PROTEIN PHOSPHATASE 2A, A POTENTIAL REGULATOR OF ACTIN DYNAMICS AND ACTIN-BASED ORGANELLE MOTILITY IN THE GREEN-ALGA ACETABULARIA

The gaint, unicellular alga Acetabularia is a well known experimental model for the study of actin-dependent intracellular organelle motilit y. In the cyst stage, however, which is equivalent to the gametophytic stage, organelles are immobile, even though an actin cytoskeleton is present. The reason for the lack of organelle motility at this stage h as not been known. To test the hypothesis that organelle motility coul d be under the control of posttranslational modification by protein ph osphorylation, we have treated cysts,vith submicromolar concentrations of okadaic acid or calyculin A, both potent inhibitors of serine/thre onine protein phosphatases (ser/thr-PPases). The effects were dramatic : Instead of linear actin bundles typical for control cysts, circular arrays of actin bundles formed in the cortical cyst cytoplasm. Concomi tant with the formation of these action rings, the cytoplasmic layers beneath the rings began to slowly rotate in a continuous and uniform c ounter-clockwise fashion. This effect suggests that protein phosphoryl ation acts on the actin cytoskeleton at two Levels: (1) It changes the assembly properties of the actin filament system to the extent that n ovel cytoskeletal configurations are formed and (2) it raises the acti vity of putative motor proteins involved in the rotational movements t o levels sufficiently high to support motility at a stage when organel le motility does not normally occur. Northern blot analysis of cyst st age-mRNA using probes specific to protein phosphatase type I (PPI) and type 2A (PP2A) reveals that PP2A is strongly expressed at this develo pmental stage whereas PP1 is not detectable, suggesting that PP2A is t he likely target to the protein phosphatase inhibitors. As a first ste p in analyzing these regulatory proteins on the molecular level, we de scribe the complete sequence of a cyst-specific PP2A cDNA clone.