Effect of phosducin silencing on the photokinetic motile response of Blepharisma japonicum.
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.
Sobierajska
Katarzyna
K
Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093, Warsaw, Poland.
Joachimiak
Ewa
E
Bregier
Cezary
C
Fabczak
Stanisław
S
Fabczak
Hanna
H
D016798:Ciliophora / Q000166:cytology / Q000502:physiology* / Q000528:radiation effects; D005136:Eye Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020555:GTP-Binding Protein Regulators / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000032:analysis / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D008027:Light; D010750:Phosphoproteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
D005136:Eye Proteins; C118981:G-protein Beta gamma; D020555:GTP-Binding Protein Regulators; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D014404:Tubulin; C065631:phosducin