2 Programmed Cell Death (apoptosis) Stereotypic death process includes: membrane blebbing nuclear fragmentation chromatin condensation and DNA framentation loss of mitochondrial integrity and release of cytochrome c Natural part of development eg., removal of webbing between digits Cancer involves mutations that block apoptosis (p53) First genes discovered in nematodes
3 Self-fertile hermaphrodites; rapid life cycle Single hermaphrodite can produce a brood of 300
4 Early Embryogenesis: making founder cells
5 Six Founder Cells (5 Somatic, 1 Germline)
6 C. elegans reproduces sexually Hermaphrodite self-fertile: germline makes ~300 sperm then oocytes Male cross-fertile: germline makes sperm only
8 C. elegans development John Sulston s drawings of nuclear positions (6 May 1980)
9 An invariant cell lineage 959 somatic cells (adult hermaphrodite) (John Sulston, Bob Horvitz, Judith Kimble )
10 Embryonic! Caenorhabditis elegans Cell Lineage! Larval! The MS! Lineage:!
11 Programmed Cell Death in C. elegans: Embryogenesis produces a hatched larva with living cells cells eliminated by programmed cell death (shortly after their births). Additional cell deaths occur during larval development. Most cell deaths are in neuronal lineages. Cell death (apoptosis) conserved in most animals. Cancer connection.
13 ced mutants: programmed cell death-defective Ed Hedgecock: unbiased Nomarski screen for mutants with defects in cellular anatomy (F2 screens) Identified two mutants, ced-1 and ced-2, with persistent cell death corpses.
14 ced mutant microscopy ced-1(-/-) and ced-2(-/-): corpses accumulate
15 Mutants defective for programmed cell death Cell death corpse accumulation: an entry point into genetic studies of programmed cell death, but not what you want to study. What genes are REQUIRED for programmed cell death??? Mutants lacking programmed cell death (not cleaning up the mess). Take advantage of ced-1/2 mutant phenotype: easy to see that programmed cell death is occurring. Screen for mutants in which no corpses are visible (in a ced-1/2 mutant background)
16 ced3 mutants fail to accumulate corpses ced-1(-/-) Horvitz lab rides again. Ellis et al, Cell 44, (1986) ced-1(-/-); ced-3(-/-)
17 Screens for mutants identify 2 genes Screen: 4000 F1 broods examined 2 alleles of ced-3 identified 1 allele of ced-4 identified in different screen (Egl sup) Not an easy screen, but succeeded in identifying two genes required for ALL cell deaths. First time such genes found in any organism.
18 Quantitation of cell deaths in ced-3 and -4 mutants
19 ced-3 (and -4) are required for pcd in all lineages
20 Model for ced gene order of action
21 New ced genes Screened 40,000 F2s for extra pharyngeal cells (at locations of I2 and NSN corpses) -- Identified multiple recessive alleles of ces-2, required only in pharynx for cell deaths. --One dominant allele of a gene called ced-9, prevents ALL.
22 Questions Geneticists Ask Does the (dominant) mutation represent a gain-offunction or an instance of haploinsufficiency? Compare phenotype of a diploid heterozygous for the mutation to a diploid heterozygous for a deficiency of the region wildtype/mutation wildtype/df
23 ced-9 (n1950) is a dominant gain-of-function allele *
24 ced-9(gof) prevents pcd of HSN neurons also In egl-1 mutants, neurons required for egg laying die. We ll come back to egl-1
25 Isolation of ced-9 loss-of-function alleles
26 ced-9 lof alleles: n1950n2077 behaves like a null; n1950n2161 is a weaker allele * * *
27 Questions Geneticists Ask Does the (recessive) mutation confer a null phenotype? Compare phenotype of a diploid homozygous for the mutation to a diploid heterozygous for the mutation and for a deficiency genex - /genex - genex - /Df
28 ced-9 lof alleles: n1950n2077 behaves like a null; n1950n2161 is a weaker allele * * *
29 ced-9 (lof) causes ectopic cell deaths
30 What is the order of action of the ced genes? Loss of CED-9 leads to all cells undergoing programmed cell death (all cells are poised to die, but for CED-9 all would!) CED-3/4 required for programmed cell deaths Does CED-9 inhibit CED-3/4 function to prevent programmed cell death?
31 Questions Geneticists Ask Do the identified genes function in a linear pathway? Compare phenotypes of two double mutant strains. For each gene, one needs alleles with contrasting phenotypes. In the case of ced genes, alleles that confer no cell death (ncd) and alleles that confer ectopic cell death (ecd) are available ced-3 ncd ced-4 ecd ced-3 ecd ced-4 ncd
32 ced-3 and -4 mutations suppress ced-9 (lof) mutations
33 ced function pathway
34 Adding more relationships to the pathway Overexpression of Ced-3 and Ced-4 causes ectopic cell death Enables another genetic test of ced-9 relationship to ced-3 and -4 Also lets us test the relationship between ced-3 and ced-4
35 verexpression of Ced-3 and Ced-4 causes ectopic cell death
36 Loss of ced-9 enhances ectopic cell death caused by overexpression of Ced-3 and Ced-4
37 Ced-3 is required for ectopic killing caused by overexpression of Ced-4, but not vice versa
38 ced-9 ced-4 ced-3 cell death
39 One more player, Egl-1 Gain-of-function egl-1 mutations cause HSNs to undergo programmed cell death. (The Horvitz lab used these egl-1 mutations to isolate some ced mutants.) Loss-of-function egl-1 mutations, isolated exactly as ced-9 (lof) alleles were isolated, prevent programmed cell death. Epistasis experiments place egl-1 upstream of all ced gene functions
40 egl-1 induced ectopic killing is suppressed by mutations that block pcd
41 Biochemistry and cell biology of Ced proteins 1. Egl-1 and Ced-9 interact (IP experiments) Radioactive Egl-1 was incubated with GST-Ced-9 (lane 1) or GST (lane 5) and bound proteins separated by electrophoresis The BH3 domain of Egl-1 is required; compare lanes 1 and 2
42 Biochem and cell biology, continued 2. Bunches of IP experiments demonstrate interaction between Egl-1 and Ced-9, between Ced-9 and Ced-4, and between Ced-3 and Ced-4 3. Ced-9 is localized to the mitochondrial outer membrane and recruits Ced-4 4. Induction of programmed cell death induces Ced-4 translocation to the nuclear membrane but not in gof Ced-9 mutants
43 Figure 1 CED-9 and CED-4 are localized to mitochondria in WT embryos. F Chen et al. Science 2000;287: Published by AAAS
44 Figure 2 CED-9 is required for the localization of CED-4 to mitochondria. F Chen et al. Science 2000;287: Published by AAAS
45 Figure 4 Overexpression of EGL-1 induces CED-4 translocation from mitochondria to nuclear membranes inced-9(+) embryos but not inced-9(n1950) embryos. Ced-4 in wt embryos overexpressing Egl-1 F Chen et al. Science 2000;287: Ced-4 in ced-9(gof) embryos overexpressing Egl-1 Published by AAAS
46 Current model
47 Model with more detail added
48 Cloning ced genes revealed similarities to mammalian proteins Ced-3 is similar to mammalian interleukin converting enzyme, a cysteine protease Ced-3 is therefore proposed to be a cysteine protease In vitro substrates for Ced-3 include actin, tubulin, and proteins involved in ATP synthesis and in DNA synthesis Ced-9 is a homolog of Bcl-2, a mammalian oncogene
49 Comparison of apoptopic pathways
50 Ced-9 is a functional homolog of mammalian bcl-2
51 Over-expression of bcl-2 mimics over-expression of ced-9 * * Wildtype: Overexpression of Ced9 and bcl2 prevents cell death * * Ced-9 (lof): Overexpression of ced9 and bcl prevents ectopic cell death
52 Parallels of CED-9 in worms and Bcl-2 in humans. Bcl-2 is a human oncogene with properties similar to CED-9 Over-expression of Bcl-2 prevents or delays cell death in B-cell and T-cell lineages. Bcl-2 expressed at high levels in blood stem cell lineages; loss of expression correlates with appearance of cell death In cancer, chromosome translocations activate Bcl-2 expression, preventing cell death in hematopoietic lineages. This results in a leukemia due to over-proliferation of some blood cell lineages. No LOF alleles.
53 Comparison of apoptopic pathways
54 3 subfamilies of Bcl-like proteins
55 Mouse mutants defective for Bcl-2 family members have altered cell death phenotypes
56 A model for mammalian cells
57 Same model, a little more detail
58 Figure 2 Function of BAX and BAK downstream of tbid and upstream of cytochrome c release. Cytochrome c microinjection M C Wei et al. Science 2001;292: Published by AAAS
59 Cytochrome c is required for oligomerization of Apaf and for activation of caspase Caspase Apaf-1
60 CED-9 is BCL-2 (a negative regulator of Caspases) Dominant mutation pays off (by way of lof). CED-3 is a pro-caspase, while CED-4 is related to vertebrate Apaf-1 (caspase activator); both found to be required in all animal cells for apoptosis. CED-9/BCL-2 are associated with mitochondrial membrane: how they are regulated and role of mitochondria remain subject of active research. One goal: trigger cancer cells to all enter apoptosis.
61 Back to checkpoints p53 is a target of checkpoint pathways and determines survival versus death In death mode, p53 interacts with and causes oligomerization of Bak This interaction causes release of cytochrome c from the mitochondrion The model is that p53 and the Bcl-2 family member Mcl1 have opposing effects on the death effector, Bak
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