- Email: [email protected]
Assembly of glial intermediate filament protein is initiated in the centriolar region
BRE 21093
Short Communications
filament probin is initbtd
Afssembdy of glkl in&madkb
V. I. KALNINS,
L. SUBRAHMANYANI
in the centriolar region
and S. FEDOROFF2
‘Department of Anatomy (Histology), University of Toronto, Toronto MS IA8 and 2Department of Anatomy, University of Saskatchewan, Saskatoon S7N OWO(Canada)
(Accepted May 21st. 1985) Key words: astrocyte -intermediate
filament assembly - centriole
Assembly of glial intermediate filament protein (GFP) into intermediate filaments (IF) was first detected by immunofluorescence in the perinuclear region of astrocytes differentiating in colony cultures before the rest of the cytoplasm was labeled. Double labeling with antisera specific for centrioles indicated that this site corresponds to the centriolar region. These studies suggest that the centriolar region plays an important role in the assembly of some types of IF as well as microtubules.
In comparison to microtubules (MT) and microfilaments very little is known about the assembly of the third major component of the cytoskeleton, the intermediate filaments (IF). Several classes of IF proteins are known to existXW5 and two of these, vimentin and glial filament protein (GFP) are present in IF of astrocytess.7,*0.30,32.36.~. In colonies of astrocyte precursor cells obtained by culturing the neopallium of mouse embryo&lo, the GFP containing IF are acquired at a specific stage of differentiation after the acquisition of the vimentin type IFlo,* as proastroblasts change into astroblastsl2. To determine where in the cell GFP containing IF first appear we examined colonies of astrocyte precursor cells at a time when relatively large number of GFP negative cells are becoming GFP positive. Using double immunofluorescence technique and sera specific for GFP*l and centrioles6.37 we demonstrate that GFP is first incorporated into IF in the centriolar region of differentiating astrocytes. To obtain colonies of astrocyte precursor cells cerebral hemispheres of l&day-old DBA/lJ mice were isolated aseptically and the meninges removed. The Correspondence:
V. I. Kalnins, Department Ont. M5S lA8, Canada.
neopallium was freed from basal ganglia, olfactory lobe and hippocampus and then geatly forced through a sterile ‘Nitex’ mesh (pore size 75 ym). The cells were suspended in a growth medium-consisting of Eagle’s Minimum Essential Medium containing a 4-fold concentration of vitamins, a double concentration of amino acids (except glutamine, which was kept at the 2 mM level) and 7.5 mM glucose and 5% horse serum (v/v). Cell viability was determined by Nigrosine dye exclusion techniquezo. Nigrosine, 5 x 104,excluding cells were plated in each 60 mm Falcon culture petri dish containing glass coverslips in a total volume of 2.5 ml growth medium. The cultures were incubated at 37 “C in a humidified atmospbre of 5 % CO, in air. After 3 days of incubation, the growth medium was removed, cell debris and non-attached cells were washed out and fresh medium was added. For immunofluorescence the cells grown on_glass coverslips for 7 days were quickly rinsed in phosphate buffered saline (PBS), fixed first for 4 min in methanol, then 2 min in acetone, both at -20 ‘C and air dried. After fixation the cells were rinsed once in PBS, treated with rabbit anti-centriole serum6%37
of Anatomy (Histology), Medical Sciences Building, University of Toronto, Toronto.
OQO6-8993/85/$03.300 1985 Ekevier Science Publishers B.V. (Biomedical Division)
323
-
.,,q,
O
O ,-.9[
O
o
.[U-
Fig. 1. [mmunofluorescence of differentiating astrocytes in colony cultures showing the same cells double labeled with s e r u m specific for centrioles (a, c. e and g) and antiserum specific for GFP (b, d, f and h). The position of centrioles in cells p~lrtially stained with antiserum to GFP is indicated by arrows.
324 (1:20) for 30 min at room temperature and washed 3 times in PBS for 10 min. This was followed by incubation with fluorescein-conjugated goat anti-rabbitIgG (Miles-Yeda Ltd., Israel) at a 1:15 dilution for 30 rain. The unbound conjugated antibodies were washed off by rinsing 5 times, 5 min each in PBS and the cells were then incubated with normal rabbit serum at 1:100 dilution for 30 min. Finally, after another 10 min wash in PBS, the cells were incubated with rabbit antiserum to GFP H, directly conjugated to tetramethyl rhodamine isothocyanate isomer R. (Becton, Dickinson and Co., U.S.A.). After washing, the preparations were mounted in PBS containing 50% glycerol and 0.02% p-phenylenediamine 19. The cells were examined in a Zeiss photomicroscope II provided with epifluorescence optics and interference filters for viewing fluorescein and rhodamine labeled cells. When 7-day-old colonies of astrocyte precursor cells obtained from the neopallium of 18-day-old mouse embryos were examined for the position of centrioles, a single brightly stained dot was seen in the perinuclear region of cells forming the colony (Fig. la, c, e, g). In the smaller more compact colonies the centrioles were located on the side of the nucleus facing the outer edge of the colony (Fig. la), whereas in the larger colonies in which the cells had moved further apart, cells with centrioles facing in many different directions were observed (Fig. lc, e). When the same colonies were examined with antiserum to GFP to determine the distribution of GFP containing IF, cells with varying amounts of finely fibrillar staining were observed. Some of the cells in these colonies had GFP containing IF distributed throughout the cytoplasm (Fig. ld, f) while others lacked them altogether (Fig. lb, d, f). In the remaining cells only a part of the cytoplasm, usually a small region on one side of the nucleus was stained (Fig. lb, d, f, h). Examination of the partially stained cells by double immunofluorescence showed that the regions which were GFP positive (Fig. lb, d, f, h) corresponded to the regions which contained the centrioles (Fig. la, c, e, g). In cells where the region occupied by the GFP containing IF was more extensive and extended further out into the cytoplasm, the centrioles were also found near the nucleus, in the region containing the highest concentration of these IF (Fig. la, b, e, f). Later over 95% of the astroblasts in such
colonies become GFP positive and have finely fibrillar staining extending throughout their cytoplasmiC. We conclude that during the differentiation of fibrous astrocytes, the assembly of GFP containing 1F is initiated in the centriolar region on one side of thc nucleus. The region containing these IF then increases gradually in size until a network extending throughout the whole cytoplasm is assembled. Whether the assembly of the vimentin type IF which appear in astrocyte precursor cells prior to the GFP containing o n e s 7.10.11.30,32.36A0 is also initiated in the centriolar region of the cell has not been determined. The observations that both types of IF aggregate in the perinuclear region in the presence of antimitotic agents 2~,30,33 and that in cells of a glioma line both IF proteins have a similar distribution and are present in the same IF 27.31, suggest that the assembly of the vimentin containing IF may also be initiated in this region. In the centriolar region the GFP may be incorporated into the vimentin type IF already present, may form IF containing only GFP or both depending on the availability of vimentin and GFP subunits at various stages of astrocyte differentiation. A close relationship between the centriote and the IF has not been observed with IF of the keratin type found in epithelial cells. In lysed cell models the assembly of new IF after addition of acid solubilized keratin could be initiated from a specific s~te near the nucleus but the position of this site bore no relationship to the posmon of the centriole s. There is considerable evidence, however, that the keratin type IF behave differently from IF of other types in the presence of antimitotic agents 22. Also during mitos~s in PtK2 cells they are not as closely associated with the centrioles as the IF of the vimentin type 2. Finally our study is different in that initiation of IF assembly during differentiation in intact cells rather than assembly of exogenous keratin m extracted cell models was examined. Our observation that the centriolar region contains sites which initiate the incorporation of GFP into IF may explain the close association between certain types of IF and centrioles 17.25,28-29-34,39 and IF and MT 13.15,16,38 observed in a number of previous studies and why both IF and MT radiate from a position near the nucleus 1,3A4,18. The close morphological proximity of IF with the centrioles have previously led to suggestions that the centriolar region of the cell may
325 serve as an o r g a n i z i n g c e n t e r for I F 17,39 as well as
gion and w h e t h e r this or o t h e r r e g i o n s s e r v e as sites
M T 4.26. This v i e w is c o n s i d e r a b l y s t r e n g t h e n e d by
of I F a s s e m b l y in fully d i f f e r e n t i a t e d cells r e m a i n s to
our o b s e r v a t i o n that the a s s e m b l y o f at least the G F P
be d e t e r m i n e d .
c o n t a i n i n g I F is initiated in this region. T h e results also indicate that the c e n t r i o l a r r e g i o n plays a l a r g e r role in the o v e r a l l o r g a n i z a t i o n of the c y t o s k e l e t o n
This w o r k was s u p p o r t e d
by M e d i c a l R e s e a r c h
t h a n p r e v i o u s l y s u s p e c t e d . W h e t h e r the a s s e m b l y of
C o u n c i l of C a n a d a G r a n t s MT-3302 to V . I . K . and
o t h e r types of I F is also initiated in the c e n t r i o l a r re-
MT-4235 to S.F.
1 Albrecht-Buehler, G. and Bushnell, A., The orientation of
centrioles in migrating 3T3 cells, Exp. Cell Res., 126 (1980) 427-437. 2 Aubin, J. E., Osborn, M., Franke, W. W. and Weber. K., Intermediate filaments of the vimentin-type and the cytokeratin-type are distributed differently during mitosis, Exp. Cell Res., 129 (1980) 149-165. 3 Borenfreund, E., Schmid, E., Bendich, A. and Franke, W. W., Constitutive aggregates of intermediate sized filaments of the vimentin and cytokeratin type in cultured hepatoma cells and their dispersal by butyrate, Exp. Cell Res., 127 (1980) 215-235. 4 Brinkley, B. R., Fistel, S. H., Marcum, J. M. and Pardue, R. L., Microtubules in cultured cells, indirect immunofluorescent staining with tubulin antibody, Int. Rev. Cytol., 63 (1980) 59-95. 5 Chiu, F. C., Norton, W. T. and Fields, K. L., The cytoskeleton of primary astrocytes in culture contains actin, glial fibrillary acid protein and the fibroblast-type filament protein vimentin, J. Neurochem., 37 (1981) 147-155. 6 Connolly, J. A. and Kalnins, V. I., Visualization of centrioles and basal bodies by fluorescent staining with non-immune rabbit sera, J. Cell Biol., 79 (1978) 526-532. 7 Dalai, D., Ruegar, D., Bignami, A., Weber, K. and Osborn, M., Vimentin, the 57000 molecular weight protein of fibroblast filaments is the major cytoskeletal component of immature glia, Eur. J. Cell Biol., 24 (1981) 191-196. 8 Eckert, B. S., Daley, R. A. and Parysek, C. M., In vivo disruption of the cytokeratin cytoskeleton in cultured epithelial cells by microinjection of anti-keratin: evidence for the presence of an intermediate filament organizing center, Cold Spring Harbor Syrup. Quant. Biol., 46 (1981) 403-412. 9 Fedoroff, S. and Doering, L., Colony culture of neural cells as a method for the study of cell lineage in the developing CNS: the astrocyte cell lineage, Curr. Top. Dev. Biol., 16 (1980) 283-304. 10 Fedoroff, S., White, R., Subrahmanyan, L. and Kalnins, V. I., Properties of putative astrocytes in colony cultures of mouse neopallium. In E. A, Costa Vidrio and S. Fedoroff (Eds.), Glial and Neuronal Cell Biology, Alan R. Liss, New York, 1981, pp. 1-19. 11 Fedoroff, S., White, R., Neal, J., Subrahmanyan, L. and Kalnins, V. I., Astrocyte cell lineage. II. Mouse fibrous astrocytes and reactive astrocytes in cultures have vimentin and GFP containing intermediate filaments, Dev. Brain Res,, 7 (1983) 303-315. 12 Fedoroff, S., Neal, J., Opas, M. and Kalnins, V. I., Astrocyte cell lineage. III. The morphology of differentiating mouse astrocytes in colony culture, J. Neurocytol., 13 (1984) 1-20.
13 Franke, W. W., Grund, C., Osborn, M. and Weber, K., The intermediate sized filaments in rat kangaroo PtK 2 cells. I. Morphology in situ, Cytobiologie, 17 (1978) 365-391. 14 Franke, W. W., Schmid, E., Winter, S., Osborn, M. and Weber, K., Widespread occurrence of intermediate sized filaments of the vimentin-type in cultured cells from diverse vertebrates, Exp. Cell Res., 123 (1979) 25-46. 15 Geiger, B. and Singer, S. J., Association of microtubules and intermediate filaments in chicken gizzard cells as detected by double immunofluorescence, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 4769-4773. 16 Goldman, R. D. and Knipe, D. M., Functions of cytoplasmic fibers in nonmuscle cell motility, Cold Spring Harbor Symp. Quant. Biol., 37 (1973) 523-534. 17 Goldman, R. D., Hill, B. F., Steinert, P., Whitman, M. A. and Zackaroff, R. V., Intermediate filament-microtubule interactions: evidence in support of a common organization center. In M. DeBrabander and J. DeMay (Eds.), Microtubules and Microtubule lnhibitors, Elsevier, New York, 1980, pp. 91-102. 18 Hynes, R. D. and Destree, A. T., Relationship between fibronectin (LETS protein) and actin, Cell, 13 (1978) 151-163. 19 Johnson, G. D. and Nodeira-Araujo, G. M. deC., Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy, J. lmmunol. Meth.. 43 (1981) 349-353. 20 Kaltenbach, J. P., Kaltenbach, M. H. and Lyons, W. B., Nigrosine as a dye for differentiating live and dead ascites cells, Exp. CellRes., 15 (1958) 112-117. 21 Lazarides, E., Intermediate filaments: a chemically heterogeneous, developmentally regulated class of proteins, Annu. Rev. Biochem., 51 (1982) 219-250. 22 Osborn, M., Franke, W. W. and Weber, K., Direct demonstration of the presence of two immunologically distinct intermediate size filament systems in the same cell by double immunofluorescence microscopy, Exp. Cell Res., 125 (1980) 37-46. 23 Osborn, M., Intermediate filaments as histologic markers: an overview, J. Invest. Dermatol., 81 (1983) 104s-109s. 24 Paetau, A., Virtanen, I., Steinman, S., Kurki, P., Linder, E., Vaheri, A., Westermark, B., Dahl, D. and Haltia, M., Glial fibrillary acidic protein and intermediate filaments in human glioma cells, Acta Neuropathol., 47 (1979) 71-74. 25 Pena, S. D. J., Opas, M., Turksen, K., Kalnins, V. I. and Carpenter, S., Immunocytochemical studies of intermediate filament aggregates and their relationship to microtubules in cultured skin fibroblasts from patients with giant axonal neuropathy, Eur. J. Cell Biol., 31 (1983) 227-234. 26 Picket-Heaps, J. D., The evolution of the mitotic apparatus: an attempt at comparative ultrastructural cytology in
326 dividing plant cells, Cytobios, 3 (1969) 257-280. 27 Quinlan, R. A. and Franke, W. W., Molecular interactions in intermediate sized filaments revealed by chemical crosslinking. Heteropolymers of vimentin and glial filament protein in cultured human glioma cells, Eur. J. Biochern., 132 (1983) 477-484. 28 Ring, D., Hubble, R., Caput, D. and Kirschner, M., Isolation of microtubule organizing centers from mouse neuroblastoma cells. In M. DeBrabander and J. DeMay (Eds.), Microtubules and Microtubule Inhibitor& Elsevier, Amsterdam, 1980, pp. 297-310. 29 Sandborn, E., Cells and Tissues by Light and Electron Microscopy, Vol. 1, Academic Press, New York, 1971). 30 Schnitzer, J., Franke, W. W. and Schachner, M., Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system, J. Cell Biol., 90 (1981) 435-447. 31 Sharp, G., Osborn, M. and Weber, K., Occurrence of two different intermediate filament proteins in the same filament in situ within a human glioma cell line, Exp. Cell Res., 141 (1982) 385-395. 32 Shaw, G., Osborn, M. and Weber, K., An immunofluorescence microscopical study of the neurofilament triplet proteins, vimentin and glial fibrillary acidic protein with the adult rat brain, Eur. J. Cell Biol., 26 (1981) 68-82. 33 Sotelo, J., Toh, B. H., Lolait, S. J., Yildiz, A., Sung, D.
34
35 36
37
38
39
40
and Holobrow, E. J., Cytoplasmic intermediate filaments in cultured glial cells, Neuropath. Appl. Neurohiol.. 6 (1980) 291-298. Starger, J. M., Brown, W. E., Goldman, A. E. and Goldman, R. D., Biochemical and immunological analysis of rapidly purified 10 nm filaments from baby hamster kidney (BHK-21) cells, J. Cell Biol., 78 (1978) 93-109. Steinert, P. M., Jones, J. C. R. and Goldman, R. D., Intermediate filaments, J. Cell Biol., 99 (1984) 22s-27s. Tapscott, S. J., Bennett, G. S., Toyama, Y., Kleinbart, F. and Holtzer, H., Intermediate filament proteins in developing chick spinal cord, Dev. Biol., 86 (1981) 40-54. Turksen, K., Aubin, J. E. and Kalnins, V. I., Identification of a centriole-associated protein by antibodies present in normal rabbit sera, Nature (Lond.), 298 (1982) 763-765. Wang, E. and Goldman, R. D., Functions of cytoplasmic fibers in intracellular movements in BHK-21 cells, I. Cell Biol., 79 (1978) 708-726. Wang, E., Connolly, J. A., Kalnins, V. I. and Choppin, P. W., Relationship between movement and aggregation of centrioles in syncytia and formation of mierotubule bundles, Proc. nat. Acad. Sei. U.S.A., 76 (1979) 5719-5723. Yen, S. H. and Fields, K., Antibodies to neurofilaments, glial filaments and fibroblast-intermediate filament-proteins bind to different cell types of the nervous system, J. Cell Biol., 88 (198 I) 115-126.
Short Communications
filament probin is initbtd
Afssembdy of glkl in&madkb
V. I. KALNINS,
L. SUBRAHMANYANI
in the centriolar region
and S. FEDOROFF2
‘Department of Anatomy (Histology), University of Toronto, Toronto MS IA8 and 2Department of Anatomy, University of Saskatchewan, Saskatoon S7N OWO(Canada)
(Accepted May 21st. 1985) Key words: astrocyte -intermediate
filament assembly - centriole
Assembly of glial intermediate filament protein (GFP) into intermediate filaments (IF) was first detected by immunofluorescence in the perinuclear region of astrocytes differentiating in colony cultures before the rest of the cytoplasm was labeled. Double labeling with antisera specific for centrioles indicated that this site corresponds to the centriolar region. These studies suggest that the centriolar region plays an important role in the assembly of some types of IF as well as microtubules.
In comparison to microtubules (MT) and microfilaments very little is known about the assembly of the third major component of the cytoskeleton, the intermediate filaments (IF). Several classes of IF proteins are known to existXW5 and two of these, vimentin and glial filament protein (GFP) are present in IF of astrocytess.7,*0.30,32.36.~. In colonies of astrocyte precursor cells obtained by culturing the neopallium of mouse embryo&lo, the GFP containing IF are acquired at a specific stage of differentiation after the acquisition of the vimentin type IFlo,* as proastroblasts change into astroblastsl2. To determine where in the cell GFP containing IF first appear we examined colonies of astrocyte precursor cells at a time when relatively large number of GFP negative cells are becoming GFP positive. Using double immunofluorescence technique and sera specific for GFP*l and centrioles6.37 we demonstrate that GFP is first incorporated into IF in the centriolar region of differentiating astrocytes. To obtain colonies of astrocyte precursor cells cerebral hemispheres of l&day-old DBA/lJ mice were isolated aseptically and the meninges removed. The Correspondence:
V. I. Kalnins, Department Ont. M5S lA8, Canada.
neopallium was freed from basal ganglia, olfactory lobe and hippocampus and then geatly forced through a sterile ‘Nitex’ mesh (pore size 75 ym). The cells were suspended in a growth medium-consisting of Eagle’s Minimum Essential Medium containing a 4-fold concentration of vitamins, a double concentration of amino acids (except glutamine, which was kept at the 2 mM level) and 7.5 mM glucose and 5% horse serum (v/v). Cell viability was determined by Nigrosine dye exclusion techniquezo. Nigrosine, 5 x 104,excluding cells were plated in each 60 mm Falcon culture petri dish containing glass coverslips in a total volume of 2.5 ml growth medium. The cultures were incubated at 37 “C in a humidified atmospbre of 5 % CO, in air. After 3 days of incubation, the growth medium was removed, cell debris and non-attached cells were washed out and fresh medium was added. For immunofluorescence the cells grown on_glass coverslips for 7 days were quickly rinsed in phosphate buffered saline (PBS), fixed first for 4 min in methanol, then 2 min in acetone, both at -20 ‘C and air dried. After fixation the cells were rinsed once in PBS, treated with rabbit anti-centriole serum6%37
of Anatomy (Histology), Medical Sciences Building, University of Toronto, Toronto.
OQO6-8993/85/$03.300 1985 Ekevier Science Publishers B.V. (Biomedical Division)
323
-
.,,q,
O
O ,-.9[
O
o
.[U-
Fig. 1. [mmunofluorescence of differentiating astrocytes in colony cultures showing the same cells double labeled with s e r u m specific for centrioles (a, c. e and g) and antiserum specific for GFP (b, d, f and h). The position of centrioles in cells p~lrtially stained with antiserum to GFP is indicated by arrows.
324 (1:20) for 30 min at room temperature and washed 3 times in PBS for 10 min. This was followed by incubation with fluorescein-conjugated goat anti-rabbitIgG (Miles-Yeda Ltd., Israel) at a 1:15 dilution for 30 rain. The unbound conjugated antibodies were washed off by rinsing 5 times, 5 min each in PBS and the cells were then incubated with normal rabbit serum at 1:100 dilution for 30 min. Finally, after another 10 min wash in PBS, the cells were incubated with rabbit antiserum to GFP H, directly conjugated to tetramethyl rhodamine isothocyanate isomer R. (Becton, Dickinson and Co., U.S.A.). After washing, the preparations were mounted in PBS containing 50% glycerol and 0.02% p-phenylenediamine 19. The cells were examined in a Zeiss photomicroscope II provided with epifluorescence optics and interference filters for viewing fluorescein and rhodamine labeled cells. When 7-day-old colonies of astrocyte precursor cells obtained from the neopallium of 18-day-old mouse embryos were examined for the position of centrioles, a single brightly stained dot was seen in the perinuclear region of cells forming the colony (Fig. la, c, e, g). In the smaller more compact colonies the centrioles were located on the side of the nucleus facing the outer edge of the colony (Fig. la), whereas in the larger colonies in which the cells had moved further apart, cells with centrioles facing in many different directions were observed (Fig. lc, e). When the same colonies were examined with antiserum to GFP to determine the distribution of GFP containing IF, cells with varying amounts of finely fibrillar staining were observed. Some of the cells in these colonies had GFP containing IF distributed throughout the cytoplasm (Fig. ld, f) while others lacked them altogether (Fig. lb, d, f). In the remaining cells only a part of the cytoplasm, usually a small region on one side of the nucleus was stained (Fig. lb, d, f, h). Examination of the partially stained cells by double immunofluorescence showed that the regions which were GFP positive (Fig. lb, d, f, h) corresponded to the regions which contained the centrioles (Fig. la, c, e, g). In cells where the region occupied by the GFP containing IF was more extensive and extended further out into the cytoplasm, the centrioles were also found near the nucleus, in the region containing the highest concentration of these IF (Fig. la, b, e, f). Later over 95% of the astroblasts in such
colonies become GFP positive and have finely fibrillar staining extending throughout their cytoplasmiC. We conclude that during the differentiation of fibrous astrocytes, the assembly of GFP containing 1F is initiated in the centriolar region on one side of thc nucleus. The region containing these IF then increases gradually in size until a network extending throughout the whole cytoplasm is assembled. Whether the assembly of the vimentin type IF which appear in astrocyte precursor cells prior to the GFP containing o n e s 7.10.11.30,32.36A0 is also initiated in the centriolar region of the cell has not been determined. The observations that both types of IF aggregate in the perinuclear region in the presence of antimitotic agents 2~,30,33 and that in cells of a glioma line both IF proteins have a similar distribution and are present in the same IF 27.31, suggest that the assembly of the vimentin containing IF may also be initiated in this region. In the centriolar region the GFP may be incorporated into the vimentin type IF already present, may form IF containing only GFP or both depending on the availability of vimentin and GFP subunits at various stages of astrocyte differentiation. A close relationship between the centriote and the IF has not been observed with IF of the keratin type found in epithelial cells. In lysed cell models the assembly of new IF after addition of acid solubilized keratin could be initiated from a specific s~te near the nucleus but the position of this site bore no relationship to the posmon of the centriole s. There is considerable evidence, however, that the keratin type IF behave differently from IF of other types in the presence of antimitotic agents 22. Also during mitos~s in PtK2 cells they are not as closely associated with the centrioles as the IF of the vimentin type 2. Finally our study is different in that initiation of IF assembly during differentiation in intact cells rather than assembly of exogenous keratin m extracted cell models was examined. Our observation that the centriolar region contains sites which initiate the incorporation of GFP into IF may explain the close association between certain types of IF and centrioles 17.25,28-29-34,39 and IF and MT 13.15,16,38 observed in a number of previous studies and why both IF and MT radiate from a position near the nucleus 1,3A4,18. The close morphological proximity of IF with the centrioles have previously led to suggestions that the centriolar region of the cell may
325 serve as an o r g a n i z i n g c e n t e r for I F 17,39 as well as
gion and w h e t h e r this or o t h e r r e g i o n s s e r v e as sites
M T 4.26. This v i e w is c o n s i d e r a b l y s t r e n g t h e n e d by
of I F a s s e m b l y in fully d i f f e r e n t i a t e d cells r e m a i n s to
our o b s e r v a t i o n that the a s s e m b l y o f at least the G F P
be d e t e r m i n e d .
c o n t a i n i n g I F is initiated in this region. T h e results also indicate that the c e n t r i o l a r r e g i o n plays a l a r g e r role in the o v e r a l l o r g a n i z a t i o n of the c y t o s k e l e t o n
This w o r k was s u p p o r t e d
by M e d i c a l R e s e a r c h
t h a n p r e v i o u s l y s u s p e c t e d . W h e t h e r the a s s e m b l y of
C o u n c i l of C a n a d a G r a n t s MT-3302 to V . I . K . and
o t h e r types of I F is also initiated in the c e n t r i o l a r re-
MT-4235 to S.F.
1 Albrecht-Buehler, G. and Bushnell, A., The orientation of
centrioles in migrating 3T3 cells, Exp. Cell Res., 126 (1980) 427-437. 2 Aubin, J. E., Osborn, M., Franke, W. W. and Weber. K., Intermediate filaments of the vimentin-type and the cytokeratin-type are distributed differently during mitosis, Exp. Cell Res., 129 (1980) 149-165. 3 Borenfreund, E., Schmid, E., Bendich, A. and Franke, W. W., Constitutive aggregates of intermediate sized filaments of the vimentin and cytokeratin type in cultured hepatoma cells and their dispersal by butyrate, Exp. Cell Res., 127 (1980) 215-235. 4 Brinkley, B. R., Fistel, S. H., Marcum, J. M. and Pardue, R. L., Microtubules in cultured cells, indirect immunofluorescent staining with tubulin antibody, Int. Rev. Cytol., 63 (1980) 59-95. 5 Chiu, F. C., Norton, W. T. and Fields, K. L., The cytoskeleton of primary astrocytes in culture contains actin, glial fibrillary acid protein and the fibroblast-type filament protein vimentin, J. Neurochem., 37 (1981) 147-155. 6 Connolly, J. A. and Kalnins, V. I., Visualization of centrioles and basal bodies by fluorescent staining with non-immune rabbit sera, J. Cell Biol., 79 (1978) 526-532. 7 Dalai, D., Ruegar, D., Bignami, A., Weber, K. and Osborn, M., Vimentin, the 57000 molecular weight protein of fibroblast filaments is the major cytoskeletal component of immature glia, Eur. J. Cell Biol., 24 (1981) 191-196. 8 Eckert, B. S., Daley, R. A. and Parysek, C. M., In vivo disruption of the cytokeratin cytoskeleton in cultured epithelial cells by microinjection of anti-keratin: evidence for the presence of an intermediate filament organizing center, Cold Spring Harbor Syrup. Quant. Biol., 46 (1981) 403-412. 9 Fedoroff, S. and Doering, L., Colony culture of neural cells as a method for the study of cell lineage in the developing CNS: the astrocyte cell lineage, Curr. Top. Dev. Biol., 16 (1980) 283-304. 10 Fedoroff, S., White, R., Subrahmanyan, L. and Kalnins, V. I., Properties of putative astrocytes in colony cultures of mouse neopallium. In E. A, Costa Vidrio and S. Fedoroff (Eds.), Glial and Neuronal Cell Biology, Alan R. Liss, New York, 1981, pp. 1-19. 11 Fedoroff, S., White, R., Neal, J., Subrahmanyan, L. and Kalnins, V. I., Astrocyte cell lineage. II. Mouse fibrous astrocytes and reactive astrocytes in cultures have vimentin and GFP containing intermediate filaments, Dev. Brain Res,, 7 (1983) 303-315. 12 Fedoroff, S., Neal, J., Opas, M. and Kalnins, V. I., Astrocyte cell lineage. III. The morphology of differentiating mouse astrocytes in colony culture, J. Neurocytol., 13 (1984) 1-20.
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