Cell Biology
International
Reports,
45
Vol. 3, No. 1, 1979
MTCROTUBUIESIN THF: CYTORI$sbB OF MOUSEE?dBRYOFIBROBLLSTS A. D. Bershadsky" V. I. Gelfand 2§ T. I. Svitkini2 and I. S. Tint2'
COLDSTABLE
1
Cancer Research Centre, Moscow, USSR 2 MOSCOWState University,
Academy of Medical MOSCOW,
Sciences,
USSR
Abstract. Treatment of cultured mouse embryo fibroblasts with Trbton X-100 after prolonged incubation at 0 C reveals a network of microtubules
cells. bules
in the cytoplasm
of cooled
This network of cold-stable microtuwas demonstrated by iaratunofluorescence microscopy, using a monospecific antibody against tubulin and by electron microscopy. The cold-stable microtubules, as well as the ordinary cytoplasmic microtubules, were sensitive to Ca ions and were not observed in the cells pre-treated with colchicine or vinblastine. The cold-stable microtubules do not seem to be in equilibriumowith the pool of depolymerized tubulin at 0 C.
Introduction Recently several methods of extraction of cultured cells with non-ionic detergents were developed which ensured the cytoplasmic microtubules are well preserved (Osborn and Weber, 1977; Small and Cells, 1978; Bershadsky et al., 'l 78). Cells extracted by these methods (cell models 3 are especially useful in immunofluorescence studies with antitubulin antibodies. Depolgmerized tubulin and other soluble proteins are readily removed from the cell models in the course of extraction and do not produce any background fluorescence. This seems to be crucial, when cells with a partially destroyed microtubular system are studied. In this case, the use of cell models instead of intact s TO whom correspondence should be addressed at the Department of Molecular Biology, Faculty of Biology, Moscow State University, Moscow 117234, USSR
0309/1651i79/010045-06/$0’2.00/0
01979
Academic
Press Inc.
(Londoni
Ltd.
46
Cell Biology
International
Reports,
Vol. 3, No. 1, 1979
cells
allows us to visualise the remaining microtubules. By using this technique we found that cytoplasm of mouse embryo fibroblasts contains ticrotubules even after prolonged incubation in the cold. Materials and Methods, Secondary cultures of mouse embryo fibroblasts were used for experiments 24 hours after plating. To extrac ‘i them the cultures were washed in three chahges of and transferred into 1% Triton X-100 in the buffer “s;* Y supplemented with 4 Y glycerol. Extraction was carried out for 30 min at a temperature which was identical to the temperature of the pre-incubation of cells in the growth medium: OoC (ice bath) or 37’C. After extraction the cultures were washed in bufferIE and fixed with 4% formaldehyde in PBS. For the lmmunofluorescent studies fixed cells were treated with acetone at 4oC and stained by the indirect method with monospecific antibodies against bovine brain tubulin (Fuller et al., 1975). For electron microscopy, extracted cultures were fisred with 2% glutaraldehyde, postfixed with Os04 and embedded in Spurrts resin (Spurr, lg69).
Results
and Discussion.
The system of microtubules visualized in the mouse embryo fibroblasts rown and extracted at 37W is shown in Fig. 3. As desc rf bed previously (Bershadsky et al., 1978), this system was identical to the microtubule system of non-extracted cells. Immunofluorescent staining of the cells incubated at OoC for 5 hrs and then extracted at the same temperature revealed quite a different pattern (Figs. 1 and 2). Instead of the well developed radial system of microtubules cytoplasm of the cooled cells contained a number of iong wavy fibrils forming a loose network. Neither the density nor the spatial distribution of these fibrils were changed if the cooling time was decreased to 10 min or increased to 76 hrs. l%actly the same fluorescence pattern was observed if Triton extraction of cold-treated cells was carried out in the glycerol-free buffer instead of in the glycerol-supplemented one. The distribution of fibrils revealedwith the antitubulin antibodies in the cooled cells was different from the distribution of micrstubules in the cells *) Abbrevia tions used: PBS - phosphate-buffered saline pH 7.2; EGTA - e thyleneglycol-bis (2.aminoe thy1 ether)-N,N’-tetraacetate; buffer M - 50 mM imidazol pH20 6.7, 50 mM KCl, 0.5 mM MgCl2, ‘l mM EGrpB, 0.1 mM ethylenediaminetetraacetate, 1 mM 2.mercaptoethenol.
Cell Biology
International
Reports,
Vol. 3, No. 1, 1979
extracted with Fig_a_.%3. Mouse embryo fibroblasts Triton X-100 and stained bg the indirect immunofluorescence method, using monospecific antibediee againat bovine brain tubalin. Bars denote 20 j&m. Figs. l-2. Cells incubated at O°C for 5 bra and then extracted at the same temperature. Note the network of cold-stable microtubules. Fig. 3. Cell grown and extracted at 37OC. Fig. 4. Electron microscopy of cooled cell after Triton extraction. Microtubule (M) and microfilament bundle (B) are seen. Bardenetes0.5 JUR.
48
Cell Biology
International
Reports,
Vol. 3, No. I, 1979
incubated at 37W. dlevertheless, these fibrils were true microtubules. This statement is based on the folwere seen, when an antilowing evidence. 1) No fibrils tubulia antibody preincubated with an excess of tubalin was used for the staining. 2) Cells grown in the pr+ sence of 10 JIM vinblastine or colchicine, then incubated at O°C for 5 hrs and extracted with Triton X-l00 as described above did not contain any fibrils. 3) Electron microscopy revealed typical microtubules with 25 nm external diameter in the cooled cells extracted with Triton (Fig. 4). The network of wavy-form microtubules was also seen in the lamellar parts of some non-extracted cooled cells after immunofluorescent staining. Therefore, the cooled cells contained the network of microtubules before the extraction procedure. However, the visualization and especially the photographic documentation of microtubules in the cooled non-extracted cells was greatly complicated by the presence of a highly fluorescent background in these cells. This background, which most probably arosed due to the increased concentration of the depolymerioed tubulin in the cell after cooling, was completely eliminated in the course of extraction. Hence, the cold-treated cells contain a system of microtubules. These microtubules differ from ordinary cytoplasmic microtubules in their stability at O°C. It is, therefore, interesting to study the sensitivity of the cold-stable microtubules to the microtubule-destroying treatment other than cold. 2 mM CaC12 in buffer M (which is equivalent to about 0.9 Iplla of free Ca++) caused the complete dissolution of cold-stable microtubules during 10 min incubation both at OoC and at 37OC, Therefore the cold-stable microtubules as well as the ordinary microtubules are sensitive to Ca++. We have shown earlier that the ordinary microtubules in the cell models were gradually dissolved during prolonged incubation in a lycerol-free buffer (Be% shadsky et al., 1978). Sim&arly, the cold-stable microtubules were dissolved when models of cooled cells were incubated in the buffer M at 37W for 5 hrs. Addition of 4 M glycerol prevented this depolymerization, as well as depolymerization of the usual cytoplasmic microtubules. On the other hand, !SlrMncubation of cooled and then Triton-extracted cells in the buffer 111 without glycerol at OOC did not produce any changes in the distribution and morphology of the cold-stable microtubules. Therefore, the cold-stable microtubules are not in equilibrium with the depolgmerized tubulin at OOC, although at 37W these microtubules are in equilibrium with the tubulin ~001.
Cell Biology
International Reports, vol. 3, No. 1, 1979
49
Several results suggest that the cold-stable microtubules do not exist in the cells before coolhg. Indeed, we have shown that the microtubule system Of cells incubated and extracted at 37’C is cold-sensitive (Bershadsky et al., 1978). This system was destroyed after 30 min incubation at O°C, although some fragments of the microtubules were seen in the cell models after the models of non-cooled cells and, cooling. Therefore, non-cooled cells before extraction contamost probably, ined few if any cold-stable microtubules before cold treatment. Possibly, these microtubules were formed as a result of the rearrangement of the ordinary cytoplasmic microtubules during cold treatment. There are some biochemical and cytological data in the literature regarding microtubules insensitive to cold. It has been shown by Grisham and Wilson (1975) that a certain microtubule assembly did occur at @C in vitro. This process could be induced in the cytosol frqqtion of the brain homogenate by the addition of GTP, and ETA. & The cold-stability of the "9+2*' microtubules of cilia and flagella is well documented (see, for example Behnke and Forer, 1967). Brinkley and Cartwright (1975) described cold-stable microtubules in the mitotic spindle of cultured cells. Tilney and Porter (1967) in their investigation of Helioeoa microtubules found so the called 'macrotubules" 34 main diameter which replaced the axopodial microtubules in cold-treated cells. The present communication is to our knowlege the first one which shows that morphologically normal microtubules exist in the cytoplasm of cooled interphase cells. The occurence of cold-st@ble cytoplasmic microtubules in cells other than mouse embryo fibroblasts, as well as the possible biological role of these microtubules requires further investigation. Acknowledaements. The authors would like to express their thanks to Professors A. S. Spirin and J. Y. Vasiliev for their support throughout this work. We are also very grateful to Mrs. G. A. Kozlov for checking the English vergion of the manuscript. Eeferences. Behnke, 0. and Forer, A. (1967) Evidence for four classes of microtubules in individual cells. Journal of Cell Science, 2, 169-192. Bershadskg, A.D., Gelfand V.I., Svitkina, T.M., and Tint, I.S. (1978) Mi crotubules in mouse embryo fibroblasts extracted with Triton X-100. Cell
50
Ceil Biology
International
Reports,
Vol. 3, NO. I, 1979
Biology International Reports, In press. Brinkley, B.R. and Cartwright J. (1975) Cold-labile and cold-stable microt&les in the mitotic spindle of mammalian cells. In: The Biology of Cytoplasmic Microtubules (D. Soifer, ed.). Annals of the New York Academy of Sciences, a, 428-439. Puller G.Y., Brinkley, B.R., and Boughter, J.M. (1975) f mmunofluorescence of mitotic spindles by using monospecific antibody against bovine brain tubulin. Science, 187, 948-950. Grisham, L.M. and Wilson, L. (1975) Evidence for subclasses of microtubules in the vertebrate central nervous system. Journal of Cell Biology, g, 14681 Osborn, M. and Weber, K. (1977) The display of microtubules in transformed cells. Cell, l2, 561-571 Small, J.V. and Cells, J.E. (1978) Filament arrangement in negatively stained cultured cells: the organization of actin, Cytobiologie, l6, 308-325 Spurr, A.R. (1969) A low viscosity epoxy resin embedding medium for electron microscopy. Journal of Bltrastructure Research, 26, 3143 Tilney, L.G. and Porter, K.R. (1967) Studies on the microtubules in Heliozoa. II. The effect of low temperature on these structures in the formation and maintenance of the axopodia. Journal of Cell Biolotm 2, 327-343.
Received:
7th
August
1978
Accepted:
21st
August
1978
International
Reports,
45
Vol. 3, No. 1, 1979
MTCROTUBUIESIN THF: CYTORI$sbB OF MOUSEE?dBRYOFIBROBLLSTS A. D. Bershadsky" V. I. Gelfand 2§ T. I. Svitkini2 and I. S. Tint2'
COLDSTABLE
1
Cancer Research Centre, Moscow, USSR 2 MOSCOWState University,
Academy of Medical MOSCOW,
Sciences,
USSR
Abstract. Treatment of cultured mouse embryo fibroblasts with Trbton X-100 after prolonged incubation at 0 C reveals a network of microtubules
cells. bules
in the cytoplasm
of cooled
This network of cold-stable microtuwas demonstrated by iaratunofluorescence microscopy, using a monospecific antibody against tubulin and by electron microscopy. The cold-stable microtubules, as well as the ordinary cytoplasmic microtubules, were sensitive to Ca ions and were not observed in the cells pre-treated with colchicine or vinblastine. The cold-stable microtubules do not seem to be in equilibriumowith the pool of depolymerized tubulin at 0 C.
Introduction Recently several methods of extraction of cultured cells with non-ionic detergents were developed which ensured the cytoplasmic microtubules are well preserved (Osborn and Weber, 1977; Small and Cells, 1978; Bershadsky et al., 'l 78). Cells extracted by these methods (cell models 3 are especially useful in immunofluorescence studies with antitubulin antibodies. Depolgmerized tubulin and other soluble proteins are readily removed from the cell models in the course of extraction and do not produce any background fluorescence. This seems to be crucial, when cells with a partially destroyed microtubular system are studied. In this case, the use of cell models instead of intact s TO whom correspondence should be addressed at the Department of Molecular Biology, Faculty of Biology, Moscow State University, Moscow 117234, USSR
0309/1651i79/010045-06/$0’2.00/0
01979
Academic
Press Inc.
(Londoni
Ltd.
46
Cell Biology
International
Reports,
Vol. 3, No. 1, 1979
cells
allows us to visualise the remaining microtubules. By using this technique we found that cytoplasm of mouse embryo fibroblasts contains ticrotubules even after prolonged incubation in the cold. Materials and Methods, Secondary cultures of mouse embryo fibroblasts were used for experiments 24 hours after plating. To extrac ‘i them the cultures were washed in three chahges of and transferred into 1% Triton X-100 in the buffer “s;* Y supplemented with 4 Y glycerol. Extraction was carried out for 30 min at a temperature which was identical to the temperature of the pre-incubation of cells in the growth medium: OoC (ice bath) or 37’C. After extraction the cultures were washed in bufferIE and fixed with 4% formaldehyde in PBS. For the lmmunofluorescent studies fixed cells were treated with acetone at 4oC and stained by the indirect method with monospecific antibodies against bovine brain tubulin (Fuller et al., 1975). For electron microscopy, extracted cultures were fisred with 2% glutaraldehyde, postfixed with Os04 and embedded in Spurrts resin (Spurr, lg69).
Results
and Discussion.
The system of microtubules visualized in the mouse embryo fibroblasts rown and extracted at 37W is shown in Fig. 3. As desc rf bed previously (Bershadsky et al., 1978), this system was identical to the microtubule system of non-extracted cells. Immunofluorescent staining of the cells incubated at OoC for 5 hrs and then extracted at the same temperature revealed quite a different pattern (Figs. 1 and 2). Instead of the well developed radial system of microtubules cytoplasm of the cooled cells contained a number of iong wavy fibrils forming a loose network. Neither the density nor the spatial distribution of these fibrils were changed if the cooling time was decreased to 10 min or increased to 76 hrs. l%actly the same fluorescence pattern was observed if Triton extraction of cold-treated cells was carried out in the glycerol-free buffer instead of in the glycerol-supplemented one. The distribution of fibrils revealedwith the antitubulin antibodies in the cooled cells was different from the distribution of micrstubules in the cells *) Abbrevia tions used: PBS - phosphate-buffered saline pH 7.2; EGTA - e thyleneglycol-bis (2.aminoe thy1 ether)-N,N’-tetraacetate; buffer M - 50 mM imidazol pH20 6.7, 50 mM KCl, 0.5 mM MgCl2, ‘l mM EGrpB, 0.1 mM ethylenediaminetetraacetate, 1 mM 2.mercaptoethenol.
Cell Biology
International
Reports,
Vol. 3, No. 1, 1979
extracted with Fig_a_.%3. Mouse embryo fibroblasts Triton X-100 and stained bg the indirect immunofluorescence method, using monospecific antibediee againat bovine brain tubalin. Bars denote 20 j&m. Figs. l-2. Cells incubated at O°C for 5 bra and then extracted at the same temperature. Note the network of cold-stable microtubules. Fig. 3. Cell grown and extracted at 37OC. Fig. 4. Electron microscopy of cooled cell after Triton extraction. Microtubule (M) and microfilament bundle (B) are seen. Bardenetes0.5 JUR.
48
Cell Biology
International
Reports,
Vol. 3, No. I, 1979
incubated at 37W. dlevertheless, these fibrils were true microtubules. This statement is based on the folwere seen, when an antilowing evidence. 1) No fibrils tubulia antibody preincubated with an excess of tubalin was used for the staining. 2) Cells grown in the pr+ sence of 10 JIM vinblastine or colchicine, then incubated at O°C for 5 hrs and extracted with Triton X-l00 as described above did not contain any fibrils. 3) Electron microscopy revealed typical microtubules with 25 nm external diameter in the cooled cells extracted with Triton (Fig. 4). The network of wavy-form microtubules was also seen in the lamellar parts of some non-extracted cooled cells after immunofluorescent staining. Therefore, the cooled cells contained the network of microtubules before the extraction procedure. However, the visualization and especially the photographic documentation of microtubules in the cooled non-extracted cells was greatly complicated by the presence of a highly fluorescent background in these cells. This background, which most probably arosed due to the increased concentration of the depolymerioed tubulin in the cell after cooling, was completely eliminated in the course of extraction. Hence, the cold-treated cells contain a system of microtubules. These microtubules differ from ordinary cytoplasmic microtubules in their stability at O°C. It is, therefore, interesting to study the sensitivity of the cold-stable microtubules to the microtubule-destroying treatment other than cold. 2 mM CaC12 in buffer M (which is equivalent to about 0.9 Iplla of free Ca++) caused the complete dissolution of cold-stable microtubules during 10 min incubation both at OoC and at 37OC, Therefore the cold-stable microtubules as well as the ordinary microtubules are sensitive to Ca++. We have shown earlier that the ordinary microtubules in the cell models were gradually dissolved during prolonged incubation in a lycerol-free buffer (Be% shadsky et al., 1978). Sim&arly, the cold-stable microtubules were dissolved when models of cooled cells were incubated in the buffer M at 37W for 5 hrs. Addition of 4 M glycerol prevented this depolymerization, as well as depolymerization of the usual cytoplasmic microtubules. On the other hand, !SlrMncubation of cooled and then Triton-extracted cells in the buffer 111 without glycerol at OOC did not produce any changes in the distribution and morphology of the cold-stable microtubules. Therefore, the cold-stable microtubules are not in equilibrium with the depolgmerized tubulin at OOC, although at 37W these microtubules are in equilibrium with the tubulin ~001.
Cell Biology
International Reports, vol. 3, No. 1, 1979
49
Several results suggest that the cold-stable microtubules do not exist in the cells before coolhg. Indeed, we have shown that the microtubule system Of cells incubated and extracted at 37’C is cold-sensitive (Bershadsky et al., 1978). This system was destroyed after 30 min incubation at O°C, although some fragments of the microtubules were seen in the cell models after the models of non-cooled cells and, cooling. Therefore, non-cooled cells before extraction contamost probably, ined few if any cold-stable microtubules before cold treatment. Possibly, these microtubules were formed as a result of the rearrangement of the ordinary cytoplasmic microtubules during cold treatment. There are some biochemical and cytological data in the literature regarding microtubules insensitive to cold. It has been shown by Grisham and Wilson (1975) that a certain microtubule assembly did occur at @C in vitro. This process could be induced in the cytosol frqqtion of the brain homogenate by the addition of GTP, and ETA. & The cold-stability of the "9+2*' microtubules of cilia and flagella is well documented (see, for example Behnke and Forer, 1967). Brinkley and Cartwright (1975) described cold-stable microtubules in the mitotic spindle of cultured cells. Tilney and Porter (1967) in their investigation of Helioeoa microtubules found so the called 'macrotubules" 34 main diameter which replaced the axopodial microtubules in cold-treated cells. The present communication is to our knowlege the first one which shows that morphologically normal microtubules exist in the cytoplasm of cooled interphase cells. The occurence of cold-st@ble cytoplasmic microtubules in cells other than mouse embryo fibroblasts, as well as the possible biological role of these microtubules requires further investigation. Acknowledaements. The authors would like to express their thanks to Professors A. S. Spirin and J. Y. Vasiliev for their support throughout this work. We are also very grateful to Mrs. G. A. Kozlov for checking the English vergion of the manuscript. Eeferences. Behnke, 0. and Forer, A. (1967) Evidence for four classes of microtubules in individual cells. Journal of Cell Science, 2, 169-192. Bershadskg, A.D., Gelfand V.I., Svitkina, T.M., and Tint, I.S. (1978) Mi crotubules in mouse embryo fibroblasts extracted with Triton X-100. Cell
50
Ceil Biology
International
Reports,
Vol. 3, NO. I, 1979
Biology International Reports, In press. Brinkley, B.R. and Cartwright J. (1975) Cold-labile and cold-stable microt&les in the mitotic spindle of mammalian cells. In: The Biology of Cytoplasmic Microtubules (D. Soifer, ed.). Annals of the New York Academy of Sciences, a, 428-439. Puller G.Y., Brinkley, B.R., and Boughter, J.M. (1975) f mmunofluorescence of mitotic spindles by using monospecific antibody against bovine brain tubulin. Science, 187, 948-950. Grisham, L.M. and Wilson, L. (1975) Evidence for subclasses of microtubules in the vertebrate central nervous system. Journal of Cell Biology, g, 14681 Osborn, M. and Weber, K. (1977) The display of microtubules in transformed cells. Cell, l2, 561-571 Small, J.V. and Cells, J.E. (1978) Filament arrangement in negatively stained cultured cells: the organization of actin, Cytobiologie, l6, 308-325 Spurr, A.R. (1969) A low viscosity epoxy resin embedding medium for electron microscopy. Journal of Bltrastructure Research, 26, 3143 Tilney, L.G. and Porter, K.R. (1967) Studies on the microtubules in Heliozoa. II. The effect of low temperature on these structures in the formation and maintenance of the axopodia. Journal of Cell Biolotm 2, 327-343.
Received:
7th
August
1978
Accepted:
21st
August
1978
