Our starting point was that amoebae treated with cycloheximide ceased fluid phase endocytosis , a property also shared with macrophages . In addition, we observed that the amoebae rounded-up and ceased movement, a behaviour surprisingly like that of the NSF temperature-sensitive mutant at the restrictive temperature ; this led us to explore further the effects of cycloheximide.
We found that, like fluid phase endocytosis, phagocytosis of latex beads is eliminated by preincubation of amoebae with cycloheximide, a phenomenon which also occurs in monocytes [23, 24]. By contrast, surface uptake measured with FM1-43 remains surprisingly unaffected: in fact, the rate of membrane internalised agrees well with the findings of Aguado-Velasco and Bretscher . This indicates that this internalised membrane is endocytosed along with very little fluid and therefore, the vesicles (if that is how the membrane is internalised) must either be flattened or have a very small radius. A similar conclusion comes from studies of FM1-43 uptake in the wild-type amoeba, NC4 . Thus the 5% fluid phase uptake remaining on cycloheximide treatment may correspond to the small amount of fluid inevitably taken up in conjunction with this membrane. Comparison with the ts mutant NSFA2 is also interesting. At the restrictive temperature, NSFA2 is unable to take up fluid phase or phagocytose: in this sense, it is like Ax2 treated with cycloheximide. However, FM1-43 uptake by NSFA2 at the restrictive temperature is reduced (by about 75%), but is not eliminated : however long the mutant is held at the restrictive temperature, there is always a small burst of dye uptake when it is added and this ceases within a few minutes (Thompson and Bretscher, unpublished observations [see Additional file 10]). This uptake may reflect a short circuit endocytic cycle independent of NSF that continues at the restrictive temperature; however, in this case there is no opportunity for the dye to back-fill into other compartments in the cell because these processes presumably require NSF. This contrasts with the effects of cycloheximide, which does not alter membrane uptake appreciably. If the above conjectures are correct, both the short circuit endocytic cycle and back-filling would continue as in the wild type. This is what we observe.
Because amoebae treated with cycloheximide round up and stop moving – much like the NSF ts mutants – we further examined those properties of a cell required for locomotion: an effective motor and a cell polarity. It is a property of all motile cells that they cap cross-linked surface antigens: amoebae are no exception. They cap Con A receptors with great efficiency: by contrast, cycloheximide-treated amoebae do not. By this test, cycloheximide converts these cells from a motile to a non-motile state, suggesting that either the motor or the intrinsic polarity is lost.
To gain further insight into what the effects of cycloheximide are, we tested actin polymerisation in response to cAMP (the "cringe" reaction). Both untreated and cycloheximide-treated amoebae showed similar filament formation in terms of their time courses, and the relative amounts of actin polymerised. Furthermore, the actin cytoskeleton can rapidly reorganise itself in response to a changing source of cAMP, despite the fact that the cells are rounded. Both lines of evidence indicate that the amoebae continue to possess an active cytoskeleton, although (as in the similar case with the NSF ts mutant (3)) some other undetected cytoskeletal defect may exist.
As actin polymerisation occurred in the direction of the cAMP source in cycloheximide-treated cells, it seemed likely that their signal transduction system would function normally in response to an external cAMP signal. That this is so is supported by the observations that PI3K and CRAC both localise towards the cAMP source. In these senses, the cycloheximide-treated cells and the NSF ts mutant share similar characteristics (David Traynor, unpublished observations).
Before turning to how cycloheximide causes these effects, it is relevant to ask how a cycloheximide resistant mutant, HH31, behaves. The rate of fluid phase uptake, as measured using FITC-dextran, was unaltered in the resistant mutant [see Additional file 1]. Additionally, when observed under the microscope, the amoebae remain motile and do not round up (data not shown). The conclusion that the target of our experiments is the ribosome and protein biosynthesis is supported by the other ribosomal inhibitors tested by Gonzalez and Satre : they all behave like cycloheximide.
Therefore, our observations indicate that continued protein synthesis is required for maintaining amoebae in a locomotory state: that may mean that a metabolically unstable protein is required. The similarities in the behaviours of the NSF mutant at the restrictive temperature and cycloheximide-treated cells, with the limited exception of membrane uptake (as discussed above), are striking: cycloheximide appears to phenocopy the NSF mutant.
There seem to be two general categories of interpretation of these observations. It could be that we are seeing a general cellular response to poor growth situations – where protein synthesis or membrane transport fails. This possibility has a precedent: a mechanism appears to exist in yeast that ties cell processes to a continuation of protein synthesis . Alternatively, it could be that an unstable protein is required to maintain the cells in a migratory state, and so is membrane transport. In this latter case, the two lines of evidence might suggest – if they are connected, which they need not be – that an unstable protein(s) is needed to maintain cell motility and that this protein is involved in, or needs to be transported in the cell by, the membrane system.
In the course of this work we noticed that, for clearer results, the length of preincubation with cycloheximide could be varied, depending on the particular assay. For example, fluid phase uptake is blocked 30 minutes after cycloheximide addition, yet 60 minutes is required to block migration. This is not surprising: each of these is a complex process and the dependence on a single factor (if that is what it is) need not be the same for each. A similar phenomenon is seen with the NSF mutant: fluid phase uptake is effectively abolished by 5 minutes at the restrictive temperature, yet cell migration continues quite happily until about 15–20 minutes after the temperature is raised (M.S. Bretscher, personal communication [see Additional file 11]). Indeed, in so far as this comparison has been explored, the assays in which Ax2 is more sensitive to cycloheximide seem to be those in which the NSF mutant is most sensitive.