Fig. 5.

Tubercidin inhibits Dictyostelium cell streaming, but does not inhibit cAR1 expression, actin polymerization, or adenylyl cyclase expression. (A) Micropipette chemotaxing assay with control cells (Upper) and cells treated with 150 μM tubercidin (Lower). The black dots show the positions of the tips of micropipettes filled with 1 μM cAMP. The control cells formed long streams, whereas the tubercidin-treated cells chemotaxed individually and formed aggregates at the micropipette tip. (B) Under-agar assay of chemotaxis in which wells contained 5 μl of differentiated amoebae (2 × 10⁷ per ml) without (Left) and with 150 μM tubercidin (Right). (Center) This well contained 1 μM cAMP. The control cells formed streams of chemotaxing amoebae, but the tubercidin-treated cells chemotaxed individually with no streams. (C) cAR1 expression: cells (2 × 10⁷ per ml), in the absence (Upper) and presence (Lower) of 100 μM tubercidin, were pulsed with 75 μM cAMP (final concentration) at 6-min intervals. Samples were taken every hour for SDS/PAGE, and gels were stained with Coomassie blue for actin or immunoblotted with anti-cAR1 antibodies. (D) The time course of the change in F-actin content of cells after stimulation by 1 μM cAMP, in the absence or presence of 100 μM tubercidin, as determined by quantifying the staining of fixed, permeablized cells with rhodamine-phalloidin. (E) Basal and Mn²⁺- and GTPγS-stimulated adenylyl cyclase activity of cells in the absence (Left) or presence (Right) of 100 μM tubercidin.