(i) Time course following the phagocytosis of 1 μm beads. Phagocytosis is abolished by 1 mM cycloheximide. (ii) Amoebae preincubated with 2 mM cycloheximide (C/D) do not phagocytose 1 μm beads. A fluorescence reading of approximately 325 units corresponds to an average of 1 bead taken up per cell, a reading of 650 implies that 2 beads have been taken up per cell and so on. Ax2 were preincubated for 30 minutes without (A/B) or with (C/D) 2 mM cycloheximide prior to incubation with fluorescent beads for 30 minutes. The cells were then washed and kept on ice until being allowed to settle on a microscope slide and viewed as sections under 60× magnification on a coverslip. A/C: Phase-contrast image; B/D: Fluorescence image. (iii) 2 mM cycloheximide abolishes capping of Con A receptors cross-linked with fluorescent Con A. Vegetative amoebae settled on glass coverslips were preincubated in the absence (A/B) or presence (C/D) of 2 mM cycloheximide for 30 minutes before being incubated with fluorescent Con A for 1 minute, rinsed and then left in KK2 Mg/ 1 mM Ca2+ for 3–5 minutes prior to fixing in 5% formaldehyde. A, C: fluorescent; B, D: phase-contrast image. All images are sections taken using a 60× objective lens unless otherwise stated. (iv) Actin polymerisation followed using TRITC-phalloidin to stain the fixed actin cytoskeleton in lysed amoebae. The dynamics and relative magnitude of actin incorporation into the triton-insoluble cytoskeleton following stimulation with 1 μM cAMP were found to be similar for control and cycloheximide-treated amoebae.
Clotworthy and Traynor BMC Cell Biology 2006 7:5 doi:10.1186/1471-2121-7-5