Cell Cycle

Eukaryotes

Introduction to Cell Cycle

– a cycle of duplication and division

– the mechanism by which all living things reproduce

– Two basic phases (Mitosis & Interphase).

– Euk cells have complex network of regulatory proteins – the cell cycle control system

– Cell growth and division must be coordinated to maintain appropriate cell size.

– Functions of cell cycle: (Embryogenesis, Tissue renewal in adults, Gamete production (meiosis & mitosis), Abnormal regulation of cell cycle (cancer / birth defects))

Phases & Regulation

1. G₁ (Gap 1) Phase –

a. cell is at highest energy level

b. Synthesis of RNA, proteins, & organelles

c. G₁/S Checkpoint:

i. Check that cell size has increased by 20% since the last M phase.

ii. Check that genetic material has no damage (DNA is intact)

iii. If cell size is not right or DNA is damaged, cell cycle is arrested until this I corrected. Then proceeds to S-phase.

2. S (Synthesis) Phase

a. DNA replication, a copy of every chromosome is made

b. 10-12 hrs (half time of entire cell cycle)

3. G₂ (Gap 2) Phase

a. Cell growth begins, synthesis of RNA, protein, & organelles

b. G₂ Checkpoint

i. Check that all chromosomes have been duplicated

ii. Check that cell size is large enough to proceed

iii. Check that genetic material has no damage (DNA is intact)

iv. Physiological conditions monitored prior to entering M-phase. If DNA replication is complete and damage is detected, the chromosome is repaired. Then the cell can proceed to M-phase.

4. M Phase

a. Chromosome condensation & segregation, cytokinesis*, <1 hr

b. M Checkpoint

i. Verify successful formation of the spindle fiber system

ii. Verify attachment of spindle fibers to kinetoechores associated with chromosome centromeres

5. G₁ Phase (cycle repeats)

6. If environmental conditions are unfavorable, cells delay G₁ progress and may enter an arrested state called G₀. Some cells remain in this phase permanently, but if stimulated to re-enter the cycle, they enter at the S-phase.

Analysis of Cell Cycle Control

- Dissected genetically in yeast

o Cells reproduce as fast as bacteria and have genome size <1% that of a mammal, can easily genetically manipulate, haploid state makes assessment of one gene copy simple. These studies have identified cdc genes (cell division cycle genes), use of temp sensitive cdc mutants to arrest cycle control and reveal checkpoint requirements

- Analyzed Biochemically in Animal Embryos

o Fertilized eggs carry lots of proteins required for cycle, Xenopus egg has 100,000x more cytoplasm than human cell. Divisions can produce 4096 cells in 7hrs. S & M phases ~ 15min ea., substances can be injected into eggs easily

- Studied in culture in vitro

o Cells isolated from mammal and grown in culture. Easy to assess one cell, however, number of divisions are limited. Immortalized cells are mutated, but easy to use.

- Stage of cycle visualized by various techniques (See below)

1) Autoradiography (³-H thymidine detected if cell is in S-phase)

2) Immunohistochemistry (Anti-BrdU antibody used to label cells in S-phase) Cell, fig 17-11

3) Flow cytometry (label DNA & get distribution for cell populations (most in G₁)). Cell, fig 17-12

Early experiments to understand cycle/ checkpoints

1. There is an M-factor that drives M phase. Supporting expts:

1) Fuse m-phase Cell + G₁ OR G₂ cell = drives M-phase in G₁ or G₂ cell.

2) Inject M-phase cell cytoplasm into oocyte (arrested in G₂ phase) and get M-phase

3) Inject Interphase cell cytoplasm into the oocyte and it remains in G₂ phase.

2. In dividing embryo, radioactive AAs were added to see which proteins being made would incorporate these. Some proteins were made and then degraded (after anaphase, ubiuqitin mediated degradation) in cyclical pattern. Named Cyclins.

Cell Cycle Control System – detailed

Central processes of cell cycle (DNA replication, mitosis, cytokinesis) are triggered by a control system. 3 triggers

1- G₁ checkpoint – Enter S (trigger DNA replication machinery – replicate DNA)

2- G₂ checkpoint – Enter M (trigger mitosis machinery – assemble mitotic spindle)

3- M checkpoint – Exit M (trigger anaphase and proceed to cytokinesis – complete cell division

Regulators of the checkpoints include a complex of cyclin with Cdk (cyclin dependent kinases). The activity of these kinases rises and falls throughout the cycle. Cdks are always present, but cyclins are continually synthesized and degraded. The oscillations lead to changes in phosphorylation of intracellular proteins that initiate or regulate major changes in the cell cycle (listed above). Assembly of the cyclin-Cdk complexes “triggers” the cell cycle.

What controls Cdk? – Cyclin levels

Cdk associates with different cyclins to trigger different events by phosphorylating specific target proteins. These target proteins may only become available during the appropriate phase.

M & S cyclins are shown here. Cyclin complexes are referred to as S-Cdk, M-Cdk, and G₁-Cdk, G₁/S-Cdk.

There are 4 classes of cyclins, but 3 required in all Euk cells:

1. G₁/S cyclins – bind Cdks at end of G₁ & commit cell to DNA replication.
2. S-cyclins – bind Cdks during S-phase & are required for initiation of DNA replication.
3. M-cyclins – promote the events of mitosis.

Diff cyclins are present at diff stages of the cell cycle

o Cyclin D – at start of G1 and stays high through M

o Cyclin E – mid G1 and drops mid S

o Cyclin A – end G1, drops start of M

o Cyclin B – mid S, drops end of M

Full activation fo the complex occurs when a Cdk activating kinase (CAK) phsophorylates an AA near active site of Cdk. Conf. change in complex allows it to phosphorylate it’s target proteins.

Cyclin destruction occurs by ubiquitination. An enzyme attaches ubiquitin to an AA sequence in the cyclin.

Trigger # 3 – example

MPF = maturation promoting factors

(in cytosol of M-phase cell)

MPF = cdc2/cdk1 (a kinase) + cyclin B/cdc13

Cdc25 activates cdc2 to complex with cyclin B

(Forming MPF) and triggering entry into Mitosis.

At the beginning of interphase cyclin rises,

peaks in mitosis, and falls at the end of mitosis.

In cells with mutant cyclin B, MPF remains

high and cells remain arrested in M-phase.

General Note:

There is only replication once per cell cycle. This is because an ↑ in Cdk causes blockage of the pre-replication complex. After Mitosis, Cdk levels ↓ and then the complex can assemble.

Overview of Cell Cycle Control

- I realize this is more detail than what was discussed, but I still thought it would be valuable to include.

- The core of the cell cycle control system is depicted here as a series of cyclin –Cdk complexes in yellow.

- The activity of each complex is also influenced by many inhibitory checkpoint mechanisms, which provide info about the extra cellular envt, cell damage, and incomplete cell cycle events.

Cell Cycle breakdown – Chromosome condensation, segregation, & cytokinesis detailed

Nuclear division (mitosis) and cell division (cytokinesis), collectively called M phase, typically occupy only a small fraction of the cell cycle. The other, much longer, part of the cycle is known as interphase. 5 stages of mitosis shown. A cell can pause in metaphase before this transition point, but once the point has been passed, the cell carries on to the end of mitosis and through cytokinesis into interphase. Note that DNA replication occurs in interphase. The part of interphase where DNA is replicated is called S phase (not shown).

1. Interphase (23 hrs of 24 hr cell cycle)

a. Interval between each mitotic division (absence of visible chromosomes)

b. 2 phases when no DNA synthesis occurs, G₁ & G₂, but intense metabolic activity, cell growth and cell differentiation + S- phase.

2. Mitosis (nuclear division)

a. Prophase

- Chromosome condensation (50x shorter, defines prophase)

o Earliest visible event in entry into Mitosis

o Mitotic spindle begins to self assemble

o Levels of packing chromatin

§ Nucleosome packing into 30 nm chromatin fiber (protein scaffold w/ loops extending – condensed metaphase chromosomes)

§ BsSMC maintenance chromosomes

· Structural maintenance of chromosomes (SMC) protein family contains proteins involved in chromosome condensation and sister chromatid cohesion. Bacillus subtilis SMC protein (BsSMC) plays a role in chromosome organization and partitioning.

· 2 Classes of BsSMCs

o Condensin – binds Chromatin and makes loops

o Cohesin – binds 2 chromatin molecules and holds them in close proximity

§ In condensation, get tangles à Topoisomerase = breaks strand of DNA, allow pass of another strand. No direction ability. Lots of Topo II w/ chromosome.

b. Prometaphase

- Nuclear envelope breakdown (in higher organisms, animals) – this is triggered when m-Cdk phosphorylates the nuclear lamina under the envelope. Now MTs can access the condensed chromosomes for the first time.

- (Migrating) – Spindle assembly & chromosome alignment – Microtubules (MTs) associate to form spindle poles, captured by kinetochores

- Kinetochore-MT attachment

c. Metaphase (once aligned)

- Chromosomes aligned at central axis of cell. There is continuous oscillatory movement of the chromosomes at the metaphase plate.

- Different microtubules involved

o Astral (radiate in all directions from centrosome) – thought to push chromosomes away from the poles.

o Kinetochore – attach end to kinetochore, forms centromere of each chromosome. Thought to pull the chromosomes toward poles.

o Overlap – interdigitate at center of spindle-interacts w/ self

d. Transition Metaphase à Anaphase

- Mitotic spindle checkpoint

- Note: treatment with drugs that destabilize MTs (colchicine or vinblastine) arrests mitosis for hours or even days.

- Transition triggered by activation of anaphase promoting complex (APC). It 1) cleaves and inactivates M-cyclin, inactivating M-Cdk, & 2) cleaves and inhibitory protein (securing) activating a protease called separase. Separase then cleaves a subunit in cohesion complex to unglue sister chromatids. Now they can move to opp. Poles.

- Chromatids start to separate

e. Anaphase

- Chromosomes move by 2 independent, but overlapping processes.

- Anaphase A

o chromosome movement to poles

o By shortening of kinetochore microtubules, chromosome is chewing its way down the MTs.

- Anaphase B

o Separation of poles themselves (Cell, pg. 1047-49)

§ 1-Sliding force (from central spindle): addition of positive end of polar MTs pushes them apart.

§ 2-Pulling force (motor proteins at the poles): acting directly on poles to move apart.

o Ways this may occur??

§ Motor molecules (dynein & kinesin) driving movement

§ Regulated disassembly of MTs, with proteins on kinetochore w/ high affinity for polymerized MTs – makes it move along.

f. Late Anaphase

g. Telophase

- Nuclear envelope reassembles around each group of chromosomes forming distinct new nuclei in emerging daughter cells.

2. Cytokinesis (Cytoplasmic division)

- Begins in anaphase and is complete by the end of telophase

- Division of a eukaryotic cell cytoplasm (after nuclear division during mitosis – or meiosis). The amt of cytoplasm & # of organelles in each daughter cell is redistributed equally.

- Exception: oogenesis – where ovum nearly takes all cytoplasm & organelles, leaving polar bodies, which die.

- In animals, cell membranes form cleavage furrows, formed by microfilaments. As cleavage occurs, midbody forms and pinches off as two new membranes. In plant cells cell plate forms, which becomes a new cell wall.

- Differences between organisms, signaling conserved, contraction forms fold in membrane.

- Cytokinesis is activated by MTs & MFs (formation of cleavage furrow is contractile ring of actin & myosin filaments).

Kinetochores

- Features

o Kinetochore = “complex structure formed from proteins on a mitotic chromosome to which MTs attach & which plays an active part in the movement of chromosomes to the poles. The kinetochore forms on the part of the chromosome known as the centromere.

o Organelles built on centromeric chromatin (In which H3 replaced with Cenp-1). Large # of proteins forms a layered structure. The location depends on previous location of a kinetochore.

o Spindles off centrioles are (1) astral MT (radiate all directions), (2) kinetochroe fiber MT (attach to kinetochore and line chromosomes), and (3) Interpolar MT (between centrioles –hold no chromosome, bind each other – stability).

- Regulation

o Checkpoint

§ In a normal mitotic spindle checkpoint, once all chromosomes are aligned, they are separated.

§ In a defective checkpoint, aneuploidy occurs where 2 chromosomes don’t separate, resulting in 2n-1 or 2n+1 chromosomes / cell (common in cancer)

o How do we know kinetochores regulate this checkpoint?

§ In a cell where 1 chromosome is not aligned and is delaying anaphase, if that chromosome’s kinetochore is destroyed with a laser microbeam è anaphase will occur even without the chromosome becoming aligned. \kinetochore signals alignment & progression of anaphase.

o How exactly do the kinetochores regulate this checkpoint?

§ Cdk1/Cyclin B activates APC/C, which degrades securin by recognizing ubiquitination tail (on the secrun/separase complex) à Separase is free and can work on removing the cohesin molecules holding the chromosomes together à So chromosomes can separate. \kinetochore causes APC to be inhibited = separase cannot remove cohesions.

§ Also, unattached kinetochores in cytoplasm catalyze the production of an APC/C inhibitor, which is inactive in the cytoplasm. As less kinetochores are available in the cytoplasm (they are bound in anaphase), the APC/C inhibitor is activated à so pathway can take place and chromosomes will be able to separate.

o APC Functions

§ Separation of chromosomes (by degrading securin & freeing separase)

§ Exiting cells from Mitosis (catalyzing degradation of cyclin B)

Meiosis Review

Features

- The process in gametogenesis or sporogenesis during which one replication of the chromosomes is followed by two nuclear divisions to produce 4 haploid cells.

- This reduces the chromosome complement from diploid to haploid prior to formation of gametes (specialized haploid reproductive cells), preventing increasing chromosome numbers when genomes are combined during sexual reproduction. During fertilization, fusion of two gametes forms a diploid with 2 sets of chromosomes (each haploid started with either a maternal or paternal homolog).

- Crossing over occurs between the maternal and paternal homologs, and then these independently assort into gametes.

- Independent assortment = the independent behavior of ea pair of homologous chromosomes during their segregation in meiosis I. The random distribution of genes on different chromosomes into gametes

- In meiosis homologs pair & synapse. Synapsed structure = a bivalent. è produces a tetrad (consists of 4 chromatids – bc both chromatids have duplicated)

- To achieve haploidy, 2 divisions are necessary.

Process:

1. FIRST DIVISION – Reduction Division

- number of chromosomes is reduced by ½

- Components of each tetrad separate yielding 2 dyads (2 sister chromatids joined at the common centromere).

- Result is 2 diploid cells

a. Prophase I

i. Members of each homologous pair of chromosomes (homologs) identify one another, align closely (pair), are held together by synapsis (formation of a synaptonemal complex) and exchange info and entire parts (gene conversion & reciprocal recombination, aka “crossing over” occurs between homologs).

ii. Recipricol recombination plays a role in establishing connections (chiasmata) between the chromosomes of a pair). This orients the homologs fro disjunction at the first division (when centromeres are separated from one another).

iii. Actually has 5 stages

1. Leptonema – chromatin condenses & homology search begins.

2. Zygonema – initial alignment of homologs (“lateral elements” visible btwn pairs in yeast à evolves to synaptonemal complex btwn homologs. At end, paired homlogs = Bivalents.

3. Pachynema – coiling / shortening of chromosomes, further development of synaptonemal complex = official synapsis. Each Bivalent contains 4 visible chromatids

4. Diplonema – W/in ea tetrad, sisters begin to separate. Remaining intertwined at chiasma = point where non sister chromatids have undergone genetic exchange via cross over.

5. Diakinesis – chrom pull farther apart, sisters loosely assoc via chiasmata. Chiasmata cont to move to ends of tetrad = terminalization. Nucleolus and nuclear envelope break down. Centromeres of tetrads become attached to spindle fibers

b. Metaphase I

i. Chromosomes are shortened & thickened

ii. Chiasmata is only thing holding sisters in tetrad together

iii. Tetrad interactions w/ spindle fibers move tetrads to metaphase plate

c. Anaphase I

i. (centromere holding sisters together does not divide in 1^st division)

ii. One half of ea tetrad (1 pair of sisters) is pulled toward each pole = “disjunction” (which half of tetrad is pulled to which pole is random – ie. Indep assort)

d. Telophase I

i. Nuclear membrane forming around separated dyads. This stage is short.

2. SECOND DIVISION – Equational Division

- There is no duplication of DNA before this division. The absence of the S phase results in haploidization.

- Each dyad splits into 2 monads (each composed of 1 chromosome from the original tetrad).

- Result is 4 haploid cells

a. Prophase II – ea. Dyad compose of one pair of sister chromatids attached by common centromere

b. Metaphase II – centromeres are directed to equatorial plate. Then centromeres divide.

c. Anaphase II – Sister chromatids of ea dyad are pulled to opp poles.

d. Telophase II – reveals one member of ea pair of homologous chromosomes present at each pole. Each chromosome is referred to as a monad.

If crossing over occurred, ea monad is comb of maternal & paternal genetic info

Conversion and reciprocal recombination (sometimes called crossing over), along with random mutation and random chromosome assortment in the divisions, produce new genotypes that are read out as phenotypes and assessed via natural selection. It has long been thought that the biological advantage of meiosis per se, and thus the conservation of its essential elements, has been the power of this genetic algorithm in solving the problem of environmental change.

Prokaryotes

Bacterial Cell Cycle

- Bacterial Cell Cycle

1) Decision to initiate chrom replication

2) Initiation of chromosome replication

3) DNA synthesis

4) Separation of replicated chromosomes

5) Septum formation & division*

- Cell Division

Septum division at predetermined position

Process of high fidelity w/ specialized proteins

1) Fts proteinsà septum formation

2) Min CDE à locating Z-ring

3) Zip A à function in separation

Basic differences in Prokaryotes (vs. Euk)

- Division in PRO is simpler because they are smaller, have less DNA and few organelles. Eukaryotic DNA is complex with histones and other proteins, which enables the DNA coil tightly into condensed bundles during cell division.

- Before the cell divides, the DNA replicates. This begins at the replication origin, which is anchored to the cell membrane.

- As the cell expands before dividing, each copy of DNA is pulled toward one side of the cell.

- When the cell is about double its original size, the cell membrane pinches inward in the middle of the cell, forming a septum which divides the cell in two.

- Because the origins of the two newly formed chromosomes are anchored to different membrane sites, each daughter cell receives one chromosome. In ideal growth conditions, the bacterial cell cycle is repeated every 30 minutes.

- The cell division of prokaryotes can be broken down into a simple cycle, consisting of three stages.

1) The growth phase (G)

2) The phase when the genome or genetic information is duplicated (S)

3) The actual division of the cell and fission of the cytoplasm, cytokinesis (C)

Determining the site of the Septasome

1) Role of FtsZ

- Forms the division plane

- GTP binding protein that associates w/ cytoplasmic membrane

- Forms membrane associated ring that extends around cell cylinder (many copies in cell- may form mult rings during divison)

- Related to tubulin (20% identity)

- FtsZ forms straight filaments when bound to GTP. Hydrolysis to GDP introduces curvature (boa constrictor effect). Z-ring then contracts and septum forms.

- Cell division apparatus & FtsZ can form the septum at 3 different places.

o 1) Medial location of Z-ring (in bacteria that divide by binary fission ie. E.coli)

o 2) Asymmetric location of z-ring (ie. Caulobacter)

o 3) Polar placement of Z-rings (cell entered sporulation ie. Bacillus) – results in mother & spore progeny

2) Role of Min CDE

a. Tells the cell where to form the division plane

b. Min CD prevents septation at unwanted sites

c. Min E protects the septation site from Min CD

d. Min CD accumulates at a pole of the cell, disperses, and then accumulates at the opposite pole. The oscillatory movement depends on MinE, which is localized near, but separate from the Z-ring. Why MinE chooses a particular location is unknown.

e. Cell division will occur where Min E is located.

Formation of the Septasome

Proteins of the septasome (in order of assembly):

1) Fts Z à associates w/ interior of cell membrane at Z-ring

2) Zip A à assoc w/ Fts Z – integral memb protein

3) Fts A à Assoc w/ FtsZ directly

4) Fts K à Assoc w/ Fts Z,A– integral memb protein

5) Fts Q à Assoc w/ FtsZ, A

6) Fts L à Assoc w/ FtsZ, A, Q

7) Fts I à Assoc w/ FtsZ, A, Q, L

8) Fts N à Assoc w/ FtsZ,A,Q

9) Fts W à ?

Protein interactions at the Septosome

- P-P interactions known btwn FtsZ + ZIP A and FtsZ+ FtsA (maybe FtsZ + FtsI)

- Fts I – FXN in formation of the septal peptidoglycan (which may push cells apart)

Properties of Other cell division proteins

- Fts Z & FtsZ A are only ones that are NOT integral Memb proteins.

- Fts I – septal peptidoglycan formation

- Fts K – resolution of chromosomal dimmers (may fxn as a checkpoint?)

Regulation of Bacterial Cell Cycle/ Division

1) Temporal

a. FtsK may be chkpt that chromosomes have replicated

b. Envt / nutrient factors

2) Spatial

a. Min CD (prevents septation at unwanted sites), but it can only prevent septum formation if Min E is not present. Min E is present where the Z-ring will form. Distribution of these two proteins affects the location of the septum formation.

b. A cell specific transcription factor (σ^E ) progresses through 3 diff subcellular locations in B. subtillus. σ^E & σ^F (these factors are sequestered from each other, ea. On one side of the septum). NOTE: the cell undergoing division at a pole = Pre-divisional sporangium. We know how S^F is activated (see below), now S^E is activated differently. Pro-S^E is located throughout cell at the interior of the cell membrane. Septum forms near one pole and the pro-S^E aggregates on the mother side of the septum. Pro-S^E is processed (cleaved) and released from the septum into mother cell as mature s^E. (This uses spatial & proteolysis & protein sequestration regulates pro-sE activity).

3) Phosphorylation/ Dephosphorylation (activation / inactivation)

a. spoIIE (phosphatase co-loacalizes w/ Z-ring & dephosphorylates spoIIAA). Z-ring forms near both poles of a cell. One Z-ring is degraded, at the other Z-ring, SpoIIE localizes & integrates into the septum. It dephosphorylates sopIIA on the side of the spore, so deph-spoIIA can activate σ^F to regulate gene TXN (there are certain genes you want ON/OFF in a mother cell vs. spore). In the end you have σ^F (F=fawn baby lamb) activated in spore and σ^E activated in mother (E=ewe mother sheep) bc diff factors recog diff consensus seq. in promoters. This regulates gene expression.

4) Proteolysis (degradation of a proteins that ends it’s activity)

a. Cleavage & activation of pro s^E. pro s^E is sequestered at cytoplasmic membrane so it can’t interact with RNAP. As the organism starts dividing, all pro s^E moves to the division plane/ polar septum, facing the mother cell. pro s^E is cleaved afer activation of specific protease. It is cleaved after cell division is finished, and released as mature s^E.

5) Protein sequestration

a. PleC &I Div J – required in Caulobacter. Cells divide into a swarmed and a stalked cell. The swarmer cell contains PleC localized to flagellum bearing cell pole. As cell differentiates into stalked cell, PleC is dispersed around the cell and DivJ is localized to the stalk bearing pole. (Ibrahim, Cell, 1997- more about CtrA, which is regulated by phosphorylation, proteolysis and temporal expression)