EXPERIMENTAL
CELL
RESEARCH
195,
269-273 (1991)
SHORT NOTE Disruption of Cytoplasmic Microtubules by Ultraviolet Radiation GLENB.ZAMANSKY,' Department
of Microbiology,
Boston
University
BRIANA.PERRINO, School
of Mrdicuw,
Ultraviolet (UV) irradiation of cultured human skin fibroblasts causes the disassembly of their microtubules. Using indirect immunofluorescence microscopy, we have now investigated whether damage to the microtubule precursor pool may contribute to the disruption of microtubules. Exposure to polychromatic UV radiation inhibits the reassembly of microtubules during cellular recovery from cold treatment. In addition, the ability of taxol to promote microtubule polymerization and bundling is inhibited in UV-irradiated cells. However, UV irradiation of taxol-pretreated cells or in situ detergent-extracted microtubules fails to disrupt the microtubule network. These data suggest that damage to dimeric tubulin, or another soluble factor(s) required for polymerization, contributes to the disassembly of microtubules in UV-irradiated human skin fibroblasts. c 1991 Academic Press. Inc.
ANDIIH-NANCHOU
X0 East Concord
Street,
Boston,
Massachusetts
02118
25 nm. They are assembled from cytoplasmic pools of tubulin dimers, consisting of partially homologous cy and p polypeptides and various microtubule-associated proteins (MAPS) [3, 71. MAPS appear to participate in the regulation of microtubule assembly and function. Polymerization of tubulin dimers into microtubules during interphase is initiated at microtubule organizing centers located within centrosomes and proceeds with microtubules growing out to the periphery of the cytoplasm [S]. We have previously demonstrated that exposure to sun lamp (290-400 nm) or polychromatic UV-A (320-400 nm) radiation causes the disassembly of microtubule networks in cultured human skin fibroblasts [ 11. Using indirect immunofluorescence microscopy, we have now investigated whether damage to the microtubule precursor pool may contribute to the disruption of microtubules. MATERIAL
AND METHODS
INTRODUCTION The extent to which ultraviolet (UV) irradiation alters non-DNA components of cells has received little attention. Because the cytoskeleton plays an important role in the regulation of several essential cellular functions, we have initiated investigations into the effects of UV radiation on the cytoskeleton of human cells [I, 21. The cytoskeleton, composed primarily of microtubules, microfilaments, and intermediate filaments, is an intricate network of structures which extend from the nucleus to the plasma membrane of eukaryotic cells. The cytoskeleton appears to serve as a critical means of transmitting external signals to the nucleus and participates in the regulation of cell growth, shape, and motility; the spatial arrangement of organelles; and secretory processes [3-61. It is therefore reasonable to expect that the UV-induced disruption of the normal assemblage of the cytoskeleton may have serious functional consequences. Microtubules, composed primarily of tubulin, are hollow structures with an outer diameter of approximately ’ To whom dressed.
correspondence
and
reprint
requests
should
he ad-
(‘ells. AG1522, a normal diploid human skin hbroblast cell strain, was grown as previously described [l]. UV irradiation o/ cells. Throughout this study Westinghouse FS40 sun lamps were used as our source of uv radiation. These lamps transmit approximately equal amounts of UV-B (290-320 nm) and IJV-A (320-400 nm) radiation through plastic culture dish covers with a peak emission in the LTV-B regLn between 310 and 315 nm. UV doses, determined as described [l], reflect total IJV levels transmitted by the lamps through the plastic covers. Approximately 18 h after plating AGE22 cells onto glass coverslips in :&mm culture dishes, cultures were rinsed with Hanks’ balanced salt solution containing 15 mM Hepes (HBSS) and UV irradiated in the presence of HBSS. Cultures exposed to taxol were incubated in medium containing 0.5-5.0 PM taxol for 6 h either before or after LTV irradiation. A stock solution of 10 mM taxol was prepared in dimethyl sulfoxide and diluted to final concentrations in culture medium. There was no effect on the organization of microtubules in control cultures exposed to similarly diluted dimethyl sulfoxide in the absence of taxol. Cell fixation and cytoskeleton staining. Cultures were fixed and prepared for microtubule staining as described by Zamansky and Chou [l]. Fixed cytoskeletons were incubated with rabbit anti-tubulin antibodies to label the microtubules. Coverslips were then washed with PBS and treated with goat anti-rabbit antibodies conjugated to rhodamine. Coverslips were mounted on glass slides prior to examination using a Nikon epifluorescence microscope. Reassembly of microtubules in uiuo. Cultures were plated as described above, rinsed with cold HBSS, and incubated in HBSS on an
269 All
Gpyright mc- 1991 rights of reproduction
0014-4827/91 $3.00 by Academic Press, Inc. in any form reserved.
SHORT ice-water bath at O-4’C for 15 min to induce microtubule disassembly. Cultures were then UV- or mock-irradiated on an ice-water bath prior to incubation in medium at 37’C for 0, 15,30, or 60 mip or for 5 h. At the appropriate time, cells were fixed and microtubules were stained as described above. IQ’ irradiation of microtubule networks prepared by detergent extraction. Cells were rinsed with M-PMBG, a cytoskeleton stabilizing buffer containing 50 mM PIPES, 3 mM MgCl,, 2 M glycerol, 10 mM EGTA (pH 6.9) and were then extracted with 0.1% NP40 in M-PMBG containing 3% polyethylene glycol (PEG, 15-20,000). Following extraction cells were UV- or mock-irradiated in M-PMBG containing 3% PEG. Following irradiation cytoskeletons were fixed in methanol at -20°C for 5 min and stained as described above.
RESULTS
The effect of UV irradiation on the repolymerization of microtubules from cytoplasmic pools of tubulin dimers is shown in Fig. 1. The microtubules of untreated AG1522 cells appear to emanate from a densely stained, perinuclear microtubule organizing center and extend to the periphery of the cytoplasm. Incubation of AG1522 cells at 4°C for 15 min results in the nearly complete depolymerization of microtubules into dimeric tubulin subunits. Repolymerization proceeds very quickly in mock-irradiated cells incubated at 37°C. Within 15 min a thin network of microtubules is observed, nucleation having occurred at a microtubule organizing center and a few filaments having grown to the cell periphery. The network becomes more dense with time and repolymerization appears to be complete within 60 min. Although UV-irradiated (7.5 kj/m’) cells appear to have a functional organizing center, a slower repolymerization process is readily apparent within the first 15-30 min postirradiation. Much like the previously observed disassembly of microtubules in UV-irradiated AG1522 cells [1], the extent of the inhibition of polymerization is dose dependent and varies from cell to cell (data not shown). Repolymerization remains incomplete 1 h postirradiation, many cells having a tangled mass of microtubules in the perinuclear region and only a thin network extending to the cell periphery. Similar results are observed at 5 h. An inhibition of microtubule reassembly also occurs after exposure to polychromatic UV-A radiation (data not shown). By exposing AG1522 cells to taxol, we have investigated the effect of UV irradiation on cells treated under conditions which promote the assembly of microtubules. As seen in Fig. 2, taxol induces a dose-dependent reorganization of microtubules in AG1522 cells. Exposure to 0.5 PALM taxol for 5 h results in a microtubule network in which individual microtubules appear to be distributed in a haphazard fashion, no longer being as-
FIG. 1. Repolymerization of microtuhules from cytoplasmic Microtubules in cells maintained at 37°C (A) or in cells incubated 37°C for 15 min (C), 30 min (E), 60 min (G) or 5 h (I). Microtubules (F), 60 min (H), or 5 h (3). Bar represents 15 pm.
271
NOTE
sociated with a microtubule organizing center. In cells exposed to 5.0 p*M taxol, we observe the bundling of short microtubules, a characteristic feature of taxoltreated cells [9, lo]. UV irradiation of taxol-pretreated cells fails to damage the reorganized microtubule network. However, UV irradiation prior to exposure to 5 pM taxol inhibits the ability of the drug to promote microtubule polymerization and bundling (Fig. 2F). Similar results were obtained with 10 pM taxol (not shown). Microtubule bundling occurs normally in mock-irradiated cells. As seen in Fig. 3, the in situ detergent extracted microtubule network of AG1522 closely resembles that observed in unirradiated control cells (Fig. 1A). However, unlike when we irradiate intact cells [ 11, we have found that exposure to UV doses as high as 15 kj/m’ fails to cause the depolymerization of detergent-extracted microtubules (Fig. 3B). DISCUSSION
Cytoplasmic microtubules exist in a dynamic state of equilibrium in which individual microtubules are either growing or rapidly shortening [21-241. Most interphase microtubules turnover with a half life of approximately 10 min. The maintenance of an intact microtubule network is therefore dependent upon a pool of tubulin dimers which are structurally competent to polymerize. In the current study, we have found that exposure to polychromatic UV radiation inhibits the reassembly of microtubules from a pool of tubulin dimers in uiuo. In addition, we have shown that the ability of taxol to promote polymerization of tubulin is greatly inhibited in cells that have been UV irradiated. On the other hand, UV irradiation of taxol-pretreated cells or detergentextracted microtubules fails to disrupt the microtubule network. Taxol appears to lower the critical concentration of tubulin required for polymerization irz vitro and in uiuo [9, 111. DeBrabander et al. [9] have shown that taxol induces the assembly of microtubules and bundles of short microtubules which are dispersed in a disorganized manner in the cytoplasm of interphase cells. These microtubules do not require a microtubule organizing center for assembly and exhibit an increased stability to exposure to cold temperature, colcemid, and nocodazole (9, 10, 121. We have found that microtubules in unirradiated AG1522 cells respond to taxol much like that of other cell types (Fig. 2). However, unlike the taxol-induced polymerization of centrosome-free microtubules and microtubule bundles in cells exposed to colcemid or
pools of tubulin dimers in AC1522 cells preincubated at 4°C for 15 min (B). Microtubules in mock-irradiated in UV-irradiated (7.5 kj/m’) cells incubated at 37°C
at 4°C for 15 min. cells reincubated at for 15 min (D), 30 min
272
SHORT
NOTE
FIG. 2. The effect oftaxol on the microtubule network in control and UV-irradiated (10 kj/m’) cells. (A) 0.5 @4taxol prior to mock-irradiation: (R) 0.5 PM taxol prior to LTV irradiation; (C) 5.0 PM taxol prior to mock-irradiation; (D) 5.0 PM taxol prior to LJV irradiation; (E) mock-irradiation prior to incubation with 5.0 &‘taxol; (F) IJV irradiation prior to incubation with 5 &f taxol. All taxol exposures were for 5 h.
nocodazole [9,13], the disruption of microtubules by UV irradiation appears to result in an altered precursor pool which can no longer be utilized for polymerization even in the presence of taxol. Although these results do not eliminate the possibility that nontubulin components of microtubules are also affected, it is important to note that the inclusion of taxol during the in vitro polymerization of tubulin permits the assembly of microtubules in the absence of MAPS and exogenous GTP or at low temperatures [ll, 141. The detergent extraction of living cells under appropriate buffer conditions results in the solubilization of plasma membrane lipids, the removal of tubulin and other soluble proteins, and the retention of an intact cytoskeleton [l&18]. Such protocols permit the in vitro study of the disassembly of microtubules which have
been polymerized in viva. In the current study, AG1522 cells were extracted in the presence of polyethylene glyco1 in order to maximize the retention of the normal complement of MAPS [ 171. However, maintenance of a stable microtubule network in mock-irradiated cultures, using a variety of extraction protocols, required the presence of glycerol. The reason for this glycerol requirement in human skin fibroblasts, but not in other cells [15-181, remains unclear. It has been shown that glycerol enhances the assembly and stability of microtubules in vitro [19,201. Despite the presence of glycerol in our extraction and irradiation buffers, the extracted microtubules were depolymerized upon exposure to Ca2+ at, concentrations greater than 100 pM or to cold temperatures, though a population of cold-resistant microtubules was observed in some extracted cells (data
273
not shown). This is in agreement with others using similar extraction protocols [17,18]. Nevertheless, exposure of extracted microtubules to an extremely high UV dose (15 kj/m’) failed to disrupt the microtubule network. Thus, taken together, the data in the current study suggest that damage to dimeric tubulin, or another soluble factor(s) required for polymerization, contributes to the disassembly of microtubules in UV irradiated human skin fibroblasts. This research was supported National Institute of Arthritis eases and by an award from cine B.IJ. Medical Research vided by the Drug Synthesis Cancer Treatment, National
in part by Grant AR38900 from the and Musculoskeletal and Skin Dis the Boston University School of MediSupport Grant Award. Taxol was proand Development Branch, Division 01 Cancer Institute.
7.
Olmsted,
j. B. (1986)
8.
Brinkley,
B. R. (1985)
9.
DeBrabander, M., (ieuens, G., Nuydens, R., Willehrords, R., and DeMey, J. (1981) Proc. Natl. Acad. Sci. USA 78, 5608-5612.
10. 11. 12.
Zamansky,
G. B. and Chou.
2.
Zamansky,
3.
Alherts, B., Bray, B., Lewis, son, J. D. (1983) in Molecular Garland Press, New York.
I. N. (1987)
J. Inucsf.
L)ermatol.
89,
603-606. G. B., and Chou,
1. N. (1990)
Photochcm.
Photobiol.
52,903-906.
4.
J., Rati, M., Roberts, K., and WatBiology of the Cell, pp. 549-609.
Penman, S., Fulton, A., Capco, D., Ben Ze’ev, A., Wittelshurger, S., and Tse, C. F. (1981) Cold Spring Harbor Symp. Quant. Viol. 46, 10131028.
$5,. Otto, A. M. (1982) Cell Biol. Int. Rep. 6, l-18. 6. Goldman, R. D., Goldman, A. E., Green, K. J., Jones, J. C. It., Jones, S. M.. and Yang, H. Y. (1986) J. (I&. Sci. Suppl. 5,69-97. Received
January
8, 1991
Annu.
SchifJ, P. B., and Horwitz, USA 77, 1561-1565. Schiff, P. B., and Horwitz, :i252. Crossin. K. L.. and Carney,
Reu. Cell Riol. 2, 421-4.57. &[I.
S. B. (1980)
Proc.
S. B. (1981)
Natl.
Acad.
Lliochcmistq
P. H. (1981)
20,
Cell 27,
Brenner, S. L., and Brinkley, B. R. (1981) Symp. Quant. Biol. 46, 241-254.
14.
Thompson,
W. C., Wilson,
1, 145172.
Cell Biol.
13.
L., and Purich,
Sci. 3247-
341-350.
Cold Spring D. I,. (1981)
Harbor Cell Mo-
til. 1, 445-454. 15.
Hershadsky, A. D., and Gelfand, SC;. (:SA 78, 361OX~613.
16.
Deery, W. ,J., Means, Rid. 98, 904-910.
A. R., and Brinkley,
17.
Schliwa, M., Euteneur, Proc. Natl. Acad. Sri.
IJ., Bulinski, J. C., and Izant, (iSA 78, 103771041.
REFERENCES 1.
Annu.
18.
Perrino,
B. A., and
Chou.
V. 1. (1981)
I. N. (1986)
Proc. Natl. B. R. (1984)
Cell
Acad. J. CclZ
J. (1981)
Riol. fnt.
Rep.
10,
,565-57'i. 1 t 1 19. 20. 21.
Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973) Acad. Ski. 1fSA 70, 765-768. Nishida, E. (1978) J. Rio&em. 84, 5077512.
22.
Schulze, 103 1. Schulze,
23.
Sammak,
E., and Kirschner, E., and Kirschner, P. J., and Borisy,
M. (1986) M. (1987) G. (1988)
J. Cell Viol. J. Cell Biol. Nature
24. Cassimeris, L., Pryer, N. K., and Salmon, Hid. 107 , 222%2'131. .-
332,
Proc. N&l.
102, 1020-
104,277-288. 724-726.
E. D. (1988)
J. c’c~ll
CELL
RESEARCH
195,
269-273 (1991)
SHORT NOTE Disruption of Cytoplasmic Microtubules by Ultraviolet Radiation GLENB.ZAMANSKY,' Department
of Microbiology,
Boston
University
BRIANA.PERRINO, School
of Mrdicuw,
Ultraviolet (UV) irradiation of cultured human skin fibroblasts causes the disassembly of their microtubules. Using indirect immunofluorescence microscopy, we have now investigated whether damage to the microtubule precursor pool may contribute to the disruption of microtubules. Exposure to polychromatic UV radiation inhibits the reassembly of microtubules during cellular recovery from cold treatment. In addition, the ability of taxol to promote microtubule polymerization and bundling is inhibited in UV-irradiated cells. However, UV irradiation of taxol-pretreated cells or in situ detergent-extracted microtubules fails to disrupt the microtubule network. These data suggest that damage to dimeric tubulin, or another soluble factor(s) required for polymerization, contributes to the disassembly of microtubules in UV-irradiated human skin fibroblasts. c 1991 Academic Press. Inc.
ANDIIH-NANCHOU
X0 East Concord
Street,
Boston,
Massachusetts
02118
25 nm. They are assembled from cytoplasmic pools of tubulin dimers, consisting of partially homologous cy and p polypeptides and various microtubule-associated proteins (MAPS) [3, 71. MAPS appear to participate in the regulation of microtubule assembly and function. Polymerization of tubulin dimers into microtubules during interphase is initiated at microtubule organizing centers located within centrosomes and proceeds with microtubules growing out to the periphery of the cytoplasm [S]. We have previously demonstrated that exposure to sun lamp (290-400 nm) or polychromatic UV-A (320-400 nm) radiation causes the disassembly of microtubule networks in cultured human skin fibroblasts [ 11. Using indirect immunofluorescence microscopy, we have now investigated whether damage to the microtubule precursor pool may contribute to the disruption of microtubules. MATERIAL
AND METHODS
INTRODUCTION The extent to which ultraviolet (UV) irradiation alters non-DNA components of cells has received little attention. Because the cytoskeleton plays an important role in the regulation of several essential cellular functions, we have initiated investigations into the effects of UV radiation on the cytoskeleton of human cells [I, 21. The cytoskeleton, composed primarily of microtubules, microfilaments, and intermediate filaments, is an intricate network of structures which extend from the nucleus to the plasma membrane of eukaryotic cells. The cytoskeleton appears to serve as a critical means of transmitting external signals to the nucleus and participates in the regulation of cell growth, shape, and motility; the spatial arrangement of organelles; and secretory processes [3-61. It is therefore reasonable to expect that the UV-induced disruption of the normal assemblage of the cytoskeleton may have serious functional consequences. Microtubules, composed primarily of tubulin, are hollow structures with an outer diameter of approximately ’ To whom dressed.
correspondence
and
reprint
requests
should
he ad-
(‘ells. AG1522, a normal diploid human skin hbroblast cell strain, was grown as previously described [l]. UV irradiation o/ cells. Throughout this study Westinghouse FS40 sun lamps were used as our source of uv radiation. These lamps transmit approximately equal amounts of UV-B (290-320 nm) and IJV-A (320-400 nm) radiation through plastic culture dish covers with a peak emission in the LTV-B regLn between 310 and 315 nm. UV doses, determined as described [l], reflect total IJV levels transmitted by the lamps through the plastic covers. Approximately 18 h after plating AGE22 cells onto glass coverslips in :&mm culture dishes, cultures were rinsed with Hanks’ balanced salt solution containing 15 mM Hepes (HBSS) and UV irradiated in the presence of HBSS. Cultures exposed to taxol were incubated in medium containing 0.5-5.0 PM taxol for 6 h either before or after LTV irradiation. A stock solution of 10 mM taxol was prepared in dimethyl sulfoxide and diluted to final concentrations in culture medium. There was no effect on the organization of microtubules in control cultures exposed to similarly diluted dimethyl sulfoxide in the absence of taxol. Cell fixation and cytoskeleton staining. Cultures were fixed and prepared for microtubule staining as described by Zamansky and Chou [l]. Fixed cytoskeletons were incubated with rabbit anti-tubulin antibodies to label the microtubules. Coverslips were then washed with PBS and treated with goat anti-rabbit antibodies conjugated to rhodamine. Coverslips were mounted on glass slides prior to examination using a Nikon epifluorescence microscope. Reassembly of microtubules in uiuo. Cultures were plated as described above, rinsed with cold HBSS, and incubated in HBSS on an
269 All
Gpyright mc- 1991 rights of reproduction
0014-4827/91 $3.00 by Academic Press, Inc. in any form reserved.
SHORT ice-water bath at O-4’C for 15 min to induce microtubule disassembly. Cultures were then UV- or mock-irradiated on an ice-water bath prior to incubation in medium at 37’C for 0, 15,30, or 60 mip or for 5 h. At the appropriate time, cells were fixed and microtubules were stained as described above. IQ’ irradiation of microtubule networks prepared by detergent extraction. Cells were rinsed with M-PMBG, a cytoskeleton stabilizing buffer containing 50 mM PIPES, 3 mM MgCl,, 2 M glycerol, 10 mM EGTA (pH 6.9) and were then extracted with 0.1% NP40 in M-PMBG containing 3% polyethylene glycol (PEG, 15-20,000). Following extraction cells were UV- or mock-irradiated in M-PMBG containing 3% PEG. Following irradiation cytoskeletons were fixed in methanol at -20°C for 5 min and stained as described above.
RESULTS
The effect of UV irradiation on the repolymerization of microtubules from cytoplasmic pools of tubulin dimers is shown in Fig. 1. The microtubules of untreated AG1522 cells appear to emanate from a densely stained, perinuclear microtubule organizing center and extend to the periphery of the cytoplasm. Incubation of AG1522 cells at 4°C for 15 min results in the nearly complete depolymerization of microtubules into dimeric tubulin subunits. Repolymerization proceeds very quickly in mock-irradiated cells incubated at 37°C. Within 15 min a thin network of microtubules is observed, nucleation having occurred at a microtubule organizing center and a few filaments having grown to the cell periphery. The network becomes more dense with time and repolymerization appears to be complete within 60 min. Although UV-irradiated (7.5 kj/m’) cells appear to have a functional organizing center, a slower repolymerization process is readily apparent within the first 15-30 min postirradiation. Much like the previously observed disassembly of microtubules in UV-irradiated AG1522 cells [1], the extent of the inhibition of polymerization is dose dependent and varies from cell to cell (data not shown). Repolymerization remains incomplete 1 h postirradiation, many cells having a tangled mass of microtubules in the perinuclear region and only a thin network extending to the cell periphery. Similar results are observed at 5 h. An inhibition of microtubule reassembly also occurs after exposure to polychromatic UV-A radiation (data not shown). By exposing AG1522 cells to taxol, we have investigated the effect of UV irradiation on cells treated under conditions which promote the assembly of microtubules. As seen in Fig. 2, taxol induces a dose-dependent reorganization of microtubules in AG1522 cells. Exposure to 0.5 PALM taxol for 5 h results in a microtubule network in which individual microtubules appear to be distributed in a haphazard fashion, no longer being as-
FIG. 1. Repolymerization of microtuhules from cytoplasmic Microtubules in cells maintained at 37°C (A) or in cells incubated 37°C for 15 min (C), 30 min (E), 60 min (G) or 5 h (I). Microtubules (F), 60 min (H), or 5 h (3). Bar represents 15 pm.
271
NOTE
sociated with a microtubule organizing center. In cells exposed to 5.0 p*M taxol, we observe the bundling of short microtubules, a characteristic feature of taxoltreated cells [9, lo]. UV irradiation of taxol-pretreated cells fails to damage the reorganized microtubule network. However, UV irradiation prior to exposure to 5 pM taxol inhibits the ability of the drug to promote microtubule polymerization and bundling (Fig. 2F). Similar results were obtained with 10 pM taxol (not shown). Microtubule bundling occurs normally in mock-irradiated cells. As seen in Fig. 3, the in situ detergent extracted microtubule network of AG1522 closely resembles that observed in unirradiated control cells (Fig. 1A). However, unlike when we irradiate intact cells [ 11, we have found that exposure to UV doses as high as 15 kj/m’ fails to cause the depolymerization of detergent-extracted microtubules (Fig. 3B). DISCUSSION
Cytoplasmic microtubules exist in a dynamic state of equilibrium in which individual microtubules are either growing or rapidly shortening [21-241. Most interphase microtubules turnover with a half life of approximately 10 min. The maintenance of an intact microtubule network is therefore dependent upon a pool of tubulin dimers which are structurally competent to polymerize. In the current study, we have found that exposure to polychromatic UV radiation inhibits the reassembly of microtubules from a pool of tubulin dimers in uiuo. In addition, we have shown that the ability of taxol to promote polymerization of tubulin is greatly inhibited in cells that have been UV irradiated. On the other hand, UV irradiation of taxol-pretreated cells or detergentextracted microtubules fails to disrupt the microtubule network. Taxol appears to lower the critical concentration of tubulin required for polymerization irz vitro and in uiuo [9, 111. DeBrabander et al. [9] have shown that taxol induces the assembly of microtubules and bundles of short microtubules which are dispersed in a disorganized manner in the cytoplasm of interphase cells. These microtubules do not require a microtubule organizing center for assembly and exhibit an increased stability to exposure to cold temperature, colcemid, and nocodazole (9, 10, 121. We have found that microtubules in unirradiated AG1522 cells respond to taxol much like that of other cell types (Fig. 2). However, unlike the taxol-induced polymerization of centrosome-free microtubules and microtubule bundles in cells exposed to colcemid or
pools of tubulin dimers in AC1522 cells preincubated at 4°C for 15 min (B). Microtubules in mock-irradiated in UV-irradiated (7.5 kj/m’) cells incubated at 37°C
at 4°C for 15 min. cells reincubated at for 15 min (D), 30 min
272
SHORT
NOTE
FIG. 2. The effect oftaxol on the microtubule network in control and UV-irradiated (10 kj/m’) cells. (A) 0.5 @4taxol prior to mock-irradiation: (R) 0.5 PM taxol prior to LTV irradiation; (C) 5.0 PM taxol prior to mock-irradiation; (D) 5.0 PM taxol prior to LJV irradiation; (E) mock-irradiation prior to incubation with 5.0 &‘taxol; (F) IJV irradiation prior to incubation with 5 &f taxol. All taxol exposures were for 5 h.
nocodazole [9,13], the disruption of microtubules by UV irradiation appears to result in an altered precursor pool which can no longer be utilized for polymerization even in the presence of taxol. Although these results do not eliminate the possibility that nontubulin components of microtubules are also affected, it is important to note that the inclusion of taxol during the in vitro polymerization of tubulin permits the assembly of microtubules in the absence of MAPS and exogenous GTP or at low temperatures [ll, 141. The detergent extraction of living cells under appropriate buffer conditions results in the solubilization of plasma membrane lipids, the removal of tubulin and other soluble proteins, and the retention of an intact cytoskeleton [l&18]. Such protocols permit the in vitro study of the disassembly of microtubules which have
been polymerized in viva. In the current study, AG1522 cells were extracted in the presence of polyethylene glyco1 in order to maximize the retention of the normal complement of MAPS [ 171. However, maintenance of a stable microtubule network in mock-irradiated cultures, using a variety of extraction protocols, required the presence of glycerol. The reason for this glycerol requirement in human skin fibroblasts, but not in other cells [15-181, remains unclear. It has been shown that glycerol enhances the assembly and stability of microtubules in vitro [19,201. Despite the presence of glycerol in our extraction and irradiation buffers, the extracted microtubules were depolymerized upon exposure to Ca2+ at, concentrations greater than 100 pM or to cold temperatures, though a population of cold-resistant microtubules was observed in some extracted cells (data
273
not shown). This is in agreement with others using similar extraction protocols [17,18]. Nevertheless, exposure of extracted microtubules to an extremely high UV dose (15 kj/m’) failed to disrupt the microtubule network. Thus, taken together, the data in the current study suggest that damage to dimeric tubulin, or another soluble factor(s) required for polymerization, contributes to the disassembly of microtubules in UV irradiated human skin fibroblasts. This research was supported National Institute of Arthritis eases and by an award from cine B.IJ. Medical Research vided by the Drug Synthesis Cancer Treatment, National
in part by Grant AR38900 from the and Musculoskeletal and Skin Dis the Boston University School of MediSupport Grant Award. Taxol was proand Development Branch, Division 01 Cancer Institute.
7.
Olmsted,
j. B. (1986)
8.
Brinkley,
B. R. (1985)
9.
DeBrabander, M., (ieuens, G., Nuydens, R., Willehrords, R., and DeMey, J. (1981) Proc. Natl. Acad. Sci. USA 78, 5608-5612.
10. 11. 12.
Zamansky,
G. B. and Chou.
2.
Zamansky,
3.
Alherts, B., Bray, B., Lewis, son, J. D. (1983) in Molecular Garland Press, New York.
I. N. (1987)
J. Inucsf.
L)ermatol.
89,
603-606. G. B., and Chou,
1. N. (1990)
Photochcm.
Photobiol.
52,903-906.
4.
J., Rati, M., Roberts, K., and WatBiology of the Cell, pp. 549-609.
Penman, S., Fulton, A., Capco, D., Ben Ze’ev, A., Wittelshurger, S., and Tse, C. F. (1981) Cold Spring Harbor Symp. Quant. Viol. 46, 10131028.
$5,. Otto, A. M. (1982) Cell Biol. Int. Rep. 6, l-18. 6. Goldman, R. D., Goldman, A. E., Green, K. J., Jones, J. C. It., Jones, S. M.. and Yang, H. Y. (1986) J. (I&. Sci. Suppl. 5,69-97. Received
January
8, 1991
Annu.
SchifJ, P. B., and Horwitz, USA 77, 1561-1565. Schiff, P. B., and Horwitz, :i252. Crossin. K. L.. and Carney,
Reu. Cell Riol. 2, 421-4.57. &[I.
S. B. (1980)
Proc.
S. B. (1981)
Natl.
Acad.
Lliochcmistq
P. H. (1981)
20,
Cell 27,
Brenner, S. L., and Brinkley, B. R. (1981) Symp. Quant. Biol. 46, 241-254.
14.
Thompson,
W. C., Wilson,
1, 145172.
Cell Biol.
13.
L., and Purich,
Sci. 3247-
341-350.
Cold Spring D. I,. (1981)
Harbor Cell Mo-
til. 1, 445-454. 15.
Hershadsky, A. D., and Gelfand, SC;. (:SA 78, 361OX~613.
16.
Deery, W. ,J., Means, Rid. 98, 904-910.
A. R., and Brinkley,
17.
Schliwa, M., Euteneur, Proc. Natl. Acad. Sri.
IJ., Bulinski, J. C., and Izant, (iSA 78, 103771041.
REFERENCES 1.
Annu.
18.
Perrino,
B. A., and
Chou.
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