Cell Biology- Chapter 18

The Cell Cycle and Control

The cell cycle is the process by which cells grow and proliferate

The eukaryotic cell cycle is divided into 4 phases

1. G1 Phase (Gap 1)

  • Growth and Preparation: The cell grows in size and synthesizes various proteins and organelles. It prepares for DNA replication.

  • Checkpoint: The cell checks for any DNA damage and ensures it has the necessary resources for DNA synthesis.

2. S Phase (Synthesis)

  • DNA Replication: The cell replicates its DNA, so each chromosome consists of two sister chromatids.

  • Centrosome Duplication: The centrosome, which helps organize the mitotic spindle, is also duplicated.

3. G2 Phase (Gap 2)

  • Further Growth and Preparation: The cell continues to grow and produce proteins. It prepares for mitosis.

  • Checkpoint: The cell checks for any DNA damage and ensures all DNA has been replicated correctly.

4. M Phase (Mitosis)

  • Mitosis: The cell divides its replicated DNA and cytoplasm to form two daughter cells. Mitosis is further divided into several stages:

    • Prophase: Chromosomes condense, and the mitotic spindle begins to form.

    • Metaphase: Chromosomes align at the cell's equatorial plate.

    • Anaphase: Sister chromatids are pulled apart to opposite poles of the cell.

    • Telophase: Nuclear membranes form around each set of chromosomes, and the chromosomes begin to decondense.

  • Cytokinesis: The cytoplasm divides, resulting in two separate daughter cells.


3 Checkpoints

  • G1-s: can the cell divide? (goes to G0 if not)

  • G2-M: are all chromosomes replicated? is all DNA damage repaired? (cells have a limit number of time to correct issues before apoptosis)

  • Metaphase checkpoint: Are all chromosomes attached to mitotic spindle? (corrections must be made before apoptosis)

Cyclin-dependent kinases control progression through cell cycle

  • do this via phosphorylation of downstream regulators

  • CdK functions…. (need more notes)

Studies in Xenopus eggs revealed the cytosolic nature of cell cycle regulators

  • Xenopus (African Clawed Frogs) have pigmented eggs

  • experiments revealed that factors controlling cell cycle projection is located in the cytosol

S and M phase Cdks are inactivated through degradation of their cyclins

  • S and M-Cdks remain active from time of activation until later in mitosis, in which they are rapidly deactivated

  • (more notes)

Full Cdk activation depends on activating phosphorylation on T-loop

  • a Cdk associate with a cyclin is only partially active

  • full activation requires phosphorylation… more notes

  • Full Cdk activation depends on dephosphorylation at inhibitory residues (more notes)

Cell cycle regulators use multiple mechanisms to pause the cell cycle

  • cdk inhibitors are the most active at the G1-S checkpoint

  • Inhibition of activating phosphatase and CAK function most strongly at the G2-M checkpoint

  • Inhibition of APC/C underlies the metaphase checkpoint

  • In all cases except at the G1-S checkpoint, a cell has a finite period of time to correct problems before it commits apoptosis

  • Cancer cells subvert the cell cycle through cell cycle deregulators

    • Genes that promote cell cycle are called proto-oncogenes, over activation leads to cancer

    • genes that suppress are tumor suppressors, under activation can lead to cancer

G,S, and M phases

Retinoblastoma is a tumor growth in the eye

  • originates from cone cell precursors in the retina

  • autosomal dominant inhertiance

  • due to a mutation in cell cycle regulator called Rb (after retinoblastoma)

  • Individuals born with an Rb mutations have an elevated risk for other cancers, as well.

Rb is an S phase suppressor targeted by Cdks

  • it’s an inhibitor of transcription factors that regulate genes involved with entry into S phase of the cell cycle

  • Cyclin dependent kinases can phosphorylate Rb, which changes it to an active state

  • Retinoblastoma mutations are loss-of-function mutations in the Rb gene

    • in familial retinoblastoma, all affected individuals are born with only one copy of Rb

    • a spontaneous mutation that eliminates the lone functional copy is a precursor to tumor formation

P53 is a cell cycle suppressor activated by DNA damage

The CDK inhibitor p21 is regulated transcriptionally by P53

P53 directs more than just cell cycle arrest

As animal species size and lifespan increases, so too does its ability to suppress cancer

  • larger animals have more cells

  • large animals don’t get cancer as much

  • there is a selection pressure to more effectively suppress cancer as animals get larger- for example, elephants have 20 copies of the P53 gene

  • Peto’s paradox

S-cdk activates the replisome and prevents immediate re-initation

  • DNA replication licensing- needs preRC

  • cdc6 and the ORC complex are the first to bind to the origin and they recruit other components of the replisome

  • when the replisome is recruited, it cannot efficiently begin replication until its associated helicase has been phosphorylated

  • S-cdk phosphorylates the helicase, but while it’s in the complex, it also phosphorylates ORC

  • Phosphorylation of ORC deactivates it

    • this is permanent, more active ORC can only be achieved via synthesis of new ORC proteins

    • origin firing only happens once per cycle because cdks are inactive

Incomplete replication and DNA damage arrests the cell cycle

(more notes)

Activated M-Cdk facilitates more M-Cdk activation in a positive feedback loop

  • M-CDks mark the transition point from G2 to mitosis

  • (more notes)

Cohesins hold sister chromatids together after S phase

  • chromosome packing is at its more dense during mitosis

  • following S phase, rings of cohesin proteins, kepe the resultant sister chromatids attached to one another

  • During prophase, most of the cohesin rings are removed, but many remain at the centromere- these won’t be removed until anaphase.

Condensins tightly pack DNA in M phase chromosomes

  • condensins are responsible for the tight packing of an individual chromosome

  • like cohesins, a ring of condensing consists of a pair of clamp-like proteins

  • Condensin organizes the linear DNA into solenoids (coiled coils) that are stacked

Two transient microtubule and microfilament structures mediate mitosis and cytokinesis

  • the mitotic spindle is composed of microtubules and is responsible for segregating sister chromatids to the opposite poles of dividing cell

  • more notes

Stages of mitosis (only need to know key features)

Prophase

  • duplicated chromosomes (sister chromatids) condense, the mitotic spindle assemble between centrosomes.

Prometaphase

  • breakdown of nuclear envelope. chromosomes now attach to spindle

Note: the centrosome replicates during interphase and migrates during mitosis

  • centrosomes form the spindle poles

  • prior to M phase, the centrioles of the centrosome separate and first form a daughter centriole and then a new centrosome

  • more notes

Stabilizing factors drive directed growth of microtubules into the mitotic spindle

  • they grow and drink from their plus end and the growth pattern is random

  • during spindle formation, microtubules grow into the region between spindle pores…more notes

3 classes of microtubules make up the mitotic spindle

  • more notes

The kinetochore is a collection of proteins that attach centromeres to the spindle

  • the microtubule is gripped…. more notes

Metaphase

  • the chromosome algin at the equator, the kinetochore microtubules attach to opposite end of spindle

Microtubules, motor proteins, and chromosomes are sufficient to assemble…more notes

Anaphase

  • sister chromatids are pulled apart

APC/C signals the degradation of cohesin

  • controller of metaphase checkpoint

  • APC/C becomes active only if all chromosomes are properly connected to the mitotic spindle

  • when active, APC/C disinhibits (via inhibition of securin) separase which catalyzes cohesin breakdown

  • without cohesins, the chromatids segregate as the kinetochore microtubules shorten.

The push and pull of anaphase

  • chromosomes pulled

  • interpolar microtubules are pushed apart

  • astral microtubules are pulled by cell cortex

Telophase

  • chromosomes arrive at the poles of spindle

  • nuclear envelope reassembles

Cytokinesis

Animal cytokinesis involves formation of a cleavage furrow

  • in most cases, mitosis is immediately followed by cytokinesis

  • cleavage is accompanied by a cleavage furrow, it becomes deeper until cell splits apart.

Cleavage furrow formation is positioned by interpolar microtubules and facilitated by RhoA

  • the major structural component is the contractile ring, a complex of actin microfilaments and motor proteins

  • Positioning of the contractile ring at the cell midline is coordinated by signals from interpolar microtubules through activation of RhoA

  • RhoA (a small G protein) facilitates assembly of the contractile ring complex

Myosin motors act on contractile ring microfilaments to split the cell in two

  • Recall that overactivation of Rho throughout the cytosol leads to formation of long, parallel filaments with associated motor proteins

  • Contractile ring filaments have…more notes

Animal cells can change shape during M phase

  • due to major recognition of microtubules and microfilaments that occur throughout mitosis and cytokinesis, the morphology of cells can temporarily change in dramatic fashion

  • In addition to cytoskeletal changes…more notes

Plant cell cytokinesis involves formation of a dividing plasma membrane and cell wall

  • too rigid for cleavage furrow

  • vesicles from Golgi are transported to the phragmoplast, at this site, new plasma membrane and cell wall forms between daughter cells.

Cell death is as important to development as cell proliferation

  • not all cells in a developing tissue contribute to final structure

Apoptosis is regulated by caspases (cleave after aspartate)

  • major events controlled by caspases

  • each caspase has multiple targets that are cleaved at conserved sequences that end with aspartate

  • Initiator caspases are activated early on in the process

  • Executioner caspases are activated by initiator caspases and drive apoptosis by cleaving their own targets- e.g.: caspase activated DNase fragments genomic DNA

Markers of apoptosis

  • DNA fragmentation - CAD breaks genome into smaller fragments

  • Annexin V staining - technique that detects phosphatidylserine

  • Active caspase 3 blotting- detect caspase 3 via molecular weight

Different caspases deliver death signals from different parts of the cell

Cytochrome c from the mitochondria facilitates caspase activation in the intrinsic pathway

  • Cytochrome C assembles with other proteins to form a complex called apoptosome, it activates initiator caspase 9

  • Bax and Bak proteins are required for cytochrome c release.

  • bcl2 inhibits Bak

Regulation of apoptosis integrates multiple signaling pathways

Pro-apoptotic signals can come from other cells (extrinsic pathway)

  • the extrinsic pathway relies on activation of plasma membrane proteins collectively termed death receptors. (DISC)

  • Fas is the best characterized

Pro survival signaling acts through inhibitors of apoptosis

  • normal animal cells require pro-survival signaling

  • these pathways suppress apoptosis by stimulating production of inhibitors

  • most promoters are present, only continuous suppression keeps cell alive

Limited survival factors prune developing nervous system

  • target cells produce a finite amount of pro-survival factors

  • as the nervous system develops postnatally, neurons die and ultimately, the number of neurons and target cells are balanced.

Mitogens stimulate S phase and cell division and promote cell proliferation

  • signal through MAPK pathways

  • PDGF factors heal bones

Growth signals increase protein synthesis and size

  • acts through Akt/tor, promotes protein synthesis and increases cell size

  • in skeletal muscle, myostatin inhibits Akt signaling through tor and suppresses muscle growth