S-Space College of Natural Sciences (자연과학대학) Dept. of Biological Sciences (생명과학부) Theses (Ph.D. / Sc.D._생명과학부)
Glutathione-mediated cAMP signal pathway during development in Dictyostelium discoideum
Dictyostelium discoideum 분화과정에서 glutathione 매개 cAMP 신호 전달체계
- 자연과학대학 생명과학부
- Issue Date
- 서울대학교 대학원
- Glutathione; Glutathione synthetase; cAR1-mediated cAMP signaling; G protein alpha 2; Dictyostelium discoideum
- 학위논문 (박사)-- 서울대학교 대학원 : 생명과학부 미생물학과, 2017. 2. 강사욱.
- Glutathione (GSH, γ-glutamyl-L-cysteinylglycine) is the most prevalent reducing thiol-containing compound in eukaryotic cells and plays indispensable roles in multicellular development. However, the function of glutathione synthetase (gshB/GSS), the final-step enzyme of GSH biosynthesis, on development of Dictyostelium discoideum has not been clearly investigated.
Herein, it was found that the overexpression of gshB (gshBoe) facilitates a significant increase of the intracellular GSH levels during both early and late development, which leads to developmental defects, such as the mound-arrest phenotype and the reduced spore formation. Moreover, in wild-type (KAx3) cells, addition of exogenous GSH led to similar defects to those characteristic of gshBoe cells. To investigate the effects of increased intracellular GSH level by gshB overexpression on Dictyostelium development in more detail, the cAMP-mediated aggregation and cell-type differentiation were examined. First, when cells were allowed to develop at low cell densities, gshBoe cells were not able to form aggregates and streams. gshBoe cells exhibited multiple pseudopodia, indicating a lack of axial polarity during aggregation. In addition, gshBoe cells exhibited abnormal fluctuating pattern in F-actin polymerization in response to cAMP stimulation.
Next, during differentiation, gshBoe cells showed remarkable differences in the cell-type proportioning with KAx3 cells. In the mounds of gshBoe cells, pre-stalk cells were increased relative to those in KAx3 mounds, whereas pre-spore cells were remarkably decreased, which was consistent with the reduced sporulation of gshBoe cells.
Based on these results, the expression pattern of the essential components of the cAMP signaling pathway (carA/cAR1/cAMP receptor 1, gpaB/Gα2/G protein alpha subunit 2, gpbA/Gβ/G protein beta subunit, and acaA/ACA/adenylyl cyclase A), which are developmental regulators, was examined in gshBoe cells. As the result, the expression level of carA and gpaB was upregulated, especially after 8 h of development. Similarly, in gshBoe cells, the intracellular cAMP concentration were significantly 4.5-fold higher than those of KAx3 cells at 8 h after the onset of development.
To further investigate the effect of gshB overexpression on the cAMP signaling pathway, the mutant strains which constitutively expressed carA and gpaA (carAoe and gpaBoe, respectively) were generated. During developmental processes, gpaBoe cells displayed the defects in multicellular aggregation and F-actin polymerization characteristic of gshBoe cells, which may be as a secondary response to abnormally elevated cAMP concentrations, but carAoe cells did not. Furthermore, gpaBoe cells exhibited phenocopies of gshBoe cells in cell-type proportioning, such as the increased pre-stalk cells and the decreased pre-spore cells. Interestingly, gpaB knockdown in gshBoe cells (gpaBas/gshBoe) partially rescued the defects of gshBoe cells. gpaBas/gshBoe cells produced fruiting bodies with small sori and were able to form aggregates and streams at low cell densities. These results suggest that the developmental defects associated with gshBoe cells may be caused by increased gpaB expression and high intracellular cAMP concentrations.
Additionally, there was the distinction in Gα2-GFP localization between KAx3 and gshBoe cells. In KAx3 cells, Gα2-GFP was uniformly expressed prior to cAMP stimulation and rapidly translocated to the plasma membrane after cAMP stimulation. However, in gshBoe cells, Gα2-GFP was strongly localized at the plasma membrane both before and after the addition of cAMP, implying that GSH could also regulate Gα2 localization.
Taken together, these findings suggest that GSH regulates developmental stage and cell-type proportioning by upregulating cAMP signaling pathway via modulation of Gα2 expression and localization during the development of Dictyostelium. These results will provide insights into the general mechanisms underlying cell development involved in GSH-mediated signaling.