Phospholipase D Signaling Regulates Microtubule Organization in the Fucoid Alga Silvetia compressa

Abstract

Recent studies in higher plants or animals have shown that phospholipase D (PLD) signaling regulates many aspects of development, including organization of microtubules (MTs), actin and the endomembrane system. PLD hydrolyzes structural phospholipids to form the second messenger phosphatidic acid (PA). To begin to understand the signaling pathways and molecules that regulate cytoskeletal and endomembrane arrays during early development in the brown alga, Silvetia compressa, we altered PLD activity by applying butyl alcohols to zygotes. 1-Butanol activates PLD and is a preferred substrate, primarily forming phosphatidyl butanol (P-butanol), which is not a signaling molecule. Treatment with 1-butanol inhibited cell division and cytokinesis but not photopolarization or germination, suggesting an MT-based effect. Immunolabeling revealed that 1-butanol treatment rapidly disrupted MT arrays and caused zygotes to arrest in metaphase. MT arrays recovered rapidly following butanol washout, but subsequent development depended on the timing of the treatment regime. Additionally, treatment with 1-butanol early in development disrupted endomembrane organization, known to require functional MTs. Interestingly, treatment with higher concentrations of 2-butanol, which also activates PLD, mimicked the effects of 1-butanol. In contrast, the control t-butanol had no effect on MTs or development. These results indicate that S. compressa zygotes utilize PLD signaling to regulate MT arrays. In contrast, PLD signaling does not appear to regulate actin arrays or endomembrane trafficking directly. This is the first report describing the signaling pathways that regulate cytoskeletal organization in the stramenopile (heterokont) lineage.

Phosphatidic Acid Regulates Microtubule Organization by Interacting with MAP65-1 in Response to Salt Stress in Arabidopsis

“…We showed that genetic and pharmacological inhibition of PA generation results in microtubule depolymerization, which is consistent with results in tobacco cells (Dhonukshe et al, 2003) and algae (Peters et al, 2007). In leek (Allium porrum) leaves, 1-butanol converts microtubules from the transverse to longitudinal orientation, presumably by promoting microtubule detachment from the membrane (Sainsbury et al, 2008).…”

Phospholipid signaling during stramenopile development

“…13,14 We recently examined the roles of PLD signaling during early development of the brown alga S. compressa. 15 A fertilized egg normally orients its growth axis in accordance with directional light (photopolarization) and germinates and grows from the rhizoid pole of that axis. 16 The first division is asymmetric and is oriented transverse to the growth axis.…”

“…17 We found that butanol treatments did not block photopolarization or germination, but cell division was inhibited. 15 This suggested that MTs, rather than actin or endomembranes, were the primary targets of drug treatment. This was a somewhat surprising finding since actin is often disrupted by 1-butanol application in plants and animals.…”

“…Following treatment, MTs initially appeared fragmented and, within two hours, became heavily bundled and resided exclusively in the cortex. 15 Treated algal zygotes ultimately arrested in mitosis, unable to form a bipolar metaphase spindle. MT arrays and development recovered quickly following 1-butanol removal, providing an easy method for synchronizing populations.…”

“…1B). Interestingly, 1-butanol also eliminates centrosomal MTs and induces formation of a bundled cortical array, 15 but the significance of these localizations is presently unclear. PA derived from the PLC-pathway likely signals to more than MTs, since R59022 blocks germination, which does not require MTs.…”