Apoptosis: Cell Cycle

ced-9 (gf) was mapped to a small region b/w markers X and Y. This region contains 15 genes. What methods can be used to identify Ced-9

• sequence DNA from ced-9 (gf) to determine which gene contains a mutation

• use RNAi to knock down each gene in WT worm to determine which RNAi can increase cell death

Not correct (in this case):

• Isolate clones for each gene from a genomic library of wild type worms and transform them into ced-9 (gf) to determine which one can produce a wild type phenotype.

• It won’t work here because the mutation in question is a gain of function with a dominant phenotype. Introducing a copy of the WT gene will not complement the activity of a gene that has more activity than normal.

Ced-9 is a homolog of the human gene Bcl-2

Homolog: genes that related b/c they have descended from the same ancestral gene

• ced-9 was isolated by positional cloning

• ced-9 encodes a protein similar to Bcl-2 (in humans) [29% identity at amino acid level)

• BcI-2 inhibits cell death in humans – similar function as Ced-9 in C. elegans

• Bcl-2 can partially complement cell death in a ced-9 (lf) mutant → conservation of cell death pathways b/w species

Pathways intiating Apoptosis

• activation of cell death from inside the cell (intrinsic/mitochondrial pathway)

• activation of cell death from outside the cell (extrinsic/death receptor pathway)

  • extrinsic → signaling thru receptor → apoptosis

• these two pathways intersect at the effector caspases (causes events of cell death)

Intrinsic/Mitochondrial Pathway

• when there’s an event that triggers apoptosis → cytochrome C release → forms complex w/ Apaf 1 + caspase 9

• initiator caspase 9: self-activates when compexed w/ cytC and Apaf-1

  • once caspase 9 is activated, it processes caspase 3

• effector caspase 3: must be activated by an upstream protease; triggers events in apoptosis

  • can’t be self-activated

• Bcl-2 protects integrity of mitochondria

Extrinsic/Death Receptor Pathway

• signal binds to death receptor

• receptor recruits adaptor proteins

• adaptor proteins help recruit pro-caspase 8 (inactive when first recruited); recruitment allows caspase 8 to self process into active form

• once caspase 8 is active, it processes caspase 3 into final form

Similarities b/w pathways

• both intersect at caspase 3

Cell Death in Arabidopsis: Background

• MEKK1, MKK1/MKK2 and MPK4 form a MAP kinase cascade to inhibit programmed cell death in Arabidopsis

• in a mekk1 lof, we’d expect to see increased cell death (can’t activate MKK1/2 → can’t activate MPK4 → more death)

• all 3 of these promote cell survival

Single Mutants of MEKK1, MKK1/2, MPK4

• MKK1 and MKK2 have overlapping functions, if you knock out one, the other can still compensate

  • would need to knock out both to see the single mutant phenotype

• single mutants have a lot more cell death

Suppressor Screen: mkk1 mkk2

• finding mutation that reverse this mutant phenotype must act in the same pathway

  • means we got another mutation in another gene in the same pathway

• mutations identified: summ = suppressor of mkk1 mkk2

Isolated Suppressors of mkk1 mkk2 (summ)

• several mutants isolated

• cell death in mkk1 mkk2 is suppressed by summ2-1

• next step: want to clone the summ2 gene; positional cloning

Positional Cloning of summ2

• Linkage analysis and fine mapping located gene to a 300 bp region

• Sequencing identified a point mutation in one of the genes.

How do they know they cloned the right genes?

• other mutants in that same gene have the phenotype

• acquired a different lof mutant that has T-DNA (plant transposon) within this suspected gene (strain summ2-8)

  • this mutant is WT for all the other genes

• crossed this mutant w/ the mkk1/2 mutant to see if the summ2-8 mutation could suppress the mkk1/2 phenotype

• WT version of summ2 promote apoptosis

How do we know we cloned the right gene? FIGURE

Epistatic Analysis

• SUMM2 is epistatic to MKK1/MKK2, which places it downstream of MKK1/MKK2

• SUMM2 encodes a protein similar to Apaf1 and Ced4 (which both promote apoptosis)

• SUMM2 is episatic to MPK4, which places it downstream of MPK4

• SUMM1 is required for cell death in mkk1 mkk2

• SUMM2 is epistatic to SUMM1, so it acts downstream of SUMM1

Identification of Cyclin in Sea Urchins

• levels of cyclin (protein) cycled regularly around cell division

• cyclin levels increased before cell division

cdc Mutants in S.pombe

• WT did not grow in restrictive temp

• cdc2 mutant grew at restrictive temperature

  • points to the WT version controlling cell division

Complementation: Genetic Analysis Tool

Two approaches

• if we cross 2 mutants w/ the same phenotype, will it reveal that the mutations are in 2 diff genes, or are they mutant alleles of the same gene? (can the mutants complement each other’s mutations in the progeny)

• can we restore the WT phenotype if we transform the mutant w/ the cloned gene of interest? (can the clone complement this mutation?)

Human version of cdc gene was found thru complementation experiments (across species)

• human gene could complement a mutation in yeast cell

Human cdc Complementation Experiment: Steps

• a temp-sensitive allele of the yeast cdk gene that lives at lower temp but dies at 36ºC (protein can no longer fold properly) → will see mutant phenotype

• transform yeast mutant w/ a human cDNA library in a yeast CEN vector, where each human gene is expressed from a yeast promoter

  • the library represents thousands of human transcripts

• select clones that are able to survive at 36ºC; these clones have the human cDNA that can rescue the death phenotype

• observed: remarkable similarity b/w yeast and human CDC2 protein (abt 63% homology in a.a seq)

  • similar function since they can substitute for the other

Human cdc Complementation Experiment: Figure

Control of the Cell Cycle: Cyclin/Cdks

• Cdc28 (S.cervisiae) and Cdc2 (S. pombe) are cyclin-dependent kinases (i.e Cdk) that regulate entry into mitosis

• cyclin/cdks regulate cell cycle progression thru 2 major checkpoints:

  • G1/S Checkpoint: entry into S phase

  • G2/M Checkpoint: entry into mitosis

  • 3rd checkpoint to exit mitosis

Control of Cdk Enzymatic Activity

Controlled by 2 independent processes:

1) Presence/absence of appropriate cyclin (a regulatory protein): this is influenced by increased expression or targeted degradation of cyclin

2) addition and removal of phosphate groups: phosphorylation of some sites inhibit Cdk activity, others activate

Cyclin-Cdk Pairs Control Entry into Phases

• diff cyclin-Cdk pairs control entry into S-phase and into M-phase

• Cdks are present throughout cell cycle

• specific cyclins are only present at certain point of the cell cycle (additional regulation thru phosphorylation of Cdk)

Cyclin/Cdk Activity in Signal Transduction Pathways

• both cyclin and Cdk activity are regulated in signal transduction pathways

• expression of cyclins can be induced to promote Cdk activity

• Cdks are targets of other kinases and phosphatasese; activity is regulated by phosphorylation/dephosphorylation

• Cdk/cyclin complexes are targets of other regulatory proteins, that activate or inhibit Cdk activity thru direct interaction

• active Cdks phosphorylate other proteins to promote events in S phase and mitosis

  • e.g. for DNA replication: Activation of DNA helicase; inactivation of a protein that regulates the origin of replication

  • e.g for mitosis: activation of Anaphase promoting Complex

Cyclin-Cdks: Cellular Signals

• Cyclin-Cdks receive cellular signals and allow the cell cycle to respond to them

• cell growth signals (e.g. growth factors): activation of the Cyclin/Cdk complex will allow the cell to proceed to the next phase

• growth inhibitory signals (e.g. DNA damage): blocking activation of the cyclin/cdk complex will be maintain the checkpoint ;cell will not proceed

Rb Protein in Mammalian Cells

• Rb protein binds to E2F transcription factor and keeps it inactive

• a cyclin-Cdk (Active G1-Cdk) appears in late G1 that phosphorylates Rb, inactivating it and lets go of E2F

• E2F is now free to transcribe genes important in DNA replication (genes encoding enzymes for replication)

  • i.e S cyclin and G1/S cyclin

• lof mutation in Rb encoding gene would result in failure to arrest in G1 phase

  • if RB isn’t working then it cannot block E2F activity, and E2F will drive expression of the genes required for S phase.

Modulating Cell # thru Cell Cycle and Apoptosis

• both cell division and death can be promoted or inhibited by internal/external signals

• the cell cycle is allowed to proceed only if conditions are favorable

• if not favorable, the cycle needs to be arrested until the problem is corrected or cell death is induced

Modulating the Cell Cycle: Examples of Regulation

• DNA damage inhibits entry to S until DNA repair is repaired

• During development, many types of cells require stimulation to divide or undergo apoptosis

p53, p21 and inhibition of E2F

In mammalian proteins again (Rb protein)

• DNA damage causes expression of p53 → p53 turns on expression of p21 → p21 binds to g1 cyclin-cdk complex to stop it from working

• Cdk cannot inactivate Rb, Rb continues to hang out to E2F (hypophosphorylated) and prevent steps for DNA synthesis

• Rb is a tumor suppressor, a lof in this gene is associated w/ cancer (i.e unregulated cell proliferation)

During development, many types of cells require stimulation to divide or undergo apoptosis

e.g. cell signaling and neuronal development

• more neurons are produced than needed

• these neurons compete for target-derived neurotrophic factors (e.g. Nerve Growth Factor) and receive survival signals only if they make appropriate connections

e.g. Apoptosis can be induced or repressed by extracellular signals (e.g. FasL system in cytotoxic T lymphocytes)

e.g. growth factor receptor signaling pathways activates cyclinD/Cdk-4/6 which inhibits and promotes E2F and Myc transcription factor

Signaling Proteins are targets for cancer-therapy

• generally, signal transduction pathways promote cell survival and signaling

• all components of signaling pathway are targets for cancer therapies

Integrating Cell Signals

• Growth factors that act thru Ras/MAPK pathway

• survival signals received at the cell surface (survival factors; attachment signals thru integrins) inhibit against apoptosis

• signal transduction pathways that regulate Cdk activity

• p53 suppresses cell cycle and can promote apoptosis thru Bax (which acts on mitochondria)