DdAlix, an Alix/AIP1 homolog in Dictyostelium discoideum, is required for multicellular development under low Ca2+ conditions

Dictyostelium discoideum requires an Alix/AIP1 homolog, DdAlix, for morphogenesis in alkaline environments

“…Dictyostelium discoideum cells were cultured in HL5 medium at 22 °C. Transformation of Dictyostelium was performed as described previously [14]. After electroporation, cells were incubated at 22 °C overnight and G418 was added to 10 μg/ml.…”

“…After electroporation, cells were incubated at 22 °C overnight and G418 was added to 10 μg/ml. After isolation of candidate colonies, expression of the exogenous protein was confirmed by Western blotting using an anti‐FLAG antibody (M2, Sigma) by the color development method using diaminobenzidine (DAB) as described previously [14]. The alx − cells expressing exogenous protein were maintained in HL5 medium in the presence of 10 μg/ml G418.…”

“…The alx − cells expressing exogenous protein were maintained in HL5 medium in the presence of 10 μg/ml G418. alx − /F‐DdAlix cells, which are alx − cells expressing FLAG‐tagged DdAlix, were also maintained in the presence of 10 μg/ml G418 [14]. The number of cells was calculated using a hematocytometer.…”

“…Developmental analyses were performed as described previously [14] with a slight modification. For experiments of pH 9.0, cells in late log phase were harvested and washed twice with CHES buffer [20 mM CHES (N‐cyclohexyl‐2‐aminoethanesulfonic acid)–NaOH and 1 mM CaCl 2 , pH 9.0].…”

“…Recently, we identified an Alix homolog, DdAlix, in Dictyostelium discoideum . Disruption of the DdAlix gene ( alx ) by homologous recombination resulted in a fruiting body formation defect under low extracellular Ca 2+ conditions using Ca 2+ ‐EGTA buffer, raising the possibility that DdAlix is involved in Ca 2+ regulation and/or signaling [14].…”