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Experimental Cell Research 92 (1975) 394-398
STUDIES
ON THE MECHANISM
ISOPROPYL
N-PHENYL
OF ACTION
OF
CARBAMATE
R. A. COSS,l R. A. BLOODGOGD,e D. L. BROWER, J. D. PICKETT-HEAPS J. R. MCINTOSH
and
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colo. 80302, USA
SUMMARY The binding of [14C]isopropyl N-phenyl carbamate (IPC) to microtubular protein isolated from chick brains, and the effect of isopropyl N-phenyl carbamate (IPC) on the in vitro reassembly of microtubules was investigated. While [l’C]colchicine binds to microtubular protein, p4CjIPC does not. Concentrations from 1 x lo-* M to 1 x lo-* M IPC do not prevent in vitro repolymerization of microtubular protein. IPC (1 x 1O-4 M) does not affect the rate of reassembly of microtubules. We conclude that IPC does not exert its effect through an interaction with microtubular protein; we suggest that IPC probably interacts with microtubule organizing centers.
Isopropyl N-phenyl carbamate (IPC) recently has been shown to be a useful drug in studies of the assembly of microtubular systems. IPC differentially affects the reappearance of three separate sets of microtubules in Ochromonas [l]; it alters the functional number of polar microtubule organizing centers and affects the orientation of the phycoplast, a set of cytoplasmic microtubules, in Oedogonium [2, 31.However, the mechanism of action of IPC is unknown. On the basis of in vitro experiments, antimitotic drugs can be divided into two classes. The first class comprises drugs which bind to microtubular protein (tubulin) and block its polymerization. Colchicine, colcemid, vinblastine sulfate, vincristine sulfate and podophyllotoxin fall into this class [4]. The second 1 Present address: The Rockefeller University, New York, N.Y. 10021, USA. e Present address: Department of Biology, Yale University, New Haven, Conn. 06520, USA. Exptl Cell Res 92 (197.5)
class includes drugs which do not bind to tubulin and do not block its in vitro polymerization. Griseofulvin belongs to this second class [5, 61. In this research, we have investigated whether IPC binds to isolated chick brain tubulin and what effects it has on the in vitro reassembly of microtubules. Our results indicate that IPC belongs to the second class of antimitotic drugs. MATERIALS AND METHODS Tubulin was isolated from chick brain tissue as previously described 171. P(c]IPC (“C-ring; 4.3 mCi/ mmole) and unlabeled IPC were provided by the Pittsburgh Plate Glass Co. The PQIPC was pharmacologically active. Ninety-six percent of the radioactivity co-migrated with unlabeled IPC as determined by thin layer chromatography on silica gel in a hexane : ether solvent (9 : 1-ViV). [l*Cjcolchicine (methoxy-14C, ring C; 15 mCi/mmole) was purchased from New England Nuclear. Unlabeled colchicine, GTP and PIPES (piperizine-N-N1-bis[2~~ane sulfonic acid]) were obtained from Sigma, and vinblastine sulfate from Eli Lilly.
Interaction of tubulin and IPC Filter assay 4 x lo-# moles of tubulin was added to: (i) 1 x lo-’ moles [r’c]IPC in PMG (buffer containing 0.1 M PIPES, 5 mM MgCl, and 1 mM GTP at pH 6.9); (ii) 1 x lo-* moles [l’c]colchicine plus 200 ~1 PMG. Control mixtures contained the same quantities of radioactive drug but did not contain any tubulin. All mixtures were incubated for 60 min at 37°C then nlaced on ice and diluted with 300 ul of cold (4°C) unlabeled IPC or colchicine in PhiG. Five ml of P&lG was added to each tube, and the contents were poured over 2 DEAE cellulose filter discs (presoaked in PMG) and gravity filtered. Each tube was washed twice with buffer and gravity filtered. The DEAE discs were then suction washed 5 times with PM (buffer containing 0.1 M PIPES and 5 mM MgCl, at pH 6.9), dried and counted.
Column assays Cold column. 8 mm x 16 cm sizing columns packed with Sephadex G-50 (Fine Grade) were used in these experiments. The elution positions of colchicine, IPC and GTP were determined with the column equilibrated with PM. The elution position of tubulin was determined with the column equilibrated with PMG. For binding assays, mixtures containing (i) 4 x 1O-8moles of tubulin and 1-10x 10-O moles [14C]colchicine, or (ii) 4 x lOma moles of tubulin and 1 x 10-r moles [r4c]IPC were incubated for 60 min at 37°C. An aliquot from each tube was then added to the column. UV absorption was monitored at 280 nm and 4 drop fractions were collected and counted in a Packard Tri-Carb liquid scintillation counter. Hot column. A sizing column of similar dimensions was packed with Sephadex G-50 equilibrated with 3.66 x 1O-B M [14c]IF’C (the molarity of IPC in the incubation mixture). The incubation mixture containing 4 x lo-@ moles of tubulin and 1 x 1O-Bmoles P4ClIPC was heated for 60 min at 37°C. A uortion of this mixture was then run on the column. UV absorption was monitored at 280 nm, and samples of 4 drops each were collected and counted.
In vitro reassembly system Electron microscouy. The revolvmerization mixture contained 10 mg/ml chick brain tubulin, 2.5 mM GTP, 0.1 M PIPES and 1 mM EGTA at vH 6.9. The cold controls were incubated for 15 min at 4°C prior to placing a drop from each tube on Formvar/ carbon coated grids and negatively staining with uranyl acetate. Warm controls were incubated for 15 min at 37°C prior to preparation for electron microscopy. The following reagents were examined for their effect on reoolvmerization of tubulin: 1 x lo-’ M IPC (0.5 % &O-H), 5 x lo-’ M IPC (2.0 % EtOH), 1 x 1O-a M IPC (2.0 % EtOH), 1 x 1O-4 M vinblastine sulfate, 1 x lo+ M IPC plus 1 x 1O-4 M vinblastine sulfate. and 2.0% EtOH. For each reagent the repolymerization mixture was incubated for 15 min at 37°C prior to preparation for electron
395
microscopy. The negatively stained grids were examined in a Philips 300 electron microscope operating at 60 kV. Viscometry. The effect of 1 x 1O-4 M IPC (aqueous) on the repolymerization of tubulin (2.2 mg/ml) in PMG was monitored using a Cannon-Manning Semimicro Kinematic Viscometer (no. 100). Microtubules were reassembled at 37°C.
RESULTS Filter assay
Table 1 is an example of the results from a typical filter assay examining the binding of [14C]colchicine and [14C]IPC to tubulin. Colchicine, as expected, binds to tubulin. No significant difference in bound cpm, however, is observed between filters washed with [14C]IPC alone or with mixtures of [14C]IPC and tubulin. Column assays Cold column. Tubulin, colchicine, IPC and
GTP were monitored individually to determine their elution positions. Colchicine, IPC and GTP elute at the bed volume, while tubulin elutes at the void volume. The relative elution positions of tubulin, colchicine and IPC are indicated by arrows in figs 1 and 2. Table 1. Binding of incubated mixturesa of [14C]IPC and tub&n, and [14C]colchicine and tub&n to DEAE cellulose filters Incubation mixture
cpm
r4c]IPC + tubulin* [14c]IPC + PMG only’ [14c]colchicine + tubulin + PMGd [14C]colchicine + PMG onlye
291 364 25 860 101
-
a Mixtures were incubated for 60 min at 37°C. * 1 x lo--’ moles [14CJIPC in PMG; 4 x lo-@ moles tubulin. z 1 x lo-’ moles [14CjIPC in PMG; 100 ~1 PMG. 1 x lo--* moles [14C]colchicine; 4 x 10-Omoles tubulin; 200 ~1 PMG. e 1 x 1O-8moles [14C]colchicine; 300 ~1 PMG. Exptl Cell Res 92 (1975)
Coss et al.
396
TUBULIN J 1500
160 UNBOUND COLCHICINE I
r
o ..“..p I
, 5
I IO
I 15
I 20
TUBULIN
60
/ 25
1
30
Fig. I. Abscissa: fraction no.; ordinate: (left) O-O, [WJcolchicine (cpm); (right) O-O, ret. units of absorption at 280 nm. Elution profiles of radioactivity and UV absorption of an incubated mixture of PQcolchicine (1 x 1O-8 moles) and tubulin (4 x 1O-s moles). PMG is the running buffer.
One or two peaks of label, depending on the concentration of colchicine, are observed when mixtures of [14C]colchicineand tubulin are run on the column. When only one peak is observed (fig. l), it is associated with the tubulin. When two peaks are observed, the second peak elutes with unbound colchicine. When mixtures of [14C]IPC and tubulin are run on the column, only one radioactive peak is present, and this elutes with free IPC and not with the tubulin (fig. 2). Hot column. Table 1 and fig. 2 imply that [14C]IPC either does not bind to tubulin or, if it does bind, it has a high rate of dissociation from tubulin. To test the latter possibility, we ran the mixture of [14C]IPC and tubulin on the same type of column used above but equilibrated with [14C]IPC at the same concentration as in the reaction mixture. If IPC binds to tubulin but has a fast off rate, then one will still observe a peak of label associated with the eluted tubulin and a trough in the label distribution associated with the position where unbound IPC normally elutes [8]. The results of this experiment are shown in fig. 3. The tubulin does not have a peak of label associated with it. Exptl Cell Res 92 (1975)
15000-
70
5000
-
Fig. 2. Abscissa: fraction no.; ordinate: (left) O-O, [W]IPC (cpm); (right) O-O, rel. units of absorption at 280 nm. Elution profiles of radioactivity and UV absorption of an incubated mixture of [“c]IPC (1 x 10-’ moles) and tubulin (4 x 10-O moles). PMG is the running buffer.
In vitro repolymerization
assays
Concentrations of 1 x 1O-4M, 5 x 1O-4M and 1 x 1O-3 M IPC do not affect the ability of tubulin to polymerize into microtubules in vitro. When observed by negative staining, the number of microtubules found per grid 1500
15
TUBULIN i
1000
O]
IO
5
IO
I5
20
25
3o”
Fig. 3. Abscissa: fraction no.; ordinate: (left) O-O, [14c]IPC @pm); (right) O-O, rel. units of absorption at 280 nm. Elution profiles of radioactivity and UV absorption of an incubated mixture of [WJIPC (1 x 10-O moles) and tubulin (4 x 10-* moles). The column was equilibrated with p4c]IPC in PMG prior to running the incubated sample. *Two bubbles occurred at the end of fraction 23 and at the beginning of fraction 24.
Interaction of tubulin and IPC
397
4. Electronmicrographof microtubulesreassembled in thepresence of 1 x KV M IPC. x 11000. 5. High magnificationof a portion of a microtubulereassembled in the presence of 1 x lO-3M IPC. The ultrastructureof thesemicrotubuleswereidenticalto microtubulesreassembled in thewarmcontrol. x 140000.
Fig. Fig.
square in the presence of IPC is similar to the number formed in the warm control (fig. 4). Further, the structure of the microtubules appears identical to that of microtubules reassembled in the absenceof the drug (fig. 5). 1 x 1O-4 M vinblastine sulfate prevented the in vitro polymerization of tubulin. It has been found that in Ochromonas, 5 x 1O-5 M vinblastine sulfate and 5 x 10e4 M IPC are Table 2. Reassembly of microtubules in vitroa in the presence of various reagents
Condition Cold control (4°C) Warmcontrol (37°C) 2.0% EtOH 1 x 1O-4M IPC (0.5% EtOH) 5 x lo-” M IPC (2.0% EtOH) 1 x lo+ M IPC (2.0% EtOH) 1 x lo-’ M vinblastinesulfate 1 x lO-4M vinblastinesulfateplus 1 x 1O-3M IPC
Microtubule reassembly in vitro +++ +++ +++ +++ +++ -
antagonistic with respect to production of macrotubules [9]. However, we find that microtubules do not form in vitro in a mixture of 1 x 10d4 M vinblastine sulfate and 1 x 1O-3 M IPC (neither have macrotubules ever been observed in vitro). The results of the repolymerization experiments are summarized in table 2. The rate of reassembly of microtubules, measured by viscometry, is unaffected by 1 x 1O-4M IPC; the specific viscosity of the reassembly mixture containing 1 x 1O-4 M IPC increases from 0.14 to 0.85, while the specific viscosity of the control mixture increasesfrom 0.11 to 0.82 during the first 15 min of incubation at 37°C. The specific viscosity of both mixtures decreases20-25 min after the start of incubation at 37°C. DISCUSSION
-
IPC does not bind to chick brain tubulin or affect the in vitro reassemblyof microtubules, a Repolymerizationbrew: 10 mg/ml chick brain indicating that IPC belongs to the class of tubulin, 2.5mM GTP, 0.1 M PIPES,1 mM EGTA, antimitotic dtigs that do not exert their efpH 6.9 Exptl Cell Res 92 (197.5)
398
Goss et al.
feet by a direct interaction with tubulin. (Subsequent to completion of this work the authors became aware of a paper by Bartels & Hilton [15]. They reported that 1.4 x 1O-3 M IPC does not prevent in vitro polymerization of pig brain tubulin, and that 14C-labeled Chloropropham [isopropyl-N-(3-chloro)phenyl carbamate] does not bind to this tubulin.) IPC may cause its effect by interfering with the structural or functional integrity of microtubule bridges, or with microtubule organizing centers (MTOCs). Although most of the reported effects of IPC on systemsin vivo argue against the bridge hypothesis as its only mechanism of action [l, 2, 3, 10, Ill, some of the effects of this drug may be exerted by interference with bridges connecting microtubules. To test this possibility, the effects of IPC on a system where bridges play a predominant role in motility was investigated; axostyles of Saccinobaculus were isolated and successfully reactivated in the presence of 1 x 1O-3 M IPC [12]. Further, IPC differentially blocks mitosis in microspores of Marsilea, with its effect being dependent on the presenceor absenceof highly structured polar bodies [13]. Hence, it appears that IPC affects MTOCs, and, more precisely, morphologically unstructured MTOCs in plants. This possibility will be tested by three experiments: (i) autoradiography of a system labeled with [3H]IPC; (ii) investigation of the binding of IPC to various isolated MTOCs; (iii) investigation of the effects of IPC on the in vitro assembly of microtubules from isolated MTOCs. Re-
Exptl Cell Res 92 (197.5)
cently, basal bodies and their interconnecting fibers have been isolated from Chlamydomonas, and both structured (basal bodies) and unstructured (interconnecting fibers) MTOCs nucleate microtubules in vitro [14]. Such a system may prove useful in these proposed experiments. The authors thank Drs W. Z. Cande and J. Snyder for providing the tubulin and Pittsburgh Plate Glass Co. for the generous gift of the labeled and unlabeled IPC used in these experiments. This work was supported by NIH grants GM 19718-02 and 5 PO7 RR00592 and NSF grant GB 25876.
REFERENCES 1. Brown, D L & Bouck, G B, J cell biol 61 (1974) 514. 2. Coss, R A & Pickett-Heaps, J D, J cell biol 59 (1973) 65~. 3. - Ibid 63 (1974) 84. 4. Wilson, L, Bramburg, J R, M&l, S B, Grisham, L M & Creswell, K R, Fed proc 33 (1974) 158. 5. Grisham, L M, Wilson, L & Bensch, K G, Nature 244 (1973) 294. 6. Grisham, L M, Bensch, K G & Wilson, L, J cell biol 59 (1973) 125~. 7. Cande, W i!, Snyder, J, Smith, D, Summers, K & McIntosh, J R, Proc natl acad sci US 71 (1974) 1559. 8. McIntosh, J R, Ph.D. dissertation. Harvard Universitv. Cambridge. Mass. f1967). Bouck; G B. Personal comnumication. 1;: Hepler, P K & Jackson, W T, J cell sci 5 (1969) -1* ILI. 11. Sanger, J M & Jackson, W T, J cell sci 8 (1971) 303. 12. Bloodaood. R A, Ph.D. dissertation. Universitv of Colorado, Boulder, Co10 (1974). 13. Hepler, P K, J cell biol 55 (1972) 112~. 14. Snell, W J, Dentler, W L, Haino, L T, Binder, L I & Rosenbaum. J L. Science 185 (1974) 357. 15. Bartels, P G & ‘Hilton, J L, Pest b&hem physiol 3 (1973) 462. Received August 27, 1974 Revised version received November 25, 1974