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Cytoplasmic microtubule assembly is altered in cytoplasts
Cell Biology lnternational Reports, Vol. 16, No. 3, 1992
CYTOPLASMIC
•ICROTUBULE ASSEMBLY CYTOPLASTS.
SUJATA GUHA
269
IS A L T E R E D
IN
ROY@ and A.N.BHISEY*
,
Cancer R e s e a r c h caInstitute, Tata Memorial C e n t r e , P a r e l , Bombay400 012, INDIA. - P r e s e n t A d d r e s s : National F a c i l i t y f o r Animal T i s s u e and Cell C u l t u r e , Paud Road, Kothrud, Pune-411 029, India. ,
Author f o r c o r r e s p o n d e n c e . ABSTRACT
Nucleation of microtubule (MT) organization of the cytoplasmic microtubule complex (CMTC) from the microtubule organizing centres (MTOC) was studied in enucleated cytoplasts of human diploid fibroblast (MRC-5) and mouse peritoneal macrophages in culture. Cytoplasts of both cell types could not organize th~ complete C M T C . Aberrant M T patterns were seen in M R C - 5 cell~ while mouse macrophages showed occurrence of few short MT. The studies suggest that nucleus may have a role in determining C M T C . INTRODUCTION
The cytoplasmic microtubular complex (CMTC) of tissue culture cells is composed of long filamentous microtubules (MT) radiating from the pericentriolar microtubule organizing centers (MTOC) towards the periphery (Osborn and Weber, 1976). Different cells exhibit different c e l l morphologies. It has been suggested that cell morphologies are inherited from one generation to the next during cell division (Albrecht-Buheler, 1977; Solomon, 1979). However, it is not clear whether this occurs by a predetermined blue print situated in the genome or it is a cytoplasmic event occuring independently of the nucleus. Several reports have shown that the C M T C organization in enucleated cells is comparable to that of the intact cells (Brown et a l . , 1980 ; Caron et a l . , 1985 ; Pittinger and Cleaveland, ]985). In addition, the M T O C have been implicated as the determinants of C M T C and hence of cellular morphology (Solomon ]980, 1981). Since tubulin is capable of self assembly, it is possible that the C M T C is formed purely as a result of M T polymerization in the cytoplasm without any nuclear influence. We report in different cell types enucleation.
this paper, which failed
0309-1651/92/030269-13/$03 00/0
variant observations in two to organize their C M T C after
© 1992 Academic Press Ltd
270
Cell Biology lnternational Reports, VoL 16, No. 3, 1992
MATERIALS AND METHODS Chemicals were obtained from the following sources : Cytochalasin B (CB), ethylene glycol-bis ( -amino-ethyl-ether N N N~-N'-tetra acetic acid (EGTA), fluorescein isothiocyanate (FITC), Piperazine N, N' bis (2 ethane sulphonic acid) (PIPES) and propidium iodide (PI) from Sigma Chemical Co., USA. Fetal calf serum from Sera Labs., UK. Nocodazole was a gift from Janssen Pharmaceutica, Beerse, Belgium. Antibody to carboxymethylated tubulin was a generous gift from Dr. K. Doenges, Institute of Cell Research, German Cancer Centre, Heidelberg. Sheep anti-rabbit IgG was prepared in the laboratory (Guha Roy and Bhisey, 1991). Nocodazole and Cytochalasin B were prepared as 1 mg/ml stock solutions in dimethylsulfoxide (DMSO) and aliquoted. Nocodazole was used at a concentration of 4 uM for 4 hours to depolymerise the C M T C . Human diploid fibroblast cell line (MRC-5) and mouse macrophages were cultured as described earlier (Guha Roy and Bhisey, 1991). Enucleation. The cells were plated on round (24 m m diameter) polylysine coated coverslips (Mazia et al., 1976) and allowed to grow for three to five days until they were semi-confluent. Sorvall SS-34 centrifuge tubes with an araldite base were constructed to support the coverslips during centrifugation. Each coverslip was placed cell side down on P V C rings resting on araldite base and submerged in about 4 ml medium containing l0 ug/ml CB. Cells were spun in Sorvall RC-5B centrifuge prewarmed to 27°C at I0,000 rpm for 15 minutes. The cells were allowed to recover in medium without CB for one hour. The same protocol was used for macrophages except for the inclusion of l ug/ml nocodazole in the enucleation medium since this produced cytoplasts more efficiently. These cells were spun at 7,500 rpm for 30 minutes. Time lapse cinematography. After centrifugation, the coverslips were assembled in Sykes Moore chambers and filmed on a Nikon inverted phase microscope fitted with a Vinten camera at 12 frames per minute (Bhisey and Feed, 1975). Immunofluorescence staining was done on cells grown on coverslips as described earlier (Guha Roy and Bhisey, 1991). RESULTS The C M T C of fibroblasts differs in density as well as in organization.
from that of macrophages The fibroblast C M T C is
Cell Biology International Reports, VoL 16, No. 3, 1992
271
very complex with an overlapping array of M T which are so densely arranged in the central region of the cell that the origin of every M T in the cell cannot be traced to the M T O C (Fig.l). Fibroblasts in which centriolar replication had been completed showed more than one MTOC. In M R C - 5 cells, the number of M T O C varied from one to four per cell. The macrophage C M T C appeared less complex. F e w M T radiated from the M T O C . The sparse nature of the C M T C made it possible to trace most M T to the M T O C (Fig.2). Nocodazole reversibly depolymerized the MT. MRC-5 cells treated with nocodazole showed heterogeneous pattern of C M T C organization : some cells were totally devoid of their C M T C , while others retained a few residual M T (Figs.3 & 4). The macrophage CMTC was uniformly depolymerized by nocodazole (Fig.5). However, when the drug was withdrawn, M T nucleation began at the M T O C and proceeded towards the cell periphery. Within ten minutes of drug withdrawl the entire C M T C in both cell types was reorganized. The role of the nucleus in M T nucleation was studied in enucleated cells. Soon after centrifugation, the morphology of the cytoplasts became aberrant, but they regained their normal shapes within 30 to "60 mins in medium without CB. Microtubule pattern in M R C - 5 cytoplasts. Except for the lack of nucleus, the cytoplasts resembled intact cells in their morphology. Enucleation was confirmed by PI staining. Staining with antitubulin revealed different classes of C M T C among cytoplasts. Counts were taken of the various CMTC patterns observed in the cytoplasts and at the same time of the patterns observed in the intact cells which served as the internal controls as they had undergone the same CB treatment as well as centrifugation. A large number (36.7 + I0.2%) of cytoplasts were found to possess brightly fluorescing M T O C but lacking any form of C M T C (Fig.6). Polymerized M T were evident in about 39% of cytoplasts but not all of these were comparable with the C M T C observed normally in intact cells. The patterns observed were as follows : (a) Cytoplasts with C M T C similar to the normal CMTC of intact cells were seen in 6.42 + 2.2~ of the total (Fig.7). (b) Cytoplasts with fewer polymerized M T which formed aberrant C M T C organizations in the central region of the cell. These M T were in the form of itangles, close to the M T O C , leaving the peripheral cytoplasm free of M T (Fig .8). Their origin from the M T O C was not obvious, such cytoplasts comprised 12.97 + 4.15% of the total. (c) Some cytoplasts did not show
272
Cell Biology International Reports, VoL 16, No. 3, 1992
°~
.
2
7
L
m
5 Fig. I-6 Immunofluorescence of M T stained with anti-tubulin antibody : Fig. 1. CMTC of MRC-5 cells. Fig. 2. Mouse macrophage C M T C with M T O C . Fig.3. M T O C in nocodazole treated M R C - 5 cells : C M T C completely depolymerized. Fig.4. Nocodazole resistant M T in M R C - 5 cells. Fig.5. Total disappearance of C M T C in nocodazole treated macrophages : MTOC with 3-4 short M T seen. Fig. 6. M R C - 5 cytoplasts devoid of M T but with prominent M T O C . Bar width I0 uM.
Cell Biology International Reports, Vol. 16, No. 3, 1992
273
(fJ I0. o o
90 80
I T
70~
60-
"'
50-
~o
40-
'"
30
z~w u 141
20
°
o.
I0
m
CYTOPLASTS
I'~
CELLS
-
-m I NORMAL CMTC (MTOC-I-)
Fig.7. Histogram different types procedure. Cells were analysed for
TANGLED CMTC 1-1 CMTC (MTOC + ) (MTOC +)
showing percentage of CMTC after were stained with v~rious C M T C types
CMTC1-1 (MTOC -)
iL FEW MT (MTOc - )
of cells and cytoplasts with recovery from enucleation antitubulin antibody. Slides and counts were taken.
MTOC, but M T polymerization had occurred in the absence of the M T O C (Fig.9) in 19.6 + 8.45% cytoplasts. Among these, in a few cytoplasts, only a few M T had polymerized. Others had neither an M T O C nor any polymerized M T (24.07 +- 7.2%). Comparing these patterns with the C M T C of intact cells on the same coverslip, the situation was reversed (Fig.7). Most of the cells (74.8 + 2.4%) had a normal C M T C complete with an M T O C (Fig.10). No aberrant tangles of M T were found. About 24% cells were devoid of C M T C although the MTOC fluoresced brightly. There were no cells without MT and M T O C but 1.5 + 0.6% of the total cells counted had a few M T polymerized in the absence of MTOC. Recovery of MRC-5 cytoplasts after nocodazole induced depolymerization. To evaluate if reorganization of C M T C after depolymerization is a nucleus dependent process, the cytoplasts were allowed to recover after enucleation and then treated with 4 uM nocodazole for four hours. This was followed by recovery in medium without nocodazole for 30 minutes. The C M T C of cytoplasts recovered from nocodazole could be classified i n t o four groups : (a) Cytoplasts with no discernible CMTC despite the presence of the M T O C . (b)
274
Cell Biology International Re oorts, Vol. 16, No. 3, 1992
9
12
.~'
~.
, ,,
13
,.
14
Figs. 8-I0 and 12,15,14 Immunofluorescence of M T stained with anti-tubulin antibody. Fig.8. 'Tangled' M T in M R C - 5 cytoplasts. Fig.9. Intact M T in M R C - 5 cytoplast without M T O C . Fig.10. Intact cell with normal C M T C . Fig.12. 'Tangled' M T in M R C - 5 cytoplast recovered after nocodazole treatment. Fig.13. Whorl-like C M T C surrounding M T O C in cytoplast recovered after nocodazole. Fig.14. Macrophage cytoplast showing very few and short MT after recovery from nocodazole. Bar width I0 urn.
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Cytoplasts that possessed an M T O C and had varying degrees of polymerized MT. (c) Cytoplasts that lacked M T and MTOC. (d) Cytoplasts that had polymerized M T inspire of the absence of an M T O C . Most of the cytoplasts (67 -+ 6.83~) had an MTOC, but lacked C M T C organization. Figure II shows the comparison of percentages of cytoplast C M T C and that of cells recovering from nocodazole treatment. Cytoplasts with varying degrees of C M T C organizations formed 29.5 + 6.67% of the total number of cytoplasts. The C M T C varied from a few polymerized M T to a C M T C comparable to that of intact cells.
t- 100 u~ ,¢[ "a. 0 I>o ,,0
90
~
60
_1
~
5o
~
40
"'
30
~'z w o
20
CYTOPLASTS 80 ]
CELLS
70
10
T
w o.
CMT=+ MTO=
Fig.ll. Histogram showing different nocodazole.
(=.T=+) MTOC +
showing patterns
MTOC -
(=.Tc - ) MTO=
--
percentage of cells and cytoplasts of CMTC after recovery from
T w o unusual forms of C M T C that were not encountered in intact cells were observed in such cytoplasts. There were cytoplasts with a tangled C M T C in the central region of the cytoplast along with the M T O C (Fig.12). All M T did not appear to be connected to the M T O C . This form of C M T C had also been observed in cytoplasts not treated with nocodazole. The second ~aberrant' C M T C consisted of M T polymerized in circular whorls around centrally localized M T O C (Fig.13). Most of the M T were independent of MTOC. Cytoplasts that lacked both M T O C and C M T C were 2.97 + 1.86% of the total and 0.78 + 0.08% of the cytoplasts had assembled a few M T in the absence of the MTOC.
Cell Biology lnternational Reports, Vol. 16, No. 3, 1992
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The macrophage cytoplast C M T C . Enucleated macrophages showed normal ruffling and were able to phagocytose heat killed E.coli. Soon after enucleation, the cytoplasts were either arborized or rounded. When placed in medium without CB, the cytoplasts took a longer time to spread than the intact cells. Motility of cytoplasts were studied by time lapse cinematography during and after recovery after enucleation in 9 different sequences involving 19 cytoplasts and 8 intact cells. The cytoplasts exhibited random motility by pseudopod formation and mass flow, similar to that of colchicine of vinblastine treated macrophages as reported earlier (Bhisey and Freed 1971, 1975). The intact cells present in the same microscope field as the cytoplasts showed ruffling and normal gliding type motility. Development of the cytoplast C M T C was analysed as a function of time. Ten minutes after transferring to control medium, few short M T were nucleated both in intact cells as well as in cytoplasts. The number and length of M T in intact cells increased with time, whereas cytoplasts had very few M T upto 60 minutes after drug withdrawl (Fig.14). In about 30% cytoplasts, M T O C could not nucleate M T at all (Table 1). Unlike M R C - 5 cytoplasts, aberrant CMTC patterns were not seen in macrophage cytoplasts. The M T O C that could nucleate a few M T were able to do so only to a limited extent.
Table
I
: Comparison of M T nucleation as a function macrophage cells and cytoplasts.
Time (rains)
~ Whole Cells With M T Without M T
of time in
% Cytoplasts With M T Without M T
20
85.6
14.4
61.5
38.5
30
86.3
13.7
66.6
33.4
45
95.5
4.5
70.0
30.0
60
93.7
6.3
72.4
27.6
DISCUSSION The M T O C have been considered to be the 'preferred site' for M T nucleation (Pickett-Heaps 1969; Brinkley 1985). This has been proved in cells recovering from M T inhibitors where nucleation is prominent at the M T O C (DeBrabender et al., 1980;
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1981) and in isolated centrosomes (Mitchison and Kirschner, 1984). The M T O C have been implicated as the center for storage and recall of information pertaining to cellular morphology (Ng and Frankel, 1977; Solomon 1980, 1981). The number of MT, their length, polarity and the lattice structure of the M T were shown to be determined by the M T O C (Anderson et al., 1982; Brinkley 1985). Although the factors that determine the morphology of eukaryotic cells are not well defined, the M T O C determine the temporal and spatial pattern of M T in the cytoplasm and thereby influence the cell shape. Demonstration of D N A in centrioles (Hall et a l . , 1989) has raised the possibility of genetic autonomy of MTOC. The microenvironment at the M T O C is suitable for M T nucleation probably due to lower critical concentrations. Using lysed cell models, the nucleation of M T from M T O C was found to be influenced by the pH of the medium (Derry and Brinkley, 1983) and the activity of a cyclic nucleotide dependent kinase (Tash et al., 1980). In case of enucleated cells, the factors regulating M T nucleation are similar to those in intact cells. The role of the nucleus, albeit unknown, is bound to be important. Although lysed cell models are used in studying M T assembly, the biochemical properties of cytoplasmic ground substance confer order and organization to the M T array (Tash et al., 1980). Unlike previous reports (Brown et al., 1980; Caron et al., 1985; Karsenti et al., 1980) that cytoplasts and intact cells have similar C M T C organization, the M R C - 5 and macrophage cytoplasts were found to lack such organization. This effect was not due to the death of the cytoplasts since these could spread out normally and a number of them showed ruffling and cell movement. The altered M T nucleation in cytoplasts was not due to the trauma of centrifugation since intact cells on the same coverslip showed normal C M T C organization. Karsenti et al. (1985) have reported the formation of peripheral M T that lie freely in the cytoplasts which lack MTOC. But they found that, in cytoplasts possessing a MTOC, the focussed M T array was always present. This did not occur in M R C - 5 cytoplasts where, inspire of the presence of the MTOC, there was either a complete lack of C M T C or an aberrant C M T C organizations were seen. The tangled M T were not all focussed at the M T O C . These observations seem to indicate that in M R C 5 cytoplasts, the removal of the nucleus somehow causes disruption of the C M T C . The M T O C is unable to direct the reorganization of the C M T C even though all other requirements for assembly are available.
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After drug induced depolymerization of cytoplast CMTC, during recovery after drug action, cytoplasts either lacked C M T C or had tangled configuration. In addition, there were cytoplasts with M T in whorls around the M T O C . These did not appear to be connected with the MTOC. The whorls may have arisen as a result of tubulin self assembly. In such cytoplasts, the M T did not seem to arise from the centrosome. However, only electron microscopy can confirm this observation. M T and IF have been shown to follow similar paths and to interact with each other (Guens et al., 1983). In our experiments, staining with anti-vimentin did not show vimentin IF in the centre of cytoplasts. But the role of other IF in generating these M T patterns in the cytoplasts cannot be ruled out. The whorl like patterns suggest that other cytoplasmic factors may be responsible for such complex M T arrangements. Zamansky et al. (1991) have observed similar tangled M T in human diploid fibroblasts recovering after U.V. irradiation. In case of macrophages, the cytoplasts did not nucleate the C M T C . Since the macrophages are small cells, a loss of part of the cytoplasm during enucleation would deprive the cell of the tubulin pool substantially as compared to the large fibroblast. Time lapse cinematography of macrophage cytoplasts have clearly shown that they are visible and exhibit ameboid movement typical of those cells lacking M T (Bhisey and Freed, 1971; 1975). Thus, it appears that, the C M T C organizations of different types of cells and their cytoplasts are dependent to variable extents on the nuclear influence. It is clear that enucleation deprives at least these two cell types of some of its information regarding the C M T C organization not only in terms of the number of M T nucleated but also the extent of C M T C . ACKNOWLEDGEMENTS
The authors gratefully acknowledge the generous gift of antitubulin antibody by Dr. K. Doenges, Institute of Cell Research, German Cancer Center, Heidelberg. Thanks are also due to the Birla Smarak Kosh, Bombay, for partial financial support to Sujata Guha-Roy. REFERENCES
Albrecht-Buehler, mirror
G. ( 1 9 7 7 ) . D a u g h t e r 3T3 c e l l s : are they i m a g e s of e a c h o t h e r ? J. Cell Biol. 72:595-603.
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Anderson, D.C., Wible, L.J., Hughes, B.J., Brinldey, B.R. (1982). Cytoplasmic polymorphonuclear leukocytes : effects stimulation and colchicine, Cell 31:719-729.
Bhisey, A.N. and Freed, J.J. (1971). in cultured macrophages Exp. Cell Res. 64:419-429.
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Bhisey, A.N. and Freed, J.J. (1975). Remnant macrophages treated with cytochalasin B in of colchicine. Exp. Cell Res. 95:376-384. Brinldey, B.R. (1985). Microtubule Rev. Cell Biol. |:145-172.
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microtubules in of chemotactic
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R.L., Wible, L.J. and Brinkley, B.R. (1980). Cytoplasmic microtubule assembly disassembly in enucleated cells and regenerating karyotypes. Cell Biol. Int. Repts. 4:453-458.
Caron,
J.M., Jones, A.L., (1985). Autoregulation cells. Nature 317:648.
Rail, L.B. of tubulin
and Kirschner, M.W. synthesis in enucleated
W.J. and Brinkely, B.R. (1983). Cytoplasmic microtubule assembly - disassembly from endogenous tubulin in Brijlysed cell models. J. Cell Biol. 96:1631-1641.
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Brabander, M., Guens, G., and De Mey, J. (1980).
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Brabander, M., Guens, G., and De Mey, J. (1981).
Nuydens,
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Willebrords,
R.
The microtubule nucleating and organising activity of kinetochores and centrosomes in living Ptk 2-cells. Microtubules and M T inhibitors, in : De Brabander, M., De Mey, J. (eds.), Amsterdam : Elsevier, North Holland, pp. 255-268.
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R.,
Willebrords,
R.
Microtubule assembly in living cells after release from nocodazole block : The effect of metabolic inhibitors, taxol and pH. Cell Biol. Int. Repts. 5:913-920.
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Roy, S. and Bhisey, A.N. (1991). Regulation of cytoplasmic microtubule complex organisation in somatic cell hybrids. Cell Biol. Int. Repts. 15:377-387.
Guens,
G., De Brabander, M., Rudyens, R. and De Mey, J. (1983). The interaction between microtubules and intermediate filaments in cultured cells treated with taxol and nocodazole. Cell Biol. Int. Repts. 7:35-47.
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L . J . , Ramanis, Z. and L u c k , D . J . L . (1989). centriolar DNA : Molecular genetics Chlamydomonas. Cell 59: 129-132.
Karsenti,
E.S.,
Koyabashi,
T.,
Mitchison,
T.
Basal body/ study in
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Kirschner,
M.W. (1980). Role of centrosome in organizing the interphase microtubule array : properties of cytoplasts containing or lacking centrosomes. J. Cell Biol. 98:17631776.
Mazia,
D., S c h a t t e n , G. and Sale, W. (1975). Adhesion of c e l l s to surfaces coated with polylysine. J. Cell Biol. 66:198200.
Mitchison,
T. and Kirschner, M. (1984). Microtubule assembly nucleated by isolated centrosomes. Nature 312:232-237.
Ng,
S.F. and Fr/nkel, J. (1977). 180 ° rotation of ciliary rows and its morphogenetic implication in Tetrahymena pyriformis. Proc. Natl. Acad. Sci. USA, 74:1115-I119.
Osborn, M. and Weber, K. (1976). Cytoplasmic microtubules in tissue culture cells appear to grow from an organising structure towards the plasma membrane. Proc. Natl. Acad. Sci. USA, 73:867-871. Pickett-Heaps, J.D. (1969). The evolution of the mitotic apparatus : an attempt at comparative ultrastructural cytology in dividing plant cells. Cytobios 3:257-280. Pittenger, M.F. and Cleveland, D.W. (1985). Retention of autoregulatory control of tubulin synthesis in cytoplasts : demonstration of a cytoplasmic mechanism that regulates the levell of tubulin expression. J. Cell Biol. 101:19491952.
Solomon, F. (1979).
Detailed neurite morphology of sister neuroblastoma cells are related. Cell 16:165-169.
Solomon, F. (1980). Neuroblastoma cells recapitulate their detailed neurite morphologies after reversible microtubule disassembly. Cell 21:333-338. Solomon, F. (1981). Specification of c e l l morphology endogenous determinants. J. Cell Biol. 90:547-553.
Tash,
J.S.,
Means,
A.R.,
Brinkley,
B.R.,
Dedma~
Cox, S.M. (1980). Cyclic nucleotide and Ca of microtubules initiation and elongation, in :
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Cell Biology International Reports, VoL 16, No. 3, 1992
281
M. De. and De Mey, J. (eds.). ,Microtubules and Microtubule I n h i b i t o r s , Amsterdam, E l s e v i e r / N o r t h }(oitand, p p . 269-279. Zamansky, G.B., Perrino, B. A. and Chou, Disruption of cytoplasmic microtubules radiation. E x p . Cell Res. 195:269-273.
Paper received 01.10.91.
I.N. (1991). by ultraviolet
Revised paper accepted 25.01.92.
CYTOPLASMIC
•ICROTUBULE ASSEMBLY CYTOPLASTS.
SUJATA GUHA
269
IS A L T E R E D
IN
ROY@ and A.N.BHISEY*
,
Cancer R e s e a r c h caInstitute, Tata Memorial C e n t r e , P a r e l , Bombay400 012, INDIA. - P r e s e n t A d d r e s s : National F a c i l i t y f o r Animal T i s s u e and Cell C u l t u r e , Paud Road, Kothrud, Pune-411 029, India. ,
Author f o r c o r r e s p o n d e n c e . ABSTRACT
Nucleation of microtubule (MT) organization of the cytoplasmic microtubule complex (CMTC) from the microtubule organizing centres (MTOC) was studied in enucleated cytoplasts of human diploid fibroblast (MRC-5) and mouse peritoneal macrophages in culture. Cytoplasts of both cell types could not organize th~ complete C M T C . Aberrant M T patterns were seen in M R C - 5 cell~ while mouse macrophages showed occurrence of few short MT. The studies suggest that nucleus may have a role in determining C M T C . INTRODUCTION
The cytoplasmic microtubular complex (CMTC) of tissue culture cells is composed of long filamentous microtubules (MT) radiating from the pericentriolar microtubule organizing centers (MTOC) towards the periphery (Osborn and Weber, 1976). Different cells exhibit different c e l l morphologies. It has been suggested that cell morphologies are inherited from one generation to the next during cell division (Albrecht-Buheler, 1977; Solomon, 1979). However, it is not clear whether this occurs by a predetermined blue print situated in the genome or it is a cytoplasmic event occuring independently of the nucleus. Several reports have shown that the C M T C organization in enucleated cells is comparable to that of the intact cells (Brown et a l . , 1980 ; Caron et a l . , 1985 ; Pittinger and Cleaveland, ]985). In addition, the M T O C have been implicated as the determinants of C M T C and hence of cellular morphology (Solomon ]980, 1981). Since tubulin is capable of self assembly, it is possible that the C M T C is formed purely as a result of M T polymerization in the cytoplasm without any nuclear influence. We report in different cell types enucleation.
this paper, which failed
0309-1651/92/030269-13/$03 00/0
variant observations in two to organize their C M T C after
© 1992 Academic Press Ltd
270
Cell Biology lnternational Reports, VoL 16, No. 3, 1992
MATERIALS AND METHODS Chemicals were obtained from the following sources : Cytochalasin B (CB), ethylene glycol-bis ( -amino-ethyl-ether N N N~-N'-tetra acetic acid (EGTA), fluorescein isothiocyanate (FITC), Piperazine N, N' bis (2 ethane sulphonic acid) (PIPES) and propidium iodide (PI) from Sigma Chemical Co., USA. Fetal calf serum from Sera Labs., UK. Nocodazole was a gift from Janssen Pharmaceutica, Beerse, Belgium. Antibody to carboxymethylated tubulin was a generous gift from Dr. K. Doenges, Institute of Cell Research, German Cancer Centre, Heidelberg. Sheep anti-rabbit IgG was prepared in the laboratory (Guha Roy and Bhisey, 1991). Nocodazole and Cytochalasin B were prepared as 1 mg/ml stock solutions in dimethylsulfoxide (DMSO) and aliquoted. Nocodazole was used at a concentration of 4 uM for 4 hours to depolymerise the C M T C . Human diploid fibroblast cell line (MRC-5) and mouse macrophages were cultured as described earlier (Guha Roy and Bhisey, 1991). Enucleation. The cells were plated on round (24 m m diameter) polylysine coated coverslips (Mazia et al., 1976) and allowed to grow for three to five days until they were semi-confluent. Sorvall SS-34 centrifuge tubes with an araldite base were constructed to support the coverslips during centrifugation. Each coverslip was placed cell side down on P V C rings resting on araldite base and submerged in about 4 ml medium containing l0 ug/ml CB. Cells were spun in Sorvall RC-5B centrifuge prewarmed to 27°C at I0,000 rpm for 15 minutes. The cells were allowed to recover in medium without CB for one hour. The same protocol was used for macrophages except for the inclusion of l ug/ml nocodazole in the enucleation medium since this produced cytoplasts more efficiently. These cells were spun at 7,500 rpm for 30 minutes. Time lapse cinematography. After centrifugation, the coverslips were assembled in Sykes Moore chambers and filmed on a Nikon inverted phase microscope fitted with a Vinten camera at 12 frames per minute (Bhisey and Feed, 1975). Immunofluorescence staining was done on cells grown on coverslips as described earlier (Guha Roy and Bhisey, 1991). RESULTS The C M T C of fibroblasts differs in density as well as in organization.
from that of macrophages The fibroblast C M T C is
Cell Biology International Reports, VoL 16, No. 3, 1992
271
very complex with an overlapping array of M T which are so densely arranged in the central region of the cell that the origin of every M T in the cell cannot be traced to the M T O C (Fig.l). Fibroblasts in which centriolar replication had been completed showed more than one MTOC. In M R C - 5 cells, the number of M T O C varied from one to four per cell. The macrophage C M T C appeared less complex. F e w M T radiated from the M T O C . The sparse nature of the C M T C made it possible to trace most M T to the M T O C (Fig.2). Nocodazole reversibly depolymerized the MT. MRC-5 cells treated with nocodazole showed heterogeneous pattern of C M T C organization : some cells were totally devoid of their C M T C , while others retained a few residual M T (Figs.3 & 4). The macrophage CMTC was uniformly depolymerized by nocodazole (Fig.5). However, when the drug was withdrawn, M T nucleation began at the M T O C and proceeded towards the cell periphery. Within ten minutes of drug withdrawl the entire C M T C in both cell types was reorganized. The role of the nucleus in M T nucleation was studied in enucleated cells. Soon after centrifugation, the morphology of the cytoplasts became aberrant, but they regained their normal shapes within 30 to "60 mins in medium without CB. Microtubule pattern in M R C - 5 cytoplasts. Except for the lack of nucleus, the cytoplasts resembled intact cells in their morphology. Enucleation was confirmed by PI staining. Staining with antitubulin revealed different classes of C M T C among cytoplasts. Counts were taken of the various CMTC patterns observed in the cytoplasts and at the same time of the patterns observed in the intact cells which served as the internal controls as they had undergone the same CB treatment as well as centrifugation. A large number (36.7 + I0.2%) of cytoplasts were found to possess brightly fluorescing M T O C but lacking any form of C M T C (Fig.6). Polymerized M T were evident in about 39% of cytoplasts but not all of these were comparable with the C M T C observed normally in intact cells. The patterns observed were as follows : (a) Cytoplasts with C M T C similar to the normal CMTC of intact cells were seen in 6.42 + 2.2~ of the total (Fig.7). (b) Cytoplasts with fewer polymerized M T which formed aberrant C M T C organizations in the central region of the cell. These M T were in the form of itangles, close to the M T O C , leaving the peripheral cytoplasm free of M T (Fig .8). Their origin from the M T O C was not obvious, such cytoplasts comprised 12.97 + 4.15% of the total. (c) Some cytoplasts did not show
272
Cell Biology International Reports, VoL 16, No. 3, 1992
°~
.
2
7
L
m
5 Fig. I-6 Immunofluorescence of M T stained with anti-tubulin antibody : Fig. 1. CMTC of MRC-5 cells. Fig. 2. Mouse macrophage C M T C with M T O C . Fig.3. M T O C in nocodazole treated M R C - 5 cells : C M T C completely depolymerized. Fig.4. Nocodazole resistant M T in M R C - 5 cells. Fig.5. Total disappearance of C M T C in nocodazole treated macrophages : MTOC with 3-4 short M T seen. Fig. 6. M R C - 5 cytoplasts devoid of M T but with prominent M T O C . Bar width I0 uM.
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273
(fJ I0. o o
90 80
I T
70~
60-
"'
50-
~o
40-
'"
30
z~w u 141
20
°
o.
I0
m
CYTOPLASTS
I'~
CELLS
-
-m I NORMAL CMTC (MTOC-I-)
Fig.7. Histogram different types procedure. Cells were analysed for
TANGLED CMTC 1-1 CMTC (MTOC + ) (MTOC +)
showing percentage of CMTC after were stained with v~rious C M T C types
CMTC1-1 (MTOC -)
iL FEW MT (MTOc - )
of cells and cytoplasts with recovery from enucleation antitubulin antibody. Slides and counts were taken.
MTOC, but M T polymerization had occurred in the absence of the M T O C (Fig.9) in 19.6 + 8.45% cytoplasts. Among these, in a few cytoplasts, only a few M T had polymerized. Others had neither an M T O C nor any polymerized M T (24.07 +- 7.2%). Comparing these patterns with the C M T C of intact cells on the same coverslip, the situation was reversed (Fig.7). Most of the cells (74.8 + 2.4%) had a normal C M T C complete with an M T O C (Fig.10). No aberrant tangles of M T were found. About 24% cells were devoid of C M T C although the MTOC fluoresced brightly. There were no cells without MT and M T O C but 1.5 + 0.6% of the total cells counted had a few M T polymerized in the absence of MTOC. Recovery of MRC-5 cytoplasts after nocodazole induced depolymerization. To evaluate if reorganization of C M T C after depolymerization is a nucleus dependent process, the cytoplasts were allowed to recover after enucleation and then treated with 4 uM nocodazole for four hours. This was followed by recovery in medium without nocodazole for 30 minutes. The C M T C of cytoplasts recovered from nocodazole could be classified i n t o four groups : (a) Cytoplasts with no discernible CMTC despite the presence of the M T O C . (b)
274
Cell Biology International Re oorts, Vol. 16, No. 3, 1992
9
12
.~'
~.
, ,,
13
,.
14
Figs. 8-I0 and 12,15,14 Immunofluorescence of M T stained with anti-tubulin antibody. Fig.8. 'Tangled' M T in M R C - 5 cytoplasts. Fig.9. Intact M T in M R C - 5 cytoplast without M T O C . Fig.10. Intact cell with normal C M T C . Fig.12. 'Tangled' M T in M R C - 5 cytoplast recovered after nocodazole treatment. Fig.13. Whorl-like C M T C surrounding M T O C in cytoplast recovered after nocodazole. Fig.14. Macrophage cytoplast showing very few and short MT after recovery from nocodazole. Bar width I0 urn.
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Cytoplasts that possessed an M T O C and had varying degrees of polymerized MT. (c) Cytoplasts that lacked M T and MTOC. (d) Cytoplasts that had polymerized M T inspire of the absence of an M T O C . Most of the cytoplasts (67 -+ 6.83~) had an MTOC, but lacked C M T C organization. Figure II shows the comparison of percentages of cytoplast C M T C and that of cells recovering from nocodazole treatment. Cytoplasts with varying degrees of C M T C organizations formed 29.5 + 6.67% of the total number of cytoplasts. The C M T C varied from a few polymerized M T to a C M T C comparable to that of intact cells.
t- 100 u~ ,¢[ "a. 0 I>o ,,0
90
~
60
_1
~
5o
~
40
"'
30
~'z w o
20
CYTOPLASTS 80 ]
CELLS
70
10
T
w o.
CMT=+ MTO=
Fig.ll. Histogram showing different nocodazole.
(=.T=+) MTOC +
showing patterns
MTOC -
(=.Tc - ) MTO=
--
percentage of cells and cytoplasts of CMTC after recovery from
T w o unusual forms of C M T C that were not encountered in intact cells were observed in such cytoplasts. There were cytoplasts with a tangled C M T C in the central region of the cytoplast along with the M T O C (Fig.12). All M T did not appear to be connected to the M T O C . This form of C M T C had also been observed in cytoplasts not treated with nocodazole. The second ~aberrant' C M T C consisted of M T polymerized in circular whorls around centrally localized M T O C (Fig.13). Most of the M T were independent of MTOC. Cytoplasts that lacked both M T O C and C M T C were 2.97 + 1.86% of the total and 0.78 + 0.08% of the cytoplasts had assembled a few M T in the absence of the MTOC.
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The macrophage cytoplast C M T C . Enucleated macrophages showed normal ruffling and were able to phagocytose heat killed E.coli. Soon after enucleation, the cytoplasts were either arborized or rounded. When placed in medium without CB, the cytoplasts took a longer time to spread than the intact cells. Motility of cytoplasts were studied by time lapse cinematography during and after recovery after enucleation in 9 different sequences involving 19 cytoplasts and 8 intact cells. The cytoplasts exhibited random motility by pseudopod formation and mass flow, similar to that of colchicine of vinblastine treated macrophages as reported earlier (Bhisey and Freed 1971, 1975). The intact cells present in the same microscope field as the cytoplasts showed ruffling and normal gliding type motility. Development of the cytoplast C M T C was analysed as a function of time. Ten minutes after transferring to control medium, few short M T were nucleated both in intact cells as well as in cytoplasts. The number and length of M T in intact cells increased with time, whereas cytoplasts had very few M T upto 60 minutes after drug withdrawl (Fig.14). In about 30% cytoplasts, M T O C could not nucleate M T at all (Table 1). Unlike M R C - 5 cytoplasts, aberrant CMTC patterns were not seen in macrophage cytoplasts. The M T O C that could nucleate a few M T were able to do so only to a limited extent.
Table
I
: Comparison of M T nucleation as a function macrophage cells and cytoplasts.
Time (rains)
~ Whole Cells With M T Without M T
of time in
% Cytoplasts With M T Without M T
20
85.6
14.4
61.5
38.5
30
86.3
13.7
66.6
33.4
45
95.5
4.5
70.0
30.0
60
93.7
6.3
72.4
27.6
DISCUSSION The M T O C have been considered to be the 'preferred site' for M T nucleation (Pickett-Heaps 1969; Brinkley 1985). This has been proved in cells recovering from M T inhibitors where nucleation is prominent at the M T O C (DeBrabender et al., 1980;
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1981) and in isolated centrosomes (Mitchison and Kirschner, 1984). The M T O C have been implicated as the center for storage and recall of information pertaining to cellular morphology (Ng and Frankel, 1977; Solomon 1980, 1981). The number of MT, their length, polarity and the lattice structure of the M T were shown to be determined by the M T O C (Anderson et al., 1982; Brinkley 1985). Although the factors that determine the morphology of eukaryotic cells are not well defined, the M T O C determine the temporal and spatial pattern of M T in the cytoplasm and thereby influence the cell shape. Demonstration of D N A in centrioles (Hall et a l . , 1989) has raised the possibility of genetic autonomy of MTOC. The microenvironment at the M T O C is suitable for M T nucleation probably due to lower critical concentrations. Using lysed cell models, the nucleation of M T from M T O C was found to be influenced by the pH of the medium (Derry and Brinkley, 1983) and the activity of a cyclic nucleotide dependent kinase (Tash et al., 1980). In case of enucleated cells, the factors regulating M T nucleation are similar to those in intact cells. The role of the nucleus, albeit unknown, is bound to be important. Although lysed cell models are used in studying M T assembly, the biochemical properties of cytoplasmic ground substance confer order and organization to the M T array (Tash et al., 1980). Unlike previous reports (Brown et al., 1980; Caron et al., 1985; Karsenti et al., 1980) that cytoplasts and intact cells have similar C M T C organization, the M R C - 5 and macrophage cytoplasts were found to lack such organization. This effect was not due to the death of the cytoplasts since these could spread out normally and a number of them showed ruffling and cell movement. The altered M T nucleation in cytoplasts was not due to the trauma of centrifugation since intact cells on the same coverslip showed normal C M T C organization. Karsenti et al. (1985) have reported the formation of peripheral M T that lie freely in the cytoplasts which lack MTOC. But they found that, in cytoplasts possessing a MTOC, the focussed M T array was always present. This did not occur in M R C - 5 cytoplasts where, inspire of the presence of the MTOC, there was either a complete lack of C M T C or an aberrant C M T C organizations were seen. The tangled M T were not all focussed at the M T O C . These observations seem to indicate that in M R C 5 cytoplasts, the removal of the nucleus somehow causes disruption of the C M T C . The M T O C is unable to direct the reorganization of the C M T C even though all other requirements for assembly are available.
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After drug induced depolymerization of cytoplast CMTC, during recovery after drug action, cytoplasts either lacked C M T C or had tangled configuration. In addition, there were cytoplasts with M T in whorls around the M T O C . These did not appear to be connected with the MTOC. The whorls may have arisen as a result of tubulin self assembly. In such cytoplasts, the M T did not seem to arise from the centrosome. However, only electron microscopy can confirm this observation. M T and IF have been shown to follow similar paths and to interact with each other (Guens et al., 1983). In our experiments, staining with anti-vimentin did not show vimentin IF in the centre of cytoplasts. But the role of other IF in generating these M T patterns in the cytoplasts cannot be ruled out. The whorl like patterns suggest that other cytoplasmic factors may be responsible for such complex M T arrangements. Zamansky et al. (1991) have observed similar tangled M T in human diploid fibroblasts recovering after U.V. irradiation. In case of macrophages, the cytoplasts did not nucleate the C M T C . Since the macrophages are small cells, a loss of part of the cytoplasm during enucleation would deprive the cell of the tubulin pool substantially as compared to the large fibroblast. Time lapse cinematography of macrophage cytoplasts have clearly shown that they are visible and exhibit ameboid movement typical of those cells lacking M T (Bhisey and Freed, 1971; 1975). Thus, it appears that, the C M T C organizations of different types of cells and their cytoplasts are dependent to variable extents on the nuclear influence. It is clear that enucleation deprives at least these two cell types of some of its information regarding the C M T C organization not only in terms of the number of M T nucleated but also the extent of C M T C . ACKNOWLEDGEMENTS
The authors gratefully acknowledge the generous gift of antitubulin antibody by Dr. K. Doenges, Institute of Cell Research, German Cancer Center, Heidelberg. Thanks are also due to the Birla Smarak Kosh, Bombay, for partial financial support to Sujata Guha-Roy. REFERENCES
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Paper received 01.10.91.
I.N. (1991). by ultraviolet
Revised paper accepted 25.01.92.