Cell Biology International Reports, VoL 16, No. 11, 1992
1055
EFFECTS OF TUBULOZOLE ON THE AMOEBOFLAGELLATE TRANSFORMATION IN PHYSARUM POLYCEPHALUM. Mark IL Adelman.
Department of Anatomy and Cell Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA.
ABSTRACT The Amoeboflagellate Transformation (AFT) of Physarum potycephalum involves rapid changes in the cytoskeleton, cell shape and cell motility. Use of pharmacologic agents to probe the role of cytoskeletal elements in the AFT are impeded because the transforming cells are very sensitive to such commonly-used drug solvents as DMSO. The anti-microtubule agent tubulozole is found to disrupt, rapidly and transiently, the AFT, inhibiting flagella formation, cell elongation and the arrangement of microtubules and microfilaments. Cells recover quickly, possibly due to precipitation of the drug; the reappearance of normal arrays of microfilaments and cytoplasmic microtubules lags behind flagella formation. INTRODUCTION Amoeboflagellate transformations (AFT) are interesting examples of relatively rapid developmental events involving striking changes in cell shape and behaviour (Fulton, 1970). Studies on the AFT inPhysarumpolycephalum (Pagh and Adelman, 1982, 1988; Uyeda and Furuya, 1985, 1989, Wright eL al., 1979, 1980) have revealed that extensive rearrangements of microfilaments and microtubules take place during the transformation but have not fully clarified their relative roles in specific changes of cell shape or motility. We (unpublished data) and others (Mir and Wright, 1978; Ohta et. al., 1991; Uyeda and Furuya, 1989; Wright, personal communication) have used a variety of pharmacologic probes in such studies, with variable success. This report describes some of the effects of the anti-microtubule agent tubulozole (Dieckmann-Schuppert and Franklin, 1990; Geuens et. al., 1985) on transforming cells. MATERIALS and METHODS Materials: Stock solutions of tubulozoles T and C (Tr, TC; Janssen) were prepared in DMSO, (Sigma) at concentrations of 10-25 mM, and stored at 4°C. 0309-1651/92/111055-6/$08.00/0
© 1992 Academic Press Ltd
1056
Cell Biology lnternationai Reports, Vol. 16, No. 11, 1992
Other reagents were as previously described (Pagh and Adelman, 1982; 1988). Cell culture and induction of the amoeboflagellate transformation: Physarum polycephalum amoebae (strain DJ 911) were cultured as described previously (Pagh and Adelman, 1982; 1988). The AFT was induced by flooding culture plates of amoebae with transformation buffer (TB = 0.10 M sucrose, 0.01 M phosphoeitrate, pH 5.6). Cells which transform more rapidly and synchronously were obtained by aspirating from a first-transformation plate the fluid, containing cells that had detached after 90'; these were collected by brief eentrifugation (5' at 500xg), and replated on agar. Such cells were incubated for 60' (at 25°C; allowing reversion to the amoeboid state) and were then induced to transform a second time with TB; we refer to this repeat amoeboflagellate transformation as the RAFF. All observations reported here were made on cells that were scraped from the agar surface immediately after starting the RAFT by re-immersion in TB; cell suspensions were incubated at 25°C, in the absence/presence of DMSO w/wo TT or TC. At indicated times, samples were fixed by adding an equal volume of 20% formalin (v/v) in a buffer consisting of 5 mM Pipes, 25 mM KC1, 25 mM NaC1, 2 mM MgC12, 2 mM EGTA, pH 6.8. Fixed cells were scored as to presence of flagellae and other morphological features ("staging"; Pagh and Adelman, 1988). Fluorescence microscopic studies: Processing for fluorescence microscopy was as described previously (Pagh and Adelman, 1988) except that cells were p e r m e a b ~ e d with Triton X-100 (0.05% w/v final). For indirect immunofluorescence detection of microtubules the primary antibody was a monoclonal anti-a-tubulin (Sigma T-9206); the solution containing FITC-labelled secondary antibody (goat anti-mouse, Sigma F-0257) also had rhodamine- phalloidin (Molecular Probes), to label microfilaments. Slides were observed on a Zeiss photomicroscope or Zeiss Axiovert, using 40X or 63X phase-DIC planapo lenses. Images were acquired with a Dage-MTI Model 66 SIT camera, processed through a simple image averaging device (Dage DSP100) and recorded on videotape. Video records were examined on an MDI 386 computer (IBM clone) equipped with Optimas software (Bioscan, Edmonds, WA), a PC-Vision+ frame-grabber card, and a secondary image monitor. Images for publication were "grabbed", processed, transferred to diskette, and converted from digitized to negative film format, from which prints were made in the conventional fashion.
RESULTS and DISCUSSION
We have probed the RAFT with a number of drugs (e.g. eytochalasins, noeodazole) known to affect eytoskeletal components, but have not got consistent results. One of the problems encountered is that the cells are very sensitive to the solvents commonly employed in such drug studies. Figure 1
Cell Biology lnternational Reports, Vol. 16, No. 11, 1992
1057
shows the effects of different levels of DMSO on the formation of flagellate cells during the RAFT; similar effects are observed with dimethylformamide and ethanol. Only at DMSO levels lower than 0.5% (v/v) do ceils appear to transform normally, and staging of cells (not shown) reveals that even DMSO levels as low as 0.25% inhibit the RAFT, as judged by the morphology of cells, even when the rate and extent of flagella formation appears normal. With DMSO at 0.1% v/v or less, we have usually found it necessary to use very high concentrations of drugs to get effects on the RAFT, and the effects were either poorly reproducible or so broad as m raise doubts about drug specificity. lOO
Figure 1: Effects of D M S O on the on the RAFT. Cells were fixed at the
UJ I--
~
so
.J ILl
o
I..-
7
4O
~
2O
0
10
20
30
40
5'0
60
indicated times after the start of the RAFT and evaluated for presence of flagella. TB contained no DMSO (open circles) or DMSO at 0.1% (open squares), 0.25% (triangles), 0.5% (filled circles), or 1% (filled squares) v/v final.
TIME (rain)
We have recently found that both tubulozole stereoisomers (TT and TC) show clear effects on the RAFT. While the data in figure 2 suggest that TT is more effective as an inhibitor than is TC, other experiments indicate both inhibitors are approximately equivalent.
'°°I
'°i1 6O
d
~
2
'1
0
5
10
i
lq5
2'0
2~5
Figure 2: Effects of T r and TC on the RAFT. DMSO (0.1% v/v), wAvo TF or TC, was added ~-1 rain after start of RAFT. Control (open circles), DMSO alone (filled circles), TI' at 1 ttM (open triangles) or 10 tiM (filled triangles), TC at 1 I~M (open squares) or 10 tiM (filled squares) final concentration.
30
TIME (mini
The effective range of each is from 1 tiM to 25 IsM. Treated cells round up rapidly and lose flagellae; double labelling studies (not shown) reveal few cytoplasmic microtubules and aetin in cortical arrays like those in stage I and II control cells (see figures 4 and 5, Pagh and Adelman, 1988). These effects of TI' and TC are transient; cells appear to recover rapidly, with the percent flagellation returning to levels near those of control cells in a matter of minutes. The recovery from "IT and TC is only partial, however, for when
1058
Cell Biology lnternational Reports, Vol. 16, No. 1 I, 1992
attempts are made to stage the transforming cells, they are found to have abnormal morphology and cytoskeleta] arrangements.
Figure 3: Morphological and Cytoskeletal Components of Cells Recovering from T r Treatment. Cells were fixed 15 minutes after the RAFT was started in the presence of TB plus 0.1% DMSO, without ("control", panels a-c) or with 5 ttM TI" (panels d-f). Panels b,e: Nomarski-DIC optics. Panels a,d fluorescence, staining for tubulin; c,f - fluorescence, staining for actin. Each panel 25 ttM wide. After 15 minutes in 5 tiM TT (figure 3) cells have recovered to >80% flagellation vs. 95% for controls (not shown). The flagellae on drug-treated eeUs are shorter than on controls and Nomarski-DIC images reveal the treated cells are more rounded; they also lack the ridge found on control cells (compare fig. 3e with 3b, also with fig.7 of Pagh and Adelman, 1988). In the treated cells the anterior cone of mierotubules is smaller than in control cells; aetin assemblies, found in the ridge and a posterior "spot" in control cells, are less extensive in treated cells and do not show the apparent close association (seen in control cells) between the aetin in the ridge and mierotubules at the base of the ridge. These results suggest that, after disruption of mierotubules by TT, cells rapidly recover the ability to assemble flagella, but that cytoplasmic mierotubules are reassembled more slowly. It is tempting to speculate that disruption of mierotubules yields cells that cannot elongate as do control cells and that rearrangement of actin filaments is also disrupted. In all such experiments, TT (and TC) effects are transient and a strong function of the time of drug addition, with maximal effects being seen if the drug is added within ~.5 minutes of the start of the RAFT. When stock solutions of TT
Cell Biology lnternational Reports, VoL 16, No. 11, 1992
1059
and TC are diluted into TB the solutions become cloudy within seconds and, in some cases, floeeulant precipitates form. It seems reasonable to presume that the transient effects of TI' and TC reflect the rapid removal of the drugs, by sequestration and/or precipitation, from the aqueous milieu of the transforming cells. Clearly, the availability of more water-soluble eytoskeletal disruptants, or conditions for rendering the drugs normally used more soluble in aqueous solutions, would greatly facilitate studies such as those reported here. ACKNOWLEDGEMENTS I thank Mrs. Olivia Walker for expert technical assistance. This work was supported by USUHS Protocols RO-7083 and BRSG-G170BG. The opinions or assertions contained herein are the private ones of the author and are not to be construed as official or reflecting the view of the DoD or the USUHS.
REFERENCES Dieckmann-Schuppert, A. and Franklin, 17,. M. (1990). Mode of Action of Tubulozoles Against Plasmodium falciparum In Vitro. Antimicrobial Agents and Chemotherapy 34: 1529-I534. Fulton, C. (1970). Amoebo-flagellates as research partners: The laboratory biology of Naegleria and Tetramitus. In Methods in Cell Physiology, vol 4, (ed. Prescott, D.M.), pp. 341-476. New York: Academic Press. Geuens, G. M. A., Nuydens, IL M., Willebrords, IL E., Van de Veire, IL M. L., GooSens, F., Dragonetti, C. H., Marcel, M. M. IL and De Brabander, M. J. (1985). Effects of Tubulozole on the Microtubule System of Cells in Culture and In Vivo. Can. Res. 45: 733-742. Mir, L. and Wright, M. (1978). Action of Antimierotubular Drugs on Physarum polycephalum. Mierobios Letters. 5: 39-44. Ohta, T., Kawano, S. and Kuroiwa, T. (1991). Migration of the Cell Nucleus During the Amoebo-flagellate Transformation of Physarum polycephalum is Mediated by an Actin-generated Force that Acts on the Centrosome. Protoplasma. 163: 114-124. Pagh, K. I. and Adelman, M. R. (1982). Identification of a MicrofilamentEnriched Motile Domain in Amoeboflagellates of Physarum polycephalum. J. Cell Sci. 54: 1-21. Pagh, K. I. and Adelman, M. R. (1988). Assembly, Disassembly, and Movements of the Microfilament-Rich Ridge During the Amoeboflagellate Transformation in Physarum Polycephalum. Cell Motility and the Cytoskeleton. 11: 223-234.
1060
Celt Biology lnternational Reports, Vol. 16, No. 11, 1992
Uyeda, T. Q. P. and Furuya, M. (1985). Cytoskeletal Changes Visualized by Fluorescence Microscopy During the Amoeba-to-Flagellate and Flagellate-to-Amoeba Transformations in Physarum potycephalum. Protoplasma. 126: 221-232. Uyeda, T. Q. p. and Furuya, M. (1989). Evidence for Active Interactions Between Microfilaments and Microtubules in Myxomycete Flagellates. J. Cell Biol. 108: 1727-1735. Wright, M., Moisand, A., and Mir, L. (1979). The Structure of the Flagellar Apparatus of the Swarm Cells of Physarumpolycephalum. Protoplasma 100: 231-250. Wright, M., Mir, L., and Moisand, A. (1980). The Structure of the Pro-Flagellar Apparatus of the Amoebae of Physarumpolycephalum: Relationship to the Flagellar Apparatus. Protoplasma 103: 69-81.
1055
EFFECTS OF TUBULOZOLE ON THE AMOEBOFLAGELLATE TRANSFORMATION IN PHYSARUM POLYCEPHALUM. Mark IL Adelman.
Department of Anatomy and Cell Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA.
ABSTRACT The Amoeboflagellate Transformation (AFT) of Physarum potycephalum involves rapid changes in the cytoskeleton, cell shape and cell motility. Use of pharmacologic agents to probe the role of cytoskeletal elements in the AFT are impeded because the transforming cells are very sensitive to such commonly-used drug solvents as DMSO. The anti-microtubule agent tubulozole is found to disrupt, rapidly and transiently, the AFT, inhibiting flagella formation, cell elongation and the arrangement of microtubules and microfilaments. Cells recover quickly, possibly due to precipitation of the drug; the reappearance of normal arrays of microfilaments and cytoplasmic microtubules lags behind flagella formation. INTRODUCTION Amoeboflagellate transformations (AFT) are interesting examples of relatively rapid developmental events involving striking changes in cell shape and behaviour (Fulton, 1970). Studies on the AFT inPhysarumpolycephalum (Pagh and Adelman, 1982, 1988; Uyeda and Furuya, 1985, 1989, Wright eL al., 1979, 1980) have revealed that extensive rearrangements of microfilaments and microtubules take place during the transformation but have not fully clarified their relative roles in specific changes of cell shape or motility. We (unpublished data) and others (Mir and Wright, 1978; Ohta et. al., 1991; Uyeda and Furuya, 1989; Wright, personal communication) have used a variety of pharmacologic probes in such studies, with variable success. This report describes some of the effects of the anti-microtubule agent tubulozole (Dieckmann-Schuppert and Franklin, 1990; Geuens et. al., 1985) on transforming cells. MATERIALS and METHODS Materials: Stock solutions of tubulozoles T and C (Tr, TC; Janssen) were prepared in DMSO, (Sigma) at concentrations of 10-25 mM, and stored at 4°C. 0309-1651/92/111055-6/$08.00/0
© 1992 Academic Press Ltd
1056
Cell Biology lnternationai Reports, Vol. 16, No. 11, 1992
Other reagents were as previously described (Pagh and Adelman, 1982; 1988). Cell culture and induction of the amoeboflagellate transformation: Physarum polycephalum amoebae (strain DJ 911) were cultured as described previously (Pagh and Adelman, 1982; 1988). The AFT was induced by flooding culture plates of amoebae with transformation buffer (TB = 0.10 M sucrose, 0.01 M phosphoeitrate, pH 5.6). Cells which transform more rapidly and synchronously were obtained by aspirating from a first-transformation plate the fluid, containing cells that had detached after 90'; these were collected by brief eentrifugation (5' at 500xg), and replated on agar. Such cells were incubated for 60' (at 25°C; allowing reversion to the amoeboid state) and were then induced to transform a second time with TB; we refer to this repeat amoeboflagellate transformation as the RAFF. All observations reported here were made on cells that were scraped from the agar surface immediately after starting the RAFT by re-immersion in TB; cell suspensions were incubated at 25°C, in the absence/presence of DMSO w/wo TT or TC. At indicated times, samples were fixed by adding an equal volume of 20% formalin (v/v) in a buffer consisting of 5 mM Pipes, 25 mM KC1, 25 mM NaC1, 2 mM MgC12, 2 mM EGTA, pH 6.8. Fixed cells were scored as to presence of flagellae and other morphological features ("staging"; Pagh and Adelman, 1988). Fluorescence microscopic studies: Processing for fluorescence microscopy was as described previously (Pagh and Adelman, 1988) except that cells were p e r m e a b ~ e d with Triton X-100 (0.05% w/v final). For indirect immunofluorescence detection of microtubules the primary antibody was a monoclonal anti-a-tubulin (Sigma T-9206); the solution containing FITC-labelled secondary antibody (goat anti-mouse, Sigma F-0257) also had rhodamine- phalloidin (Molecular Probes), to label microfilaments. Slides were observed on a Zeiss photomicroscope or Zeiss Axiovert, using 40X or 63X phase-DIC planapo lenses. Images were acquired with a Dage-MTI Model 66 SIT camera, processed through a simple image averaging device (Dage DSP100) and recorded on videotape. Video records were examined on an MDI 386 computer (IBM clone) equipped with Optimas software (Bioscan, Edmonds, WA), a PC-Vision+ frame-grabber card, and a secondary image monitor. Images for publication were "grabbed", processed, transferred to diskette, and converted from digitized to negative film format, from which prints were made in the conventional fashion.
RESULTS and DISCUSSION
We have probed the RAFT with a number of drugs (e.g. eytochalasins, noeodazole) known to affect eytoskeletal components, but have not got consistent results. One of the problems encountered is that the cells are very sensitive to the solvents commonly employed in such drug studies. Figure 1
Cell Biology lnternational Reports, Vol. 16, No. 11, 1992
1057
shows the effects of different levels of DMSO on the formation of flagellate cells during the RAFT; similar effects are observed with dimethylformamide and ethanol. Only at DMSO levels lower than 0.5% (v/v) do ceils appear to transform normally, and staging of cells (not shown) reveals that even DMSO levels as low as 0.25% inhibit the RAFT, as judged by the morphology of cells, even when the rate and extent of flagella formation appears normal. With DMSO at 0.1% v/v or less, we have usually found it necessary to use very high concentrations of drugs to get effects on the RAFT, and the effects were either poorly reproducible or so broad as m raise doubts about drug specificity. lOO
Figure 1: Effects of D M S O on the on the RAFT. Cells were fixed at the
UJ I--
~
so
.J ILl
o
I..-
7
4O
~
2O
0
10
20
30
40
5'0
60
indicated times after the start of the RAFT and evaluated for presence of flagella. TB contained no DMSO (open circles) or DMSO at 0.1% (open squares), 0.25% (triangles), 0.5% (filled circles), or 1% (filled squares) v/v final.
TIME (rain)
We have recently found that both tubulozole stereoisomers (TT and TC) show clear effects on the RAFT. While the data in figure 2 suggest that TT is more effective as an inhibitor than is TC, other experiments indicate both inhibitors are approximately equivalent.
'°°I
'°i1 6O
d
~
2
'1
0
5
10
i
lq5
2'0
2~5
Figure 2: Effects of T r and TC on the RAFT. DMSO (0.1% v/v), wAvo TF or TC, was added ~-1 rain after start of RAFT. Control (open circles), DMSO alone (filled circles), TI' at 1 ttM (open triangles) or 10 tiM (filled triangles), TC at 1 I~M (open squares) or 10 tiM (filled squares) final concentration.
30
TIME (mini
The effective range of each is from 1 tiM to 25 IsM. Treated cells round up rapidly and lose flagellae; double labelling studies (not shown) reveal few cytoplasmic microtubules and aetin in cortical arrays like those in stage I and II control cells (see figures 4 and 5, Pagh and Adelman, 1988). These effects of TI' and TC are transient; cells appear to recover rapidly, with the percent flagellation returning to levels near those of control cells in a matter of minutes. The recovery from "IT and TC is only partial, however, for when
1058
Cell Biology lnternational Reports, Vol. 16, No. 1 I, 1992
attempts are made to stage the transforming cells, they are found to have abnormal morphology and cytoskeleta] arrangements.
Figure 3: Morphological and Cytoskeletal Components of Cells Recovering from T r Treatment. Cells were fixed 15 minutes after the RAFT was started in the presence of TB plus 0.1% DMSO, without ("control", panels a-c) or with 5 ttM TI" (panels d-f). Panels b,e: Nomarski-DIC optics. Panels a,d fluorescence, staining for tubulin; c,f - fluorescence, staining for actin. Each panel 25 ttM wide. After 15 minutes in 5 tiM TT (figure 3) cells have recovered to >80% flagellation vs. 95% for controls (not shown). The flagellae on drug-treated eeUs are shorter than on controls and Nomarski-DIC images reveal the treated cells are more rounded; they also lack the ridge found on control cells (compare fig. 3e with 3b, also with fig.7 of Pagh and Adelman, 1988). In the treated cells the anterior cone of mierotubules is smaller than in control cells; aetin assemblies, found in the ridge and a posterior "spot" in control cells, are less extensive in treated cells and do not show the apparent close association (seen in control cells) between the aetin in the ridge and mierotubules at the base of the ridge. These results suggest that, after disruption of mierotubules by TT, cells rapidly recover the ability to assemble flagella, but that cytoplasmic mierotubules are reassembled more slowly. It is tempting to speculate that disruption of mierotubules yields cells that cannot elongate as do control cells and that rearrangement of actin filaments is also disrupted. In all such experiments, TT (and TC) effects are transient and a strong function of the time of drug addition, with maximal effects being seen if the drug is added within ~.5 minutes of the start of the RAFT. When stock solutions of TT
Cell Biology lnternational Reports, VoL 16, No. 11, 1992
1059
and TC are diluted into TB the solutions become cloudy within seconds and, in some cases, floeeulant precipitates form. It seems reasonable to presume that the transient effects of TI' and TC reflect the rapid removal of the drugs, by sequestration and/or precipitation, from the aqueous milieu of the transforming cells. Clearly, the availability of more water-soluble eytoskeletal disruptants, or conditions for rendering the drugs normally used more soluble in aqueous solutions, would greatly facilitate studies such as those reported here. ACKNOWLEDGEMENTS I thank Mrs. Olivia Walker for expert technical assistance. This work was supported by USUHS Protocols RO-7083 and BRSG-G170BG. The opinions or assertions contained herein are the private ones of the author and are not to be construed as official or reflecting the view of the DoD or the USUHS.
REFERENCES Dieckmann-Schuppert, A. and Franklin, 17,. M. (1990). Mode of Action of Tubulozoles Against Plasmodium falciparum In Vitro. Antimicrobial Agents and Chemotherapy 34: 1529-I534. Fulton, C. (1970). Amoebo-flagellates as research partners: The laboratory biology of Naegleria and Tetramitus. In Methods in Cell Physiology, vol 4, (ed. Prescott, D.M.), pp. 341-476. New York: Academic Press. Geuens, G. M. A., Nuydens, IL M., Willebrords, IL E., Van de Veire, IL M. L., GooSens, F., Dragonetti, C. H., Marcel, M. M. IL and De Brabander, M. J. (1985). Effects of Tubulozole on the Microtubule System of Cells in Culture and In Vivo. Can. Res. 45: 733-742. Mir, L. and Wright, M. (1978). Action of Antimierotubular Drugs on Physarum polycephalum. Mierobios Letters. 5: 39-44. Ohta, T., Kawano, S. and Kuroiwa, T. (1991). Migration of the Cell Nucleus During the Amoebo-flagellate Transformation of Physarum polycephalum is Mediated by an Actin-generated Force that Acts on the Centrosome. Protoplasma. 163: 114-124. Pagh, K. I. and Adelman, M. R. (1982). Identification of a MicrofilamentEnriched Motile Domain in Amoeboflagellates of Physarum polycephalum. J. Cell Sci. 54: 1-21. Pagh, K. I. and Adelman, M. R. (1988). Assembly, Disassembly, and Movements of the Microfilament-Rich Ridge During the Amoeboflagellate Transformation in Physarum Polycephalum. Cell Motility and the Cytoskeleton. 11: 223-234.
1060
Celt Biology lnternational Reports, Vol. 16, No. 11, 1992
Uyeda, T. Q. P. and Furuya, M. (1985). Cytoskeletal Changes Visualized by Fluorescence Microscopy During the Amoeba-to-Flagellate and Flagellate-to-Amoeba Transformations in Physarum potycephalum. Protoplasma. 126: 221-232. Uyeda, T. Q. p. and Furuya, M. (1989). Evidence for Active Interactions Between Microfilaments and Microtubules in Myxomycete Flagellates. J. Cell Biol. 108: 1727-1735. Wright, M., Moisand, A., and Mir, L. (1979). The Structure of the Flagellar Apparatus of the Swarm Cells of Physarumpolycephalum. Protoplasma 100: 231-250. Wright, M., Mir, L., and Moisand, A. (1980). The Structure of the Pro-Flagellar Apparatus of the Amoebae of Physarumpolycephalum: Relationship to the Flagellar Apparatus. Protoplasma 103: 69-81.
