Gaillard: Programmed Cell Death in Chlamydomonas

Anne R. Gaillard, Matt R. Breuer, and Ally R. Keathley

Final Project Report for Summer 2015

Programmed cell death (PCD) is a deliberate, highly organized, and genetically controlled mechanism of cell suicide. PCD has been documented in a wide variety of organisms, including multicellular organisms such as animals, plants, and fungi, as well as in unicellular organisms, such as unicellular algae, yeast, protozoa, and bacteria. PCD contrasts greatly with cell death by necrosis, which is an accidental, catastrophic death that occurs due to sudden environmental trauma. Using the unicellular green alga Chlamydomonas as a model organism, the overall goals of the Summer 2015 FAST project were: 1) to determine whether the mechanism of PCD in unicellular organisms is similar to that of apoptosis in animals, and 2) to address the question of why PCD evolved in unicellular organisms.

The FAST team made excellent progress on the first objective. The team subjected log phase Chlamydomonas cells to heat stress at 42°C for 30-120 minutes to induce PCD, and then observed the cells for PCD-characteristic events. In parallel, the team performed an acute heat stress at 85°C to induce necrosis. In cells exposed to 42°C, slow, progressive cell death occurred, and cell shrinkage, vacuolization, a sharp increase in intracellular reactive oxygen species (ROS), externalization of plasma membrane phosphatidylserine (PS), and DNA laddering were observed. These events did not occur for cells subjected to 85°C heat stress. In contrast, cells exposed to 85°C exhibited rapid death, cell swelling, cell lysis, no increase in intracellular ROS, no externalization of PS, and nonspecific DNA degradation. These results demonstrate that the events of PCD in Chlamydomonas are distinct from those of passive, necrotic cell death, and that Chlamydomonas PCD exhibits many hallmark features of apoptosis.

Once the team had characterized PCD in Chlamydomonas and shown that PCD occurs only in response to moderate stress, but not in response to acute trauma, experiments were conducted to address the second objective. The team first tested the hypothesis that cells undergoing PCD release beneficial substances into the surrounding medium that enhance growth of non-PCD cells. Chlamydomonas cells were incubated at 25°C, 42°C, and 85°C for two hours, and the medium surrounding the cells was extracted and added to fresh Chlamydomonas cells. Cell growth was then measured for 5-7 days. The team also tested the hypothesis that cells undergoing PCD release substances that only benefit other cells of the same species. The team is continuing to conduct experimental trials, but the data set is not large enough yet to form overall conclusions.
In another approach to address the second objective, the team tested the hypothesis that the rate of PCD in a population of Chlamydomonas cells is regulated by the level of genetic variation among the population. In other words, the team wanted to determine whether the seemingly altruistic event of PCD was more likely to occur if the cells were surrounded by kin. Chlamydomonas cells were again subjected to 42°C, but this time three different populations of cells were used: a pure Chlamydomonas reinhardtii culture, a pure Chlamydomonas eugametos culture, and a mixed culture of half Chlamydomonas reinhardtii and half Chlamydomonas eugametos. The team predicted that the mixed culture would exhibit a significantly lower rate of PCD in response to heat stress compared to the pure cultures. However, the results of the study show that the rate of PCD is not significantly different among the three populations; thus, the results do not support the team’s hypothesis. The team is planning to conduct more experimental trials, but with different Chlamydomonas species. It is possible that the hypothesis was not supported because the genetic variation between Chlamydomonas reinhardtii and Chlamydomonas eugametos is not sufficiently high.
Overall, the FAST team had a very productive and successful summer, and the team is extremely grateful for the funding that EURECA provided. The team is planning on several conference presentations of this data, as well as publication of the study. One member of the team, Allyson Keathley, has decided to expand her work on this project and conduct an Honor’s thesis.