleached area was unmoved.
conclusion of microtubule experiment
- Kinetochores are microtubule-associated motor proteins.
- They walk along the kinetochore microtubule pulling the chromosome along. The end of the microtubule then depolymerizes into tubulin subunits.
by the end of telophase,
telophase is end of mitosis
- Separation of the genetic material is complete.
chromosomes in telophase
The chromosomes become less tightly coiled and the chromatin again begins to form.
nuclear structures in telophase
New nuclear membranes and nucleoli begin to form around each group of daughter chromosomes.
microtubules in telophase
Lengthening of the polar microtubules elongates the cell further.
cytokinesis
- The final part of the M phase is cytokinesis where two complete daughter cells are formed.
- This involves the formation of barrier a between the two new cells.
cleavage furrow
In animal cells, this forms from a cleavage furrow that requires actin and myosin filaments.
cell plate
In plant cells, a cell plate forms as vesicles derived from the Golgi fuse to make a new plant cell wall.
anaphase microtubule polarity
Microtubules have a polarity:
“plus” end is the free end.
“minus” end is the end attached to the centrosome.
A closer look at the action of kinetochore microtubules during anaphase
During anaphase,
- kinetochore microtubules shorten
- daughter chromosomes move toward poles
- these forces are generated mainly at the kinetochores
Depolymerization of kinetochore microtubules shortens the microtubule, which pulls the kinetochores toward the poles. (the kinetochores walk along the kinetochore microtubules, pulling the chromosomes along.)
two forces in anaphase
Outward pull: astral microtubules
Outward push: polar microtubules
astral microtubules in anaphase
outward pull
- Dyneins are involved (minus-end-directed motor proteins; have “three dots” with two “feet”)
- one “Dot” is tethered to the cell cortex
- “Feet” walk along astral microtubules, pulling them toward cell periphery.
polar microtubules in anaphase
outward push
- Kinesins (plus-end-directed motor proteins) “carry” (not really carrying) one microtubule as they walk along another polar microtubule, pushing behind them the ones they are walking along.
- “Walking” is opposite direction of movement of polar microtubules.
How kinesin walks along microtubules from minus end to plus end.
remember: w/ phosphate group (w/ ATP) means attached to MT
1. starts off: Trailing head (h2) has ADP bound and is unbound to MT. Leading head (h1) is bound to MT
2. ATP binds to leading head, causing trailing head (h2) to step past leading head (h1)
3. ADP released from previously trailing head (t2) (now in the lead), ATP hydrolyzed to ADP on the previously leading head (h1) (now trailing). Phosphate group released and head releases its grip on MT.
Each head spends about 50% of time in bound state and 50% of time in unbound state.
what drives chromosomes to the metaphase plate
Polar ejection force pushes chromosomes away from the poles toward the equator.
what drives chromosomes to the metaphase plate: experiment
- using laser, cut chromosome in prometaphase when only one of its sister chromatids is attached to a kinetochore MT (before a second MT attaches to the other sister chromatid’s kinetochore)
- result: unattached arm moves (pushed) away from pole; attached arm is pulled by MT towards pole
meta vs anaphase movements
- Kinesin “walks” on a polar microtubule and “carries” the chromosome arms
pushing them toward the metaphase plate. - Meanwhile, kinetochore “walks” along kinetochore microtubule
pulling the chromosomes toward the spindle pole.
microtubules
- long, hollow cylinders made of tubulin
- 25 nm outer diameter
- more rigid than actin or intermediate filaments
- long and straight
- typically have one end attached to a single microtubule-organizing center (centrosome)
- They constantly change shape (and length) through cycles of polymerization / depolymerization.
protofilaments
microtubules
- There are thirteen strands or protofilaments arranged in a circle around a hollow lumen.
- Each protofilament is made of chains of heterodimers.
There is a plus end and a minus end.
These correspond to the two polypeptides, α and β tubulin
that make up each dimer: “plus” end is β, “minus” end is α .
microtubules and GTP
- Each monomer (the α/β tubulin dimer) has a bound GTP.
- When it is added to the polymer the GTP (T) is hydrolyzed to GDP (D).
- Hydrolysis of the bound nucleotide reduces the binding affinity of the newly added subunit for neighboring subunits and makes it more likely to dissociate from the end.
- Therefore, it is the T form that adds and the D form that leaves.
monomer concentrations
(remember monomer is alpha+beta dimer)
The higher the monomer concentration (T form) the more likely addition will be greater than subtraction and the microtubule will grow.
Therefore, the “on” rate will be dependent upon the concentration (availability) of free monomers.
At lower monomer concentrations, subtraction will be faster than addition and it will shrink.
At higher monomer concentrations, addition will be faster than subtraction and it will grow.
The concentration where addition and subtraction are equal is called…
the critical concentration.
Plus end vs minus end
Monomers add more easily to the plus end than they do to the minus end. Therefore, the critical concentration is lower for the plus end (monomers add more easily to that end) than it is for the minus end.
GTP cap
If monomer addition (T-on) is faster than hydrolysis of nucleotide (D-off), a GTP CAP will form at the growing end.
De-polymerization will be inhibited and the growing end will be stabilized.
This will occur when the concentration of T-form monomers is higher than the critical concentration for that end.
Another way of looking at the critical concentration.
At any given concentration of monomer, addition at the minus end is slower, so hydrolysis predominates, and the minus end will more likely have a subunit with a “D” nucleotide.
On the other hand, plus end addition is faster, so the plus end will have a “T” nucleotide.
Thus, Cc(T) (plus) is lower than Cc(D) (minus).
In other words, since the monomers are being added to the plus end faster, the concentration where they are added and removed at the same rate (the critical concentration) for the plus end is lower (fewer are needed to add on more monomers).
graphical way of looking at criticial concentration
- Graph of subunit concentration (x) vs. elongation rate (y) for plus and minus ends.
- Slope is steeper for plus end because subunits add added more easily there.
- x-int is where the monomers are being added and removed at the same rate to that end—this is the critical concentrations (Cc).
- More subunits are needed to grow the minus end since they are added more slowly there—therefore the Cc(D) is higher.
- A lower monomer concentration is needed to grow the plus end since they are added more easily there—therefore the Cc(T) is lower.
- When the actual concentration is in between Cc(T) and Cc(D), the monomers are being added to the plus end but removed from the minus end—this is the treadmilling range (xD - xT)
treadmilling
- When the free monomer concentration is above the Cc for the plus end
but below the Cc for the minus end, subunits undergo a net assembly at
the plus end and net disassembly at the minus end at an identical rate. - The polymer maintains a constant length, even though there is a net flux
of subunits through the polymer.
treadmilling MT diagram
- Tubulin monomers labeled with green fluorescent dye.
- Filament seems to slide from left to right, but adding subunits to the right (plus end) and losing subunits from the left (minus end).
- plus end is “dynamically
unstable”; it grows, shrinks
then grows again.
asexual reprod
Asexual reproduction
Generates a new individual that is essentially genetically identical to the parent. It involves a cell or cells that were generated by mitosis.
Genetic variation is generated from random mutations or environmental effects.
This can be efficient for environmental adaptation if offspring
production is rapid, e.g. bacterial reproduction.
sexual reprod
Sexual reproduction mixes and shuffles genes from two parents to form a new individual.
- new genetic combinations are created.
- these new combinations produce genetic variation.
- this allows for the potential for greater adaptation of
organisms to their environment.
Homologous chromosomes
come from the two parents
Like mitosis, meiosis
is preceded by the replication of chromosomes.
unlike mitosis, meiosis
- Unlike mitosis, meiosis takes place in two consecutive cell divisions, called meiosis I and meiosis II.
- The two cell divisions result in four daughter cells, rather than the two daughter cells in mitosis.
- Each daughter cell has only half as many chromosomes as the parent cell.
prophase I
In early prophase I (after chromosome duplication in interphase) each chromosome pairs with its homologue and crossing over occurs.
- The result is that gene alleles are swapped between homologous
chromosomes. This swapping is a source of genetic variation
among offspring because these new combinations will be sorted
out into different gametes.
chiasmata
X-shaped regions called chiasmata are sites of crossover between non-sister chromatids.
metaphase I
- In metaphase I, pairs of homologues line up at the metaphase plate, with one chromosome facing each pole.
- Each pair of duplicated homologous chromosomes is called a tetrad.
synapsis
(tetrads in metaphase I)
when synapsing occurs, the chromosomes don’t lie side by side but rather on top of each other w/ a protein called the synaptonemal complex holding them together
microtubules in metaphase I
Microtubules from one pole are attached to the kinetochore of one chromosome of each tetrad; microtubules from the other pole are attached to the kinetochore of the other chromosome.
anaphase I
- In anaphase I, pairs of homologous chromosomes separate.
- One chromosome of each pair moves toward opposite poles, guided by the spindle apparatus.
- Sister chromatids remain attached at the centromere and move as one unit toward the pole.
Telophase I and Cytokinesis
- In the beginning of telophase I, each half of the cell has a haploid set of chromosomes; each chromosome still consists of two (non-identical due to prophase I crossover) sister chromatids.
- Cytokinesis usually occurs simultaneously, forming two haploid daughter cells.
prophase II
In prophase II, a spindle apparatus forms in each cell.
In late prophase II, chromosomes (each still composed of two chromatids) move toward the metaphase plates.
metaphase II
- In metaphase II, the (non-identical due to prophase I crossover) sister chromatids are arranged at the metaphase plate.
- The kinetochores of sister chromatids attach to microtubules extending from opposite poles.
anaphase II
- In anaphase II, the sister chromatids separate.
- The cohesin molecules keeping the sister chromatids attached are cleaved.
- The sister chromatids of each chromosome now move as two newly individual chromosomes toward opposite poles.
Telophase II and Cytokinesis
In telophase II, the chromosomes arrive at opposite poles.
Nuclear membrane reforms.
Cytokinesis separates the cytoplasm.
mitosis vs meiosis: chromosome sets
- Mitosis conserves the number of chromosome sets, producing cells that are genetically identical to the parent cell
- Meiosis reduces the number of chromosomes sets from two (diploid) to one (haploid), producing cells that differ genetically from each other and from the parent cell
mitosis and meiosis similarity
The mechanism for separating sister chromatids is virtually identical in meiosis II and mitosis
events are unique to meiosis
Three events are unique to meiosis, and all three occur in meiosis l:
- Synapsis and crossing over in prophase I.
- At the metaphase plate, there are paired homologous chromosomes (tetrads) instead of individual replicated chromosomes
- At anaphase I, it is homologous chromosomes
instead of sister chromatids, that separate
Three Sources of Genetic Variation
1) Distribution of genetic material between chromosomes during “crossing over” of prophase I.
2) Random arrangement of chromosomes at Metaphase I (Independent Assortment).
3) The random nature of fertilization.
possible combo numbers
- Random fertilization adds to genetic variation because any sperm can fuse with any ovum (unfertilized egg).
- Independent assortment can generate 2^23 or 8.4 million possible chromosome combinations in each gamete.
- Random fertilization (any egg can theoretically combine with any sperm) will then produce a zygote with any of about 2^23 X 2^23 or 70 trillion diploid combinations.
- These calculations don’t even account for the variability coming from crossing over events.
