Figure Q18–1 Appearance of paws in Apaf1–/– and Casp9–/– newborn mice relative to normal newborn mice (problem +/+ –/– 18–6). (From h. Yoshida et al., Cell 94:739– Casp9 750, 1998. With permission from Elsevier.) 18–7 When human cancer cells are exposed to ultraviolet (UV) light at 90 mJ/cm2, most of the cells undergo apoptosis within 24 hours. Release of cytochrome c from mitochondria can be detected as early as 6 hours after exposure of a population of cells to UV light, and it continues to increase for more than 10 hours thereafter. Does this mean that individual cells slowly release their cytochrome c over this time period? Or, alternatively, do individual cells release their cytochrome c rapidly but with different cells being triggered over the longer time period?
To answer this fundamental question, you have fused the gene for green fluorescent protein (GFP) to the gene for cytochrome c, so that you can observe the behavior of individual cells by confocal fluorescence microscopy. In cells that are expressing the cytochrome c–GFP fusion, fluorescence shows the punctate pattern typical of mitochondrial proteins. You then irradiate these cells with UV light and observe individual cells for changes in the punctate pattern. Two such cells (outlined in white) are shown in Figure Q18–2A and B. Release of cytochrome c–GFP is detected as a change from a punctate to a diffuse pattern of fluorescence. Times after UV exposure are indicated as hours:minutes below the individual panels. Which model for cytochrome c release do these observations support? Explain your reasoning.
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Figure Q18–2 time-lapse video fluorescence microscopic analysis of cytochrome c–GFp release from mitochondria of individual cells (problem 18–7). (A) Cells observed for 6 minutes, 10 hours after UV irradiation. (B) Cells observed for 8 minutes, 17 hours after UV irradiation. One cell in (A) and one in (B), each outlined in white, have released their cytochrome c–GFp during the time frame of the observation, which is shown as hours:minutes below each panel. (From
J.C. Goldstein et al., Nat. Cell Biol. 2:156–162, 2000. With permission from Macmillan publishers Ltd.) 18–8 Fas ligand is a trimeric, extracellular protein that binds to its receptor, Fas, which is composed of three identical transmembrane subunits (Figure Q18–3). The binding of Fas ligand alters the conformation of Fas so that it binds an adaptor protein, which then recruits and activates caspase-8, triggering a caspase cascade that leads to cell death. In humans, the autoimmune lymphoproliferative syndrome (ALPS) is associated with dominant mutations in Fas that include point mutations and C-terminal 1034 Chapter 18: Cell Death truncations. In individuals that are heterozygous for such mutations, lymphocytes do not die at their normal rate and accumulate in abnormally large numbers, causing a variety of clinical problems. In contrast to these patients, individuals that are heterozygous for mutations that eliminate Fas expression entirely have no clinical symptoms.
A. Assuming that the normal and dominant forms of Fas are expressed to the same level and bind Fas ligand equally, what fraction of Fas–Fas ligand complexes on a lymphocyte from a heterozygous ALPS patient would be expected to be composed entirely of normal Fas subunits?
B. In an individual heterozygous for a mutation that eliminates Fas expression, what fraction of Fas–Fas ligand complexes would be expected to be composed entirely of normal Fas subunits?
C. Why are the Fas mutations that are associated with ALPS dominant, while those that eliminate expression of Fas are recessive?