- Email: [email protected]
Colchicine binding of cell extracts from colchicine-resistant mutants of Chlamydomonas reinhardi
474
Preliminary
notes
of the filter, and in the scanning electron microscope this layer is composed of uniform, dense layers of tibrils. Both Klebe [8] and Pearlstein [12] also noted that cells could bind to non-serum treated collagen gels, and that this binding could be removed by treatment of their gels with 8 M urea at room temperature. They attributed this result to the removal of endogenous serum-binding factor in their collagen preparations. Maroudas [ 111, however, on theoretical grounds has raised the possibility that the 8 M urea treatment could partially denature the collagen gels, altering their fibrillar structure and thus masking any cell attachment mechanisms that might recognize native collagen tibrils. Our data strongly support this interpretation that there may indeed be at least two types of cellular binding to collagen; one directly to the native molecules or fibrils and one involving a serum-protein intermediate. We thank Dr Richard Hynes for performing assays for LETS protein. This is publication no. 747 of the Robert W. Lovett Memorial Group for the Study of Diseases Causing Deformities. Supported by grants from the NIH (EY02261, AM3564 and DE04220). Dr Linsenmayer is a recipient of the Research Career Development Award from the NIH (AMOO031) and Dr Toole is an Established Investigator of the American Heart Association (no. 73 138).
References
5. 6. 7. 8. 9. IO. Il. 12.
Culp, L A, J cell bio163 (1974) 71. Gulp, L A & Black, P H, Biochemistry 11 (1972) 2161. Dodson, J W & Hay, E D, Exp cell res 65 (1971) 215. Hauschka, S D &White, N K, Research in muscle develonment and the muscle spindle. Excerpta medica international congress series (1972) 63. Hay, E D & Revel, J P, Fine structure of the avian cornea (eh A Wolsky & P S Chen) vol. I. S Karger, Basel. Hynes, R 0, Proc natl acad sci US 70 (1973) 3170. Hynes, R 0 & Bye, J M, Cell 3 (1974) 113. Kiebe, R J, Nature 250 (1974) 248. Kleinman, H K, McGoodwin, E B & Klebe, R J, Biochem biophys res commun 72 (1976) 426. Linsenmayer, T F, Smith, G N & Hay, E D, Proc natl acad sci US 74 (1977) 39. Maroudas, N G, Nature 267 (1977) 183. Pearlstein, E, Nature 262 (1976) 497.
13. Trelstad, R L & Columbre, A J, J cell biol 50 (1971) 840. 14. Vaheri, A & Ruoslahti, E, Int j cancer 13 (1974) 579. 15. Walther, B T, Ohman, R & Roseman, S, Proc natl acad sci US 70 (1973) 1569. 16. Yamada, K M, Schlesinger, D H, Kennedy, D W & Pastan, I, Biochemistry 16 (1977) 5553. 17. Yamada, K M &Weston, J A, Cell 5 (1975) 75. Received April 17, 1978 Revised version received July 21, 1978 Accepted July 26, 1978
Printed in Sweden Copyright @ 1978 by Academic Pres. Inc. All rights of reproduction in any form reserved 0014.4827/78/1162-0474$02.00/O
Colchicine binding of cell extracts from colchicine-resistant mutants of Chlamydomonas reinhardi D. J. FLANAGAN of Biology, University YOI 5DD, UK
and J. R. WARR, Department of York, Heslington,
York,
The colchicine-binding activity of a high speed supematant from fourteen colchicine- and/or vinblastine-resistant mutants of Chlamydomonas reinhardi has been compared to that of wild type. Four of the mutants have reduced binding per unit protein. The low level of binding of one of these mutants is unusually stable. Three other mutants have normal initial binding levels, but show altered kinetics of decay of binding activity. Most of the mutants with altered colchicine-binding activitv oroduce abnormally large cells. Seven other mutants showed only slight or no differences in colchicine binding from wild type. Summary.
Several single gene mutations in Chlamyconfer changes in the levels of resistance to the antimicrotubular agents, colchicine and vinblastine [l-3]. Colchicine binding should provide an experimental approach to study the basis of resistance in these mutants, but a problem arises with the low level of the colchicinebinding activity in Chlamydomonas cell extracts. Plant material has a very low level of colchicine-binding activity in general [4], and only labile colchicine-binding activity can be detected in Chlamydomonas extracts [5]. It has not been proved that bind-
domonas reinhardi
Preliminary
Table 1. Colchicine Chlamydomonas
binding
of high speed supernatants
from
mutant
notes
475
and wild type
Means of determinations on three preparations of each mutant and twelve of wild type. The table lists only the binding of samples which have not been preincubated (see also figs 1 and 2). Figures for “Other mutants” are means of pooled data for toll, co&, col, and coll,. Colchicine bound (dpm/lO” cells)
~ol$icine (dpm/mg protein) Wild type vin, col, col,, col,, Other mutants
1 914 944* 1 109* 1 002* 967* 1 744
1.00 1.21” 0.82” 1.35b 1.60* 1.20*
1 910 1 199* 909* 1 353” 1 547 2 095
a Significant difference from wild type at the 2.5 % probability level. * Significant difference from wild type at the 0.1% probability level.
ing is specifically to tubulin, but the ob- period. When bound colchicine is then servation that it is not influenced by high plotted against preincubation time, the firstconcentrations of lumicolchicines suggests order decay line may be extrapolated back that this may be so. The present com- to the start of the incubation period to determine the initial binding capacity. However, munication compares this weak colchicinebinding activity in high speed supernatants colchicine-binding activity to a high speed from colchicine-resistant mutants with that supernatant from wild-type Chfamydofrom wild type cells. monas reinhardi has not been found to decay in a simple exponential fashion during preincubation at 25°C [5]. During the Materials and Methods early stages of preincubation the binding acThe origin of the mutant strains used here is described tivity is relatively stable and may actually in [3]. Cell culture conditions and techniques of colchicine binding have been described in [5]. In each exrise slightly before entering exponential periment, suffkient cells were harvested to provide at decay after about 45 min. The presence of a least 5 mg protein per 1 ml of supematant. shoulder means that simple extrapolation of the decay curve to estimate initial binding Results capacity is not feasible. We therefore ilBefore describing the properties of individlustrate our comparisons of wild type and ual mutants attention is drawn to a general mutant extracts by quoting data on colchicine binding for samples which have not feature of our results. Colchicine-binding activity from animal sources decays with previously been preincubated and by prefirst-order kinetics [6]. To allow for loss senting graphs of the decay of the colof binding activity which occurs during the chicine-binding activity after different preincubation period, it is normal practice in incubation periods. most studies with animal material to preFour of our mutants co&, col13, col16and incubate aliquots for differing times in the vin, show clear-cut differences in colabsence of colchicine and then measure the chicine-binding activity from wild type. colchicine bound during a fixed incubation Table 1 gives the values for binding activity
476
Preliminary notes
100
I 60
40
I 180
Fig. 1. Abscissa: preincubation period (min) prior to incubation with colchicine for 45 min; ordinate: colchicine bound (dpm/mg protein). A-A, Vin,; O-O, wild type. Colchicine binding of high speed supernatant from mutant vin,.
of samples which have not been preincubated and figs 1 and 2 show the decay of the activity for two mutants. The shape of the decay curve obtained was consistent in each of three separate experiments for each mutant carried out on different days. Mutant vin, shows the most striking differences from wild type. The colchicine bound per unit protein is consistently less than half that of wild type. Cells of this mutant are slightly larger than wild type and therefore the colchicine bound per cell is just above half that bound per wild-type cell. This very low level of colchicine-binding activity found in vin, is remarkably stable in contrast to wild type (fig. 1). Mutants co&, col13 and col16 all show significantly lower levels of colchicine binding than wild type on a unit protein basis, although in contrast to vin, the activity is not particularly stable. The kinetics of decay of binding activity of col13are shown in fig. 2; co16and col16 give similar decay curves to that shown for col13. col16has exceptionally large cells and consequently does not have EXP Cd
Rer
116 11978)
significantly different colchicine binding from wild type expressed on a per cell basis. Three other mutants (co&, co& and col15) can also be distinguished from wild type. Although the binding of mutant samples which have not been preincubated is only very slightly less than wild type, the decay of binding follows different kinetics. The decay curve for each of these mutants (illustrated, for example, in fig. 3) lacks a shoulder. The mutant-binding activity immediately starts to decay in an approximately exponential fashion in contrast to that of wild type which, as mentioned above, hardly decays at all for 45 min. All of these mutants produce larger than normal cells (soluble protein contents of 2.5, 1.7 and 2.1 times wild type respectively). Since their colchicine binding per unit protein is close to wild type levels, their values for colchicine binding per cell are above wild type. Several of our mutants show only very slight or no differences in colchicine binding from wild type. These mutants are colI,
ZOOO_!~ . I \ 10m--A,*
500-
.
\
zoo-
100 0
I 60
Fig. 2. Abscissa and ordinate,
\
. .
I 120
I 180
see fig. 1. A-A, col& O-O, wild-type control. Colchicine binding of high speed supematant from mutant col13.
Preliminary
notes
477
of colchicine binding per unit protein. This does not, of course, necessarily arise from a change in the structure of a colchicinebinding protein in the mutant. A relatively high proportion of the colchicine-binding protein could be in a form which no longer binds to colchicine (possibly intact microtubules?). Alternatively the observation could arise from a change in the ratio of colchicine-binding protein(s) to proteins of different kinds within the cell. However, in the case of three of our mutants, vin,, col, and col13, although the cells are large, the colchicine binding per cell is still abnorFig. 3. Abscissa and ordinate, see fig. 1. A-A, col,; O-O, wild-type control. mally low, so that a simple model of a fixed Colchicine binding of high speed supernatant from amount of colchicine-binding material per mutant co&. cell with an excess of non-colchicine-binding proteins in the mutant is inadequate. The mutants which appear to have colZ, co13, col, and coll,. Two other mutants, col, and collZ appear to show slightly altered colchicine-binding decay kinetics reduced binding activity, but this requires (col,, co& and col,,) also need not involve further confirmation. changes in the structure of colchicine-binding protein(s). Non-specific changes in Discussion other components of the cell extract may It should be emphasised that the nature of destabilize the colchicine-binding activity. the colchicine-binding protein(s) being Whatever the basis of the instability, it is instudied here is not established. The nature teresting to note that all these mutants proof colchicine-binding material from plant duce very large cells and it is interesting to sources in general is obscure. Hart & Sab- speculate that the instability in the colnis [4] have recently thoroughly reviewed chicine-binding protein(s) could play a the topic and drawn attention to non-spe- causative role in delaying these mutants at a cific binding to proteins other than tubulins. late stage of the cell cycle. We have previously found that lumicolSome of our mutants, toll, col,, co&, co& chicines do not compete with colchicine and col17 have no, or only very slight difbinding to Chlamydomonas high speed ferences in binding compared with wild supernatants, but that podophyllotoxin type. We have previously found col17 to be does so. These observations provide some cross-resistant to caffeine and to have a support for the view that the binding is to slightly reduced [3H]colchicine uptake [3]. tubulin, but do not prove it [5]. The ex- This mutant also shows a consistently retremely labile nature of colchicine binding duced level of [3H]caffeine uptake [7]. It in Chlamydomonas has so far prevented seems very likely that the primary defect of further clarification of this point. col17involves membrane permeability. At least four of our mutants, vin,, co&, We gratefully acknowledge financial support from the coll:, and ~01,~appear to have altered levels Science Research Council of the United Kingdom. 31-7x181x
tin
Cell Rc\ 116 1197X1
478
Preliminary
notes
regulating the concentration of insulin re1. Adams, M & Warr, J R, Exp cell res 71 (1972) 473. ceptors [3] since treatment of cells with db2. Warr, J R & Gibbons, D, Exp cell res 85 (1974) 117. CAMP increases their number. Serum com3. Warr, J R, Flanagan, D & Quinn, D, Exp cell res ponents may modify the level of receptors 111(1978) 37. 4. Hart, J W & Sabnis, D D, Curr adv pl sci 26 (1976) of mitogen factors (somatomedin and in1095. sulin) in chick embryo fibroblasts [4]. 5. Flanagan, D & Warr, J R, FEBS lett 80 (1977) 14. 6. Wilson, L, Biochemistry 9 (1970) 4999. Finally, a cell-cycle dependence has been 7. Warr, J R & Quinn, D. Unpublished data. demonstrated for melanocyte stimulating Received April 26, 1978 hormone and nerve growth factor receptors Revised version received July 11, 1978 [5,6]. In the present work, we have studied Accepted July 3 1, 1978 the effect of serum, growth status and the cell cycle on insulin-binding sites of normal cultured mouse BALB/3T3 fibroblasts.
References
Printed in Sweden Copyright @ 1978 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/78/l 162.0478$02.00/O
Studies on the regulation of insulin receptors in cultured BALB/3T3 fibroblasts P. THOMOPOULOS,’ M. C. WILLINGHAM and I. PASTAN, Laboratory of Molecular Biology, National Cancer Institute, National Bethesda, MD 20014, USA
Institutes
of
Health,
Summary. The level of [‘251]insulin binding to BALBI 3T3 tibroblasts was low in growing cells and high in stationary cells. Since frequent changes of medium (every 2 h) did not modify the hormone binding of the stationary cells, it is unlikely that serum factors directly regulate the number of insulin receptors. Cells were grown to different densities by plating them in different concentrations of serum. Insulin binding was low in dense cultures maintained actively growing by high serum concentration, while binding was high in sparse cultures which were growth-arrested due to serum depletion. Thus, cell density does not directly regulate the insulin receptors. The growth status of the cells is the only factor that explains consistently the variations of insulin binding in these and previous [ 1, 21 experiments. Synchronization of the cells by two different methods did not show a reproducible cellcycle dependence for the insulin receptors.
Materials
and Methods
Porcine insulin (lot 7 GU 48L) was purchased from Elanco. Na-lZ51 (carrier-free) was from Amershaml Searle, r3H]thymidine (6.7 Cilmmol) from New England Nuclear, bovine serum albumin (fraction V) from Armour, and methyl cellulose from Fisher. All chemicals were of reagent grade. Normal BALB/3T3 mouse fibroblasts were grown as described previously [ 11.For binding studies, the fibroblasts were detached from the dishes by incubation with Ca2+-and Mg2+-free Dulbecco’s PBS (pH 7.4) and then sedimented by centrifugation (5 min at 250 g). The cells were resuspended in assay buffer, sedimented (250 g), and resuspended in the same buffer at a final concentration of 0.4X 106-2X lo6 cells/ml. For the cell-cvcle exueriments the cells were synchronized by two methods. In the first method the cells were mated at 5~ 103 cells/cm2 in Dulbecco-Voat’s medium with 10% calf serum. On day 2, the dishes were changed to the same medium with 5 % calf plasma and the number of cells/dish was measured daily. Under these conditions the cell growth was arrested after 3-l days of culture in plasma, before confluency was reached. At that moment calf serum was added to a 20% final concentration and the number of cells/ dish. the 13Hlthvmidine uotake and the r’251]insulin binding were studied at the-indicated time poinis. In the second method. the cells were cultured in suspension ( lo6 cells/ml) ‘in Dulbecco-Vogt’s medium containing 10% calf serum and 1.17% methyl cellulose in agar-layered plastic dishes ([7] and M. C. Willineham. unoublished observations). The cultures were ilncubated’at 37°C under 95 % air - 5 % CO, humidified atmosphere. Medium was added every 24 h to keep the methyl cellulose gel from drying. At day 4, the dishes were chilled at 4°C to liquefy the gel, the methyl cellulose suspension was removed, diluted with 4 vol of cold medium, and the cells were sedimented by centrifugation (10 min at 550 g at 4°C). The cell pellet was resuspended in warm medium, sedimented (250 g
Insulin receptors in cultured mouse fibroblasts show variations according to the conditions of the culture. We [l] and others [2] have demonstrated that the levei of [lz51]insulin binding is low in non-confluent cells and increases in confluent cells. The mechanisms regulating this phenomenon are not completely understood. The intracellular 1 On leave from U-35 INSERM, Hopital Hem-i Monlevel of CAMP might be one of the factors dor, Creteil, France.
Preliminary
notes
of the filter, and in the scanning electron microscope this layer is composed of uniform, dense layers of tibrils. Both Klebe [8] and Pearlstein [12] also noted that cells could bind to non-serum treated collagen gels, and that this binding could be removed by treatment of their gels with 8 M urea at room temperature. They attributed this result to the removal of endogenous serum-binding factor in their collagen preparations. Maroudas [ 111, however, on theoretical grounds has raised the possibility that the 8 M urea treatment could partially denature the collagen gels, altering their fibrillar structure and thus masking any cell attachment mechanisms that might recognize native collagen tibrils. Our data strongly support this interpretation that there may indeed be at least two types of cellular binding to collagen; one directly to the native molecules or fibrils and one involving a serum-protein intermediate. We thank Dr Richard Hynes for performing assays for LETS protein. This is publication no. 747 of the Robert W. Lovett Memorial Group for the Study of Diseases Causing Deformities. Supported by grants from the NIH (EY02261, AM3564 and DE04220). Dr Linsenmayer is a recipient of the Research Career Development Award from the NIH (AMOO031) and Dr Toole is an Established Investigator of the American Heart Association (no. 73 138).
References
5. 6. 7. 8. 9. IO. Il. 12.
Culp, L A, J cell bio163 (1974) 71. Gulp, L A & Black, P H, Biochemistry 11 (1972) 2161. Dodson, J W & Hay, E D, Exp cell res 65 (1971) 215. Hauschka, S D &White, N K, Research in muscle develonment and the muscle spindle. Excerpta medica international congress series (1972) 63. Hay, E D & Revel, J P, Fine structure of the avian cornea (eh A Wolsky & P S Chen) vol. I. S Karger, Basel. Hynes, R 0, Proc natl acad sci US 70 (1973) 3170. Hynes, R 0 & Bye, J M, Cell 3 (1974) 113. Kiebe, R J, Nature 250 (1974) 248. Kleinman, H K, McGoodwin, E B & Klebe, R J, Biochem biophys res commun 72 (1976) 426. Linsenmayer, T F, Smith, G N & Hay, E D, Proc natl acad sci US 74 (1977) 39. Maroudas, N G, Nature 267 (1977) 183. Pearlstein, E, Nature 262 (1976) 497.
13. Trelstad, R L & Columbre, A J, J cell biol 50 (1971) 840. 14. Vaheri, A & Ruoslahti, E, Int j cancer 13 (1974) 579. 15. Walther, B T, Ohman, R & Roseman, S, Proc natl acad sci US 70 (1973) 1569. 16. Yamada, K M, Schlesinger, D H, Kennedy, D W & Pastan, I, Biochemistry 16 (1977) 5553. 17. Yamada, K M &Weston, J A, Cell 5 (1975) 75. Received April 17, 1978 Revised version received July 21, 1978 Accepted July 26, 1978
Printed in Sweden Copyright @ 1978 by Academic Pres. Inc. All rights of reproduction in any form reserved 0014.4827/78/1162-0474$02.00/O
Colchicine binding of cell extracts from colchicine-resistant mutants of Chlamydomonas reinhardi D. J. FLANAGAN of Biology, University YOI 5DD, UK
and J. R. WARR, Department of York, Heslington,
York,
The colchicine-binding activity of a high speed supematant from fourteen colchicine- and/or vinblastine-resistant mutants of Chlamydomonas reinhardi has been compared to that of wild type. Four of the mutants have reduced binding per unit protein. The low level of binding of one of these mutants is unusually stable. Three other mutants have normal initial binding levels, but show altered kinetics of decay of binding activity. Most of the mutants with altered colchicine-binding activitv oroduce abnormally large cells. Seven other mutants showed only slight or no differences in colchicine binding from wild type. Summary.
Several single gene mutations in Chlamyconfer changes in the levels of resistance to the antimicrotubular agents, colchicine and vinblastine [l-3]. Colchicine binding should provide an experimental approach to study the basis of resistance in these mutants, but a problem arises with the low level of the colchicinebinding activity in Chlamydomonas cell extracts. Plant material has a very low level of colchicine-binding activity in general [4], and only labile colchicine-binding activity can be detected in Chlamydomonas extracts [5]. It has not been proved that bind-
domonas reinhardi
Preliminary
Table 1. Colchicine Chlamydomonas
binding
of high speed supernatants
from
mutant
notes
475
and wild type
Means of determinations on three preparations of each mutant and twelve of wild type. The table lists only the binding of samples which have not been preincubated (see also figs 1 and 2). Figures for “Other mutants” are means of pooled data for toll, co&, col, and coll,. Colchicine bound (dpm/lO” cells)
~ol$icine (dpm/mg protein) Wild type vin, col, col,, col,, Other mutants
1 914 944* 1 109* 1 002* 967* 1 744
1.00 1.21” 0.82” 1.35b 1.60* 1.20*
1 910 1 199* 909* 1 353” 1 547 2 095
a Significant difference from wild type at the 2.5 % probability level. * Significant difference from wild type at the 0.1% probability level.
ing is specifically to tubulin, but the ob- period. When bound colchicine is then servation that it is not influenced by high plotted against preincubation time, the firstconcentrations of lumicolchicines suggests order decay line may be extrapolated back that this may be so. The present com- to the start of the incubation period to determine the initial binding capacity. However, munication compares this weak colchicinebinding activity in high speed supernatants colchicine-binding activity to a high speed from colchicine-resistant mutants with that supernatant from wild-type Chfamydofrom wild type cells. monas reinhardi has not been found to decay in a simple exponential fashion during preincubation at 25°C [5]. During the Materials and Methods early stages of preincubation the binding acThe origin of the mutant strains used here is described tivity is relatively stable and may actually in [3]. Cell culture conditions and techniques of colchicine binding have been described in [5]. In each exrise slightly before entering exponential periment, suffkient cells were harvested to provide at decay after about 45 min. The presence of a least 5 mg protein per 1 ml of supematant. shoulder means that simple extrapolation of the decay curve to estimate initial binding Results capacity is not feasible. We therefore ilBefore describing the properties of individlustrate our comparisons of wild type and ual mutants attention is drawn to a general mutant extracts by quoting data on colchicine binding for samples which have not feature of our results. Colchicine-binding activity from animal sources decays with previously been preincubated and by prefirst-order kinetics [6]. To allow for loss senting graphs of the decay of the colof binding activity which occurs during the chicine-binding activity after different preincubation period, it is normal practice in incubation periods. most studies with animal material to preFour of our mutants co&, col13, col16and incubate aliquots for differing times in the vin, show clear-cut differences in colabsence of colchicine and then measure the chicine-binding activity from wild type. colchicine bound during a fixed incubation Table 1 gives the values for binding activity
476
Preliminary notes
100
I 60
40
I 180
Fig. 1. Abscissa: preincubation period (min) prior to incubation with colchicine for 45 min; ordinate: colchicine bound (dpm/mg protein). A-A, Vin,; O-O, wild type. Colchicine binding of high speed supernatant from mutant vin,.
of samples which have not been preincubated and figs 1 and 2 show the decay of the activity for two mutants. The shape of the decay curve obtained was consistent in each of three separate experiments for each mutant carried out on different days. Mutant vin, shows the most striking differences from wild type. The colchicine bound per unit protein is consistently less than half that of wild type. Cells of this mutant are slightly larger than wild type and therefore the colchicine bound per cell is just above half that bound per wild-type cell. This very low level of colchicine-binding activity found in vin, is remarkably stable in contrast to wild type (fig. 1). Mutants co&, col13 and col16 all show significantly lower levels of colchicine binding than wild type on a unit protein basis, although in contrast to vin, the activity is not particularly stable. The kinetics of decay of binding activity of col13are shown in fig. 2; co16and col16 give similar decay curves to that shown for col13. col16has exceptionally large cells and consequently does not have EXP Cd
Rer
116 11978)
significantly different colchicine binding from wild type expressed on a per cell basis. Three other mutants (co&, co& and col15) can also be distinguished from wild type. Although the binding of mutant samples which have not been preincubated is only very slightly less than wild type, the decay of binding follows different kinetics. The decay curve for each of these mutants (illustrated, for example, in fig. 3) lacks a shoulder. The mutant-binding activity immediately starts to decay in an approximately exponential fashion in contrast to that of wild type which, as mentioned above, hardly decays at all for 45 min. All of these mutants produce larger than normal cells (soluble protein contents of 2.5, 1.7 and 2.1 times wild type respectively). Since their colchicine binding per unit protein is close to wild type levels, their values for colchicine binding per cell are above wild type. Several of our mutants show only very slight or no differences in colchicine binding from wild type. These mutants are colI,
ZOOO_!~ . I \ 10m--A,*
500-
.
\
zoo-
100 0
I 60
Fig. 2. Abscissa and ordinate,
\
. .
I 120
I 180
see fig. 1. A-A, col& O-O, wild-type control. Colchicine binding of high speed supematant from mutant col13.
Preliminary
notes
477
of colchicine binding per unit protein. This does not, of course, necessarily arise from a change in the structure of a colchicinebinding protein in the mutant. A relatively high proportion of the colchicine-binding protein could be in a form which no longer binds to colchicine (possibly intact microtubules?). Alternatively the observation could arise from a change in the ratio of colchicine-binding protein(s) to proteins of different kinds within the cell. However, in the case of three of our mutants, vin,, col, and col13, although the cells are large, the colchicine binding per cell is still abnorFig. 3. Abscissa and ordinate, see fig. 1. A-A, col,; O-O, wild-type control. mally low, so that a simple model of a fixed Colchicine binding of high speed supernatant from amount of colchicine-binding material per mutant co&. cell with an excess of non-colchicine-binding proteins in the mutant is inadequate. The mutants which appear to have colZ, co13, col, and coll,. Two other mutants, col, and collZ appear to show slightly altered colchicine-binding decay kinetics reduced binding activity, but this requires (col,, co& and col,,) also need not involve further confirmation. changes in the structure of colchicine-binding protein(s). Non-specific changes in Discussion other components of the cell extract may It should be emphasised that the nature of destabilize the colchicine-binding activity. the colchicine-binding protein(s) being Whatever the basis of the instability, it is instudied here is not established. The nature teresting to note that all these mutants proof colchicine-binding material from plant duce very large cells and it is interesting to sources in general is obscure. Hart & Sab- speculate that the instability in the colnis [4] have recently thoroughly reviewed chicine-binding protein(s) could play a the topic and drawn attention to non-spe- causative role in delaying these mutants at a cific binding to proteins other than tubulins. late stage of the cell cycle. We have previously found that lumicolSome of our mutants, toll, col,, co&, co& chicines do not compete with colchicine and col17 have no, or only very slight difbinding to Chlamydomonas high speed ferences in binding compared with wild supernatants, but that podophyllotoxin type. We have previously found col17 to be does so. These observations provide some cross-resistant to caffeine and to have a support for the view that the binding is to slightly reduced [3H]colchicine uptake [3]. tubulin, but do not prove it [5]. The ex- This mutant also shows a consistently retremely labile nature of colchicine binding duced level of [3H]caffeine uptake [7]. It in Chlamydomonas has so far prevented seems very likely that the primary defect of further clarification of this point. col17involves membrane permeability. At least four of our mutants, vin,, co&, We gratefully acknowledge financial support from the coll:, and ~01,~appear to have altered levels Science Research Council of the United Kingdom. 31-7x181x
tin
Cell Rc\ 116 1197X1
478
Preliminary
notes
regulating the concentration of insulin re1. Adams, M & Warr, J R, Exp cell res 71 (1972) 473. ceptors [3] since treatment of cells with db2. Warr, J R & Gibbons, D, Exp cell res 85 (1974) 117. CAMP increases their number. Serum com3. Warr, J R, Flanagan, D & Quinn, D, Exp cell res ponents may modify the level of receptors 111(1978) 37. 4. Hart, J W & Sabnis, D D, Curr adv pl sci 26 (1976) of mitogen factors (somatomedin and in1095. sulin) in chick embryo fibroblasts [4]. 5. Flanagan, D & Warr, J R, FEBS lett 80 (1977) 14. 6. Wilson, L, Biochemistry 9 (1970) 4999. Finally, a cell-cycle dependence has been 7. Warr, J R & Quinn, D. Unpublished data. demonstrated for melanocyte stimulating Received April 26, 1978 hormone and nerve growth factor receptors Revised version received July 11, 1978 [5,6]. In the present work, we have studied Accepted July 3 1, 1978 the effect of serum, growth status and the cell cycle on insulin-binding sites of normal cultured mouse BALB/3T3 fibroblasts.
References
Printed in Sweden Copyright @ 1978 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/78/l 162.0478$02.00/O
Studies on the regulation of insulin receptors in cultured BALB/3T3 fibroblasts P. THOMOPOULOS,’ M. C. WILLINGHAM and I. PASTAN, Laboratory of Molecular Biology, National Cancer Institute, National Bethesda, MD 20014, USA
Institutes
of
Health,
Summary. The level of [‘251]insulin binding to BALBI 3T3 tibroblasts was low in growing cells and high in stationary cells. Since frequent changes of medium (every 2 h) did not modify the hormone binding of the stationary cells, it is unlikely that serum factors directly regulate the number of insulin receptors. Cells were grown to different densities by plating them in different concentrations of serum. Insulin binding was low in dense cultures maintained actively growing by high serum concentration, while binding was high in sparse cultures which were growth-arrested due to serum depletion. Thus, cell density does not directly regulate the insulin receptors. The growth status of the cells is the only factor that explains consistently the variations of insulin binding in these and previous [ 1, 21 experiments. Synchronization of the cells by two different methods did not show a reproducible cellcycle dependence for the insulin receptors.
Materials
and Methods
Porcine insulin (lot 7 GU 48L) was purchased from Elanco. Na-lZ51 (carrier-free) was from Amershaml Searle, r3H]thymidine (6.7 Cilmmol) from New England Nuclear, bovine serum albumin (fraction V) from Armour, and methyl cellulose from Fisher. All chemicals were of reagent grade. Normal BALB/3T3 mouse fibroblasts were grown as described previously [ 11.For binding studies, the fibroblasts were detached from the dishes by incubation with Ca2+-and Mg2+-free Dulbecco’s PBS (pH 7.4) and then sedimented by centrifugation (5 min at 250 g). The cells were resuspended in assay buffer, sedimented (250 g), and resuspended in the same buffer at a final concentration of 0.4X 106-2X lo6 cells/ml. For the cell-cvcle exueriments the cells were synchronized by two methods. In the first method the cells were mated at 5~ 103 cells/cm2 in Dulbecco-Voat’s medium with 10% calf serum. On day 2, the dishes were changed to the same medium with 5 % calf plasma and the number of cells/dish was measured daily. Under these conditions the cell growth was arrested after 3-l days of culture in plasma, before confluency was reached. At that moment calf serum was added to a 20% final concentration and the number of cells/ dish. the 13Hlthvmidine uotake and the r’251]insulin binding were studied at the-indicated time poinis. In the second method. the cells were cultured in suspension ( lo6 cells/ml) ‘in Dulbecco-Vogt’s medium containing 10% calf serum and 1.17% methyl cellulose in agar-layered plastic dishes ([7] and M. C. Willineham. unoublished observations). The cultures were ilncubated’at 37°C under 95 % air - 5 % CO, humidified atmosphere. Medium was added every 24 h to keep the methyl cellulose gel from drying. At day 4, the dishes were chilled at 4°C to liquefy the gel, the methyl cellulose suspension was removed, diluted with 4 vol of cold medium, and the cells were sedimented by centrifugation (10 min at 550 g at 4°C). The cell pellet was resuspended in warm medium, sedimented (250 g
Insulin receptors in cultured mouse fibroblasts show variations according to the conditions of the culture. We [l] and others [2] have demonstrated that the levei of [lz51]insulin binding is low in non-confluent cells and increases in confluent cells. The mechanisms regulating this phenomenon are not completely understood. The intracellular 1 On leave from U-35 INSERM, Hopital Hem-i Monlevel of CAMP might be one of the factors dor, Creteil, France.