Visualization of the interaction between Gβγ and tubulin during light-induced cell elongation of Blepharisma japonicum

Abstract

Blepharisma japonicum ciliates display reversible cell elongation in response to lasting bright illumination. This light-induced phenomenon has been ascribed to the active sliding of the cortical microtubules of the ciliate. The detailed intracellular signaling pathway that activates the microtubule network in response to light, resulting in cell elongation, is unknown. We have previously reported that light stimulation initiates sequential molecular events consisting of a decrease in the phosphorylation of ciliate Pdc, followed by increased binding of Pdc to membrane-localised Gbetagamma and the subsequent translocation of the Pdc-Gbetagamma complex to the cytoplasm. In this study, we used selected agents known to influence protein phosphorylation to test whether alterations in Pdc phosphorylation levels by light affect ciliate shape. Behavioural analysis indicated that cell treatment with okadaic acid, an inhibitor of protein phosphatase activity, heavily abolished the effect of light on cell elongation, whereas the presence of H-89, a specific inhibitor of cAMP-dependent protein kinase (PKA) activity, had no appreciable effect on the cell length. Phosphorylation assays showed that cell incubation with H-89 mimicked light by promoting Pdc dephosphorylation and its colocalization with Gbetagamma. However, as demonstrated by FRET-AP, Pdc-Gbetagamma complex formation and changes in the length of the cell did not occur under the same conditions. Moreover, fluorescence microscopy showed localization of Gbetagamma and beta-tubulin in the same cell compartment and demonstrated that a direct interaction between these proteins occurs in cells adapted to darkness or exposed to prolonged illumination (> or = 10 min). In contrast, an opposite effect, i.e. a transient decrease in the interaction between Gbetagamma and beta-tubulin and distinct Pdc dephosphorylation, was observed in cells illuminated for short time. Under these conditions, Pdc preferentially occupies the cell submembrane region and interacts with Gbetagamma. In cells illuminated for a longer time (> or = 10 min) and despite the constant light intensity, Pdc was progressively rephosphorylated and then dissociated from Gbetagamma, relocalizing within the cell cytoplasm. The results obtained in this study suggest that alterations in Pdc phosphorylation may be involved in light-induced elongation of the Blepharisma cell body, which affects the interaction of Gbetagamma with beta-tubulin and cell cytoskeleton remodelling.

Folate malabsorption is associated with down-regulation of folate transporter expression and function at colon basolateral membrane in rats

“…Slides were put in primary diluted antibody (rabbit polyclonal anti-rat RFC and PCFT (1:200) for 2 h at 37°C followed by secondary antibodies as goat anti-rabbit IgG-horseradish peroxidase (1:200)). Presence of antibody at specific site(s) was revealed using freshly prepared 3,3′-diaminobenzene ( 29 ) and H 2 O 2 at room temperature for 3–5 min and counter-staining with haematoxylin.…”

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

“…15,17 Our recent study showed that the modification of Blepharisma body length during cellular illumination may be due to changes in Pdc dephosphorylation. 20 The Pdc-mediated behavioural responses initiated by light-activated G-proteins may include the functional coupling of G-protein subunits and tubulin, which subsequently controls microtubule assembly and stability. 20, 21 Because both photokinesis and cellular elongation occur under the same light conditions in our model, it would be of great interest to examine whether similar lightdependent molecular processes are responsible.…”