Superoxide dismutase C and Ras affects Dictyostelium Contractile vacuole morphogenesis and function

Dictyostelium cells lacking SodC displayed severely compromised CV morphogenesis and function. The sodC- cells were vulnerable to hypotonic stress and showed poorly developed CV tubules, significant increase in the number of CV bladders, and a prolonged charging phase. Alleviation of the intracellular superoxide level in sodC- cells with the cytoplasmic Superoxide dismutase A (SodA) or superoxide scavenger XTT significantly reduced the number of CV bladders to the level of the wild type, but the discharging step was still hampered. Introduction of redox insensitive RasG(C118A) mutant in sodC- cells or inhibition of RasG target PI3K with LY204002 also significantly reduced the number of the CV bladders. In contrast to the superoxide scavenging, obstruction of the RasG downstream PI3K signaling aggravated the discharging step. It is plausible that other superoxide sensitive small GTPase’s such as Rab8 is likely persistently activated in LY204002 treated or RasG(C118A) expressing sodC- cells. In addition, we report that sodC- cells not only suffer from high level of RasG activity, but also from low level of RacC. Furthermore, sodC- cells treated with amiloride EIPA, which inhibits sodium proton exchange, rescued aberrant regulation of Ras, but not RacC. The EIPA treated sodC- cells displayed a significant increase in directional cell migration, reduction of the bladder number to the wild type, and significantly improved bladder charging and discharging. We also found that heavy chain of Myosin II is essential for normal architecture of CV system as mhcA- cells displayed defects in both, morphogenesis and function of contractile vacuole. Thus, rectifying the dysregulation of RasG and PI3K signaling, global suppression of superoxide level with XTT or SodA expression in sodC- cells rescued CV morphogenesis. In contrast, inhibition of NHE proteins with EIPA in sodC- cells induced near complete rescue of both CV formation and function. EIPA may not only affect the RasG/PI3K signaling, but may also modulate other unidentified signaling components of the functional CV cycle.