On the effects of cycloheximide on cell motility and polarisation in Dictyostelium discoideum

Abstract

Background: Cycloheximide is a protein synthesis inhibitor that acts specifically on the 60S subunit of eukaryotic ribosomes. It has previously been shown that a short incubation of Dictyostelium discoideum amoebae in cycloheximide eliminates fluid phase endocytosis.

Results: We found that treatment with cycloheximide also causes the amoebae to retract their pseudopodia, round up and cease movement. Furthermore, fluid phase endocytosis, phagocytosis and capping cease in the presence of 2 mM cycloheximide, although membrane uptake, as measured using FM1-43, is unaffected. In the presence of cycloheximide, aggregation-competent amoebae sensitive to cAMP, although round, can still localise CRAC, ABP120, PI3K and actin polymerisation in response to a micropipette filled with cAMP. The behaviour of wild-type amoebae in the presence of cycloheximide is surprisingly similar to that of amoebae having a temperature-sensitive version of NSF at the restrictive temperature.

Conclusion: Our results may suggest that, upon cycloheximide treatment, either a labile protein required for polarised membrane recycling is lost, or a control mechanism linking protein synthesis to membrane recycling is activated.

Figure 1

(i) Fluid phase uptake monitored using FITC-dextran. 2 mM cycloheximide inhibits fluid phase uptake by >95%, as shown in this time course. The data obtained for each experiment were normalised with respect to the 30 minute time point for Ax2 in the absence of cycloheximide. (ii) Photomicrographs of amoebae incubated with FITC-dextran. Ax2 were preincubated in the absence (A/ B) or presence (C/ D) of 2 mM cycloheximide for 30 minutes, followed by incubation with FITC-dextran. The amoebae were subsequently fixed in 5% formaldehyde and mounted on a coverslip. A, C: Phase-contrast; B, D: Fluorescence. (iii) Time course showing how membrane uptake, as monitored using FM1-43, is unaffected by cycloheximide. FM1-43 was added at a concentration of 5 μM. The data obtained for each experiment were normalised with respect to the 30 minute time point for Ax2 in the absence of cycloheximide. (iv) Internal membrane bound structures stained with FM1-43. Ax2 were preincubated for 30 minutes without (A/B) or with (C/D) 2 mM cycloheximide prior to incubation with FM1-43 for 60 minutes. The cells were subsequently washed and kept on ice before being placed on a coverslip. A, C: Phase-contrast; B, D: Fluorescence. All images are sections taken using a 60× objective lens unless otherwise stated.

Figure 2

(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.

Figure 3

CRAC-GFP (A/B), PI3K2-CFP (C/D), ABP120-GFP (E/F) and Actin-GFP (G/H) can translocate in response to a micropipette filled with 1 μM cAMP in the presence of cycloheximide. Polarisation of these markers was followed in the absence (A, C, E, G) or presence (B, D, F, H) of 2 mM cycloheximide. The position of the cAMP-filled micropipette is indicated by the white "<" symbol. The micropipette always comes in from the right-hand side. Fluorescent images were taken as sections using a 60× objective lens. Scale bar represents 10 μm. For movies, see Additional files.