BACKGROUND
using dictyostelium discoideum amoebae, gonzalez and satre  <cit>  investigated the effect of protein synthesis inhibition on fluid phase uptake.
they found that cycloheximide, which acts specifically on the 60s subunit of eukaryotic ribosomes  <cit> , was the most potent inhibitor.
treatment of amoebae with cycloheximide for  <dig> minutes essentially eliminated fluid phase uptake, as measured using fitc-dextran.
this, coupled with their observation that several other inhibitors of protein synthesis have a similar effect, suggested that a labile protein is essential for endocytosis.
we examined dictyostelium amoebae after treatment with cycloheximide and confirmed the observations of gonzalez and satre  <cit> .
furthermore, we noticed that under these conditions the cells withdrew their pseudopodia and rounded up.
the loss of nsf  also leads to dictyostelium amoebae rounding up and ceasing to move or endocytose fluid phase  <cit> .
nsf is an essential atpase required for snare complex dissociation after membrane fusion events  <cit> .
as snare proteins are widely required to mediate specific vesicle fusion with the target membrane  <cit> , it is believed that nsf is essential for many vesicular trafficking events, including those at several different stages of the endocytic cycle.
the close similarities that were apparent led us to investigate the extent to which the effects of cycloheximide mirrored the mutant nsf phenotype.
besides examining different aspects of endocytosis, we also studied the ability of the amoebae to polarise various markers.
starving amoebae use camp pulses as a chemoattractant during aggregation.
binding of camp to its g-protein coupled receptor leads to the translocation of pi3k  to the membrane at the leading edge of a moving cell.
by contrast, pten  is translocated from the cytoplasm to the plasma membrane towards the rear of the cell.
the combined uneven distribution of these two enzymes is thought to lead to a local increase in pip <dig> and pip <dig> levels at the leading edge of the cell, enabling specific ph domain containing proteins, such as crac  to be recruited there.
these steps have been followed in cycloheximide-treated cells using cfp-labelled pi3k  <cit>  and gfp-labelled crac  <cit> .
in addition, we also sought to find out whether cycloheximide affects the ability of the cell's cytoskeleton to respond to camp signals by following actin polymerisation and its localisation using gfp-tagged actin  <cit>  and the actin-binding domain of abp <dig>   <cit> .
in short, this work explores a possible link between protein synthesis and cell movement.
RESULTS
fluid phase uptake but not membrane uptake is inhibited by cycloheximide
gonzalez and satre  <cit>  showed that inhibiting protein synthesis in dictyostelium inhibits fluid phase uptake.
we first confirmed this observation with our strain of ax2: the degree of inhibition was dose dependent  and 1).
the rate of fluid phase uptake over the first  <dig> minutes ranged from approximately  <dig>  to  <dig>  μl/ <dig> ×  <dig> cells/ minute for ax <dig> in the absence of cycloheximide.
this compares reasonably well with the value of approximately  <dig>  μl/ <dig> ×  <dig> cells/ minute obtained by vogel et al  <cit> .
in the presence of  <dig> mm cycloheximide, this was reduced to between  <dig>  and  <dig>  nl/ <dig> ×  <dig> cells/ minute.
we next addressed the question of whether bulk membrane uptake is affected by cycloheximide treatment as well.
although some membrane is taken up by macropinocytic vesicles, this is only a small portion of the total surface area taken up  <cit> .
membrane uptake can be monitored using the dye fm1- <dig>  <cit> .
this dye is only weakly fluorescent in aqueous solutions, but fluoresces intensely when partitioned into membranes.
fm1- <dig> cannot cross membranes, so when membrane is endocytosed in vesicles, the fluorescence is trapped in the cell and cannot be washed away  <cit> .
hence, fm1- <dig> provides a convenient measure of total surface membrane uptake.
when we looked at the effect of cycloheximide on fm1- <dig> uptake, we found, unexpectedly, that it had no effect  and 1).
the initial rate of membrane uptake, when compared with the surface fluorescence obtained for a population of cells as described in the methods section, indicated that one plasma membrane equivalent could be internalised in approximately 8– <dig> minutes.
capping requires protein synthesis
capping is the process by which cross-linked antigens or receptors on the cell surface are swept to the rear in migrating cells  <cit> .
we used fluorescent tetrameric con a  to cross-link con a receptors and observed capping of the fluorescence towards the rear of approximately 90% of migrating cells in the absence of cycloheximide .
99% of cycloheximide-treated cells failed to cap .
fig.
<dig> shows a representative sample of treated and untreated amoebae.
phagocytosis is sensitive to cycloheximide
phagocytosis has long been known to involve actin cytoskeleton remodelling  <cit> , and is also thought to require local membrane exocytosis  <cit> .
phagocytosis was monitored by measuring the uptake of  <dig> μm diameter carboxylate-modified fluorescent microspheres in the presence or absence of cycloheximide.
the amoebae were preincubated for  <dig> minutes in various concentrations of cycloheximide prior to addition of the fluorescent beads, and were subsequently shaken for the time intervals shown, before being washed and the fluorescence measured ).
amoebae in buffer alone were able to phagocytose several beads each over  <dig> minutes, whilst the cycloheximide-treated amoebae generally contained none ).
fluorescent abp120-transformed amoebae without cycloheximide
fluorescent actin-transformed amoebae with cycloheximide
fluorescent actin-transformed amoebae without cycloheximide
cell motility is blocked by cycloheximide treatment
we observed that vegetative dictyostelium amoebae lost their ability to translocate upon cycloheximide treatment and wanted to investigate whether aggregation competent cells were still able to chemotax towards camp.
we found that the cells were unable to move but could form small pseudopods in the direction of chemoattractant .
also, nih image was used in conjunction with standard techniques to quantify the differences in speed and persistence between cycloheximide-treated and untreated cells chemotaxing towards a micropipette filled with camp.
in the absence of cycloheximide, amoebae chemotaxed towards the source of camp with an average speed of  <dig>  ±  <dig>  μm/ minute .
this compares well with the value of  <dig>  ±  <dig>  recorded for wild-type cells moving up a gradient  <cit> .
the persistence value for the chemotaxis of these amoebae was  <dig>  ±  <dig> , over  <dig> successive images recorded at  <dig> s intervals .
a value of  <dig> represents chemotaxis in a straight line towards the source of camp.
in the presence of cycloheximide, speed was reduced to  <dig>  ±  <dig>  μm/ minute .
for the  <dig> cells for whom persistence values could be calculated for part of the frame sequence, it was found to be reduced to  <dig>  ±  <dig> .
we speculated that this loss of speed and persistence could be due to a defect in the cells' ability to translocate polarising signalling molecules such as pi3k and crac to the leading edge.
the ability of amoebae these proteins was therefore investigated using cfp- and gfp-tagged proteins respectively.
conditioned amoebae were stimulated with a micropipette containing camp and we found that the amoebae were able to localise each of the markers towards the chemoattractant, even though the cells were round .
we then considered whether aberrant actin remodelling might be partly responsible for the phenotypes observed.
fluorescent crac-transformed amoebae with cycloheximide
fluorescent crac-transformed amoebae without cycloheximide
fluorescent pi3k-transformed amoebae with cycloheximide
fluorescent pi3k-transformed amoebae without cycloheximide
the rate of actin polymerisation does not depend on protein synthesis
the dynamics and magnitudes of actin polymerisation were followed using gfp-tagged abp <dig> and actin  and additional files).
we observed that actin did indeed polymerise at the edge of the cell adjacent to, and in response to, the camp-filled micropipette, irrespective of whether the cells had been preincubated in the presence of cycloheximide or not.
the course of actin polymerisation after camp addition was also followed fluorometrically using tritc-phalloidin to label the triton-insoluble actin cytoskeleton.
the kinetics of actin assembly and the relative amounts polymerised were similar whether cycloheximide was present or not  ).
fluorescent abp120-transformed amoebae with cycloheximide
discussion
our starting point was that amoebae treated with cycloheximide ceased fluid phase endocytosis  <cit> , a property also shared with macrophages  <cit> .
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  <cit> ; 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  <cit> .
by contrast, surface uptake measured with fm1- <dig> remains surprisingly unaffected: in fact, the rate of membrane internalised agrees well with the findings of aguado-velasco and bretscher  <cit> .
this indicates that this internalised membrane is endocytosed along with very little fluid and therefore, the vesicles  must either be flattened or have a very small radius.
a similar conclusion comes from studies of fm1- <dig> uptake in the wild-type amoeba, nc <dig>  <cit> .
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 nsfa <dig> is also interesting.
at the restrictive temperature, nsfa <dig> is unable to take up fluid phase or phagocytose: in this sense, it is like ax <dig> treated with cycloheximide.
however, fm1- <dig> uptake by nsfa <dig> at the restrictive temperature is reduced , but is not eliminated  <cit> : 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 .
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 .
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 ) 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 .
before turning to how cycloheximide causes these effects, it is relevant to ask how a cycloheximide resistant mutant, hh <dig>  behaves.
the rate of fluid phase uptake, as measured using fitc-dextran, was unaltered in the resistant mutant .
additionally, when observed under the microscope, the amoebae remain motile and do not round up .
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  <cit> : 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 , 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  <cit> .
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 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  <dig> minutes after cycloheximide addition, yet  <dig> minutes is required to block migration.
this is not surprising: each of these is a complex process and the dependence on a single factor  need not be the same for each.
a similar phenomenon is seen with the nsf mutant: fluid phase uptake is effectively abolished by  <dig> minutes at the restrictive temperature, yet cell migration continues quite happily until about 15– <dig> minutes after the temperature is raised .
indeed, in so far as this comparison has been explored, the assays in which ax <dig> is more sensitive to cycloheximide seem to be those in which the nsf mutant is most sensitive.
CONCLUSIONS
our results show that movement, fluid phase uptake, phagocytosis and capping require continued protein synthesis, whilst localised actin polymerisation, membrane uptake and pi3k and crac translocation to the leading edge do not.
our results may suggest that, upon cycloheximide treatment, either a labile protein required for motility and perhaps involving polarised membrane recycling is lost, or a control mechanism linking protein synthesis to membrane recycling is activated.

 using dictyostelium discoideum amoebae, gonzalez and satre investigated the effect of protein synthesis inhibition on fluid phase uptake. they found that cycloheximide, which acts specifically on the 60s subunit of eukaryotic ribosomes, was the most potent inhibitor. treatment of amoebae with cycloheximide for minutes essentially eliminated fluid phase uptake, as measured using fitc-dextran. this, coupled with their observation that several other inhibitors of protein synthesis have a similar effect, suggested that a labile protein is essential for endocytosis. we examined dictyostelium amoebae after treatment with cycloheximide and confirmed the observations of gonzalez and satre. furthermore, we noticed that under these conditions the cells withdrew their pseudopodia and rounded up. the loss of nsf also leads to dictyostelium amoebae rounding up and ceasing to move or endocytose fluid phase. nsf is an essential atpase required for snare complex dissociation after membrane fusion events. as snare proteins are widely required to mediate specific vesicle fusion with the target membrane, it is believed that nsf is essential for many vesicular trafficking events, including those at several different stages of the endocytic cycle. the close similarities that were apparent led us to investigate the extent to which the effects of cycloheximide mirrored the mutant nsf phenotype. besides examining different aspects of endocytosis, we also studied the ability of the amoebae to polarise various markers. starving amoebae use camp pulses as a chemoattractant during aggregation. binding of camp to its g-protein coupled receptor leads to the translocation of pi3k to the membrane at the leading edge of a moving cell. by contrast, pten is translocated from the cytoplasm to the plasma membrane towards the rear of the cell. the combined uneven distribution of these two enzymes is thought to lead to a local increase in pip and pip levels at the leading edge of the cell, enabling specific ph domain containing proteins, such as crac to be recruited there. these steps have been followed in cycloheximide-treated cells using cfp-labelled pi3k and gfp-labelled crac. in addition, we also sought to find out whether cycloheximide affects the ability of the cell's cytoskeleton to respond to camp signals by following actin polymerisation and its localisation using gfp-tagged actin and the actin-binding domain of abp. in short, this work explores a possible link between protein synthesis and cell movement.  fluid phase uptake but not membrane uptake is inhibited by cycloheximide gonzalez and satre showed that inhibiting protein synthesis in dictyostelium inhibits fluid phase uptake. we first confirmed this observation with our strain of ax2: the degree of inhibition was dose dependent and 1). the rate of fluid phase uptake over the first minutes ranged from approximately to μl/ × cells/ minute for ax in the absence of cycloheximide. this compares reasonably well with the value of approximately μl/ × cells/ minute obtained by vogel et al. in the presence of mm cycloheximide, this was reduced to between and nl/ × cells/ minute. we next addressed the question of whether bulk membrane uptake is affected by cycloheximide treatment as well. although some membrane is taken up by macropinocytic vesicles, this is only a small portion of the total surface area taken up. membrane uptake can be monitored using the dye fm1-. this dye is only weakly fluorescent in aqueous solutions, but fluoresces intensely when partitioned into membranes. fm1- cannot cross membranes, so when membrane is endocytosed in vesicles, the fluorescence is trapped in the cell and cannot be washed away. hence, fm1- provides a convenient measure of total surface membrane uptake. when we looked at the effect of cycloheximide on fm1- uptake, we found, unexpectedly, that it had no effect and 1). the initial rate of membrane uptake, when compared with the surface fluorescence obtained for a population of cells as described in the methods section, indicated that one plasma membrane equivalent could be internalised in approximately 8– minutes. capping requires protein synthesis capping is the process by which cross-linked antigens or receptors on the cell surface are swept to the rear in migrating cells. we used fluorescent tetrameric con a to cross-link con a receptors and observed capping of the fluorescence towards the rear of approximately 90% of migrating cells in the absence of cycloheximide. 99% of cycloheximide-treated cells failed to cap. fig. shows a representative sample of treated and untreated amoebae. phagocytosis is sensitive to cycloheximide phagocytosis has long been known to involve actin cytoskeleton remodelling, and is also thought to require local membrane exocytosis. phagocytosis was monitored by measuring the uptake of μm diameter carboxylate-modified fluorescent microspheres in the presence or absence of cycloheximide. the amoebae were preincubated for minutes in various concentrations of cycloheximide prior to addition of the fluorescent beads, and were subsequently shaken for the time intervals shown, before being washed and the fluorescence measured ). amoebae in buffer alone were able to phagocytose several beads each over minutes, whilst the cycloheximide-treated amoebae generally contained none ). fluorescent abp120-transformed amoebae without cycloheximide fluorescent actin-transformed amoebae with cycloheximide fluorescent actin-transformed amoebae without cycloheximide cell motility is blocked by cycloheximide treatment we observed that vegetative dictyostelium amoebae lost their ability to translocate upon cycloheximide treatment and wanted to investigate whether aggregation competent cells were still able to chemotax towards camp. we found that the cells were unable to move but could form small pseudopods in the direction of chemoattractant. also, nih image was used in conjunction with standard techniques to quantify the differences in speed and persistence between cycloheximide-treated and untreated cells chemotaxing towards a micropipette filled with camp. in the absence of cycloheximide, amoebae chemotaxed towards the source of camp with an average speed of ± μm/ minute. this compares well with the value of ± recorded for wild-type cells moving up a gradient. the persistence value for the chemotaxis of these amoebae was ±, over successive images recorded at s intervals. a value of represents chemotaxis in a straight line towards the source of camp. in the presence of cycloheximide, speed was reduced to ± μm/ minute. for the cells for whom persistence values could be calculated for part of the frame sequence, it was found to be reduced to ±. we speculated that this loss of speed and persistence could be due to a defect in the cells' ability to translocate polarising signalling molecules such as pi3k and crac to the leading edge. the ability of amoebae these proteins was therefore investigated using cfp- and gfp-tagged proteins respectively. conditioned amoebae were stimulated with a micropipette containing camp and we found that the amoebae were able to localise each of the markers towards the chemoattractant, even though the cells were round. we then considered whether aberrant actin remodelling might be partly responsible for the phenotypes observed. fluorescent crac-transformed amoebae with cycloheximide fluorescent crac-transformed amoebae without cycloheximide fluorescent pi3k-transformed amoebae with cycloheximide fluorescent pi3k-transformed amoebae without cycloheximide the rate of actin polymerisation does not depend on protein synthesis the dynamics and magnitudes of actin polymerisation were followed using gfp-tagged abp and actin and additional files). we observed that actin did indeed polymerise at the edge of the cell adjacent to, and in response to, the camp-filled micropipette, irrespective of whether the cells had been preincubated in the presence of cycloheximide or not. the course of actin polymerisation after camp addition was also followed fluorometrically using tritc-phalloidin to label the triton-insoluble actin cytoskeleton. the kinetics of actin assembly and the relative amounts polymerised were similar whether cycloheximide was present or not ). fluorescent abp120-transformed amoebae with cycloheximide discussion 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. by contrast, surface uptake measured with fm1- 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 must either be flattened or have a very small radius. a similar conclusion comes from studies of fm1- uptake in the wild-type amoeba, nc. 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 nsfa is also interesting. at the restrictive temperature, nsfa is unable to take up fluid phase or phagocytose: in this sense, it is like ax treated with cycloheximide. however, fm1- uptake by nsfa at the restrictive temperature is reduced, 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. 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. 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 ) 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. before turning to how cycloheximide causes these effects, it is relevant to ask how a cycloheximide resistant mutant, hh behaves. the rate of fluid phase uptake, as measured using fitc-dextran, was unaltered in the resistant mutant. additionally, when observed under the microscope, the amoebae remain motile and do not round up. 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, 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 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 minutes after cycloheximide addition, yet minutes is required to block migration. this is not surprising: each of these is a complex process and the dependence on a single factor need not be the same for each. a similar phenomenon is seen with the nsf mutant: fluid phase uptake is effectively abolished by minutes at the restrictive temperature, yet cell migration continues quite happily until about 15– minutes after the temperature is raised. indeed, in so far as this comparison has been explored, the assays in which ax is more sensitive to cycloheximide seem to be those in which the nsf mutant is most sensitive.  our results show that movement, fluid phase uptake, phagocytosis and capping require continued protein synthesis, whilst localised actin polymerisation, membrane uptake and pi3k and crac translocation to the leading edge do not. our results may suggest that, upon cycloheximide treatment, either a labile protein required for motility and perhaps involving polarised membrane recycling is lost, or a control mechanism linking protein synthesis to membrane recycling is activated. 
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. it has previously been shown that a short incubation of dictyostelium discoideum amoebae in cycloheximide eliminates fluid phase endocytosis. furthermore, fluid phase endocytosis, phagocytosis and capping cease in the presence of mm cycloheximide, although membrane uptake, as measured using fm1- is unaffected. in the presence of cycloheximide, aggregation-competent amoebae sensitive to camp, although round, can still localise crac, abp pi3k and actin polymerisation in response to a micropipette filled with camp. cycloheximide is a protein synthesis inhibitor that acts specifically on the 60s subunit of eukaryotic ribosomes. 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.
 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.  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 mm cycloheximide, although membrane uptake, as measured using fm1- is unaffected. in the presence of cycloheximide, aggregation-competent amoebae sensitive to camp, although round, can still localise crac, abp 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.  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. 