Effect of phosducin silencing on the photokinetic motile response of Blepharisma japonicum

Photochem Photobiol Sci. 2011 Jan;10(1):19-24. doi: 10.1039/c0pp00221f. Epub 2010 Oct 26.

Abstract

The coloured ciliate Blepharisma japonicum changes swimming velocity (positive photokinesis) and elongates its body in response to a prolonged illumination. We have recently proposed that alterations in the phosphorylation level of the ciliate phosducin (Pdc) may be involved in light-induced cell elongation, which in turn affects the interaction of βγ-dimer of G-proteins (Gβγ) with β-tubulin and subsequent cytoskeletal remodelling. The cellular mechanism that governs the photokinetic effect in this ciliate has not been elucidated. In the present study, we utilise real-time PCR to demonstrate that the levels of ciliate Pdc mRNA are significantly reduced in Pdc-RNAi-treated cells compared to cells fed with bacteria carrying the empty vector (control cells). Using western immunoblotting, we confirmed that these cells treated with Pdc-RNAi expressed a substantially lower level of the Pdc protein. The assay also revealed that in ciliates treated with Pdc-RNAi and exposed to light, the cytosolic level of Gβ (~36 kDa) was reduced, whereas the level of Gβ localized to the membrane (~32 kDa) was increased compared to control cells. In addition, behavioural analysis of the cells indicated a substantial reduction of photokinesis. The findings in this study provide additional characterization of the functional properties of the ciliate Pdc protein and we discuss a likely role for this phosphoprotein in the photokinetic phenomenon of the ciliate protist Blepharisma.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ciliophora / cytology
  • Ciliophora / physiology*
  • Ciliophora / radiation effects
  • Eye Proteins / antagonists & inhibitors*
  • Eye Proteins / genetics
  • Eye Proteins / metabolism
  • GTP-Binding Protein Regulators / antagonists & inhibitors*
  • GTP-Binding Protein Regulators / genetics
  • GTP-Binding Protein Regulators / metabolism
  • GTP-Binding Protein beta Subunits / analysis
  • GTP-Binding Protein beta Subunits / metabolism
  • GTP-Binding Protein gamma Subunits / analysis
  • GTP-Binding Protein gamma Subunits / metabolism
  • Kinetics
  • Light
  • Phosphoproteins / antagonists & inhibitors*
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Phosphorylation
  • RNA Interference
  • RNA, Small Interfering / metabolism
  • Tubulin / metabolism

Substances

  • Eye Proteins
  • G-protein Beta gamma
  • GTP-Binding Protein Regulators
  • GTP-Binding Protein beta Subunits
  • GTP-Binding Protein gamma Subunits
  • Phosphoproteins
  • RNA, Small Interfering
  • Tubulin
  • phosducin