Gamma-tubulin colocalizes with microtubule arrays and tubulin paracrystals in dividing vegetative cells of higher plants

Protoplasma (2000) 210:179-187 PROTOPLASMA �9 Springer-Verlag 2000 Printed in Austria

Gamma-tubulin colocalizes with microtubule arrays and tubulin paracrystals in dividing vegetative cells of higher plants

E. Panteris 1, e. Apostolakos 1, R. Griif 2, and B. Galatis 1,.

Department of Botany, Faculty of BioIogy, University of Athens, Athens and 2 Zellbiologie, Adolf-Butenandt-Institut, Universitfit Mttnchen, Munich

Received April 16, 1999 Accepted August 24, 1999

Summary. The distribution of 7-tubulin throughout cell division is studied in several taxa of higher plants. 7-Tubutin is present along the whole length of microtubules (Mts) in every cell stage-specific Mt array such as the preprophase band, the preprophase-prophase perinuclear Mts, the kinetochore Mt bundles, the phragmoplast, and the telophase-interphase transition Mt arrays, y-Tubulin follows with precision the Mt pattern, being absent from any other, Mt-free, cell site. In cells treated with anti-Mt drugs, y-tubulin is present only on degrading or on reappearing Mt arrays, while it is totally absent from cells devoid of Mts. y-Tubulin is also present in tubulin paracrystals, which are formed in colchicine-treated cells. These observations support the view that in higher plants 7-tubulin may not be a microtubule-organizing-center-specific protein, but it may play a certain structural and/or functional role being related to c~- and ~3-tubulin.

Keywords: 7-Tubulin; Microtubule organization; Plant cell division; Tubulin paracrystals.

Abbreviations: Mt microtubule; MTOC microtubule-organizing center; PPB preprophase band.

Introduction

y-Tubulin, since its first discovery (C. Oakley and Oakley 1989), has been found in several eukaryotes such as animals, plants, and fungi (see Joshi and Palevitz 1996, Balczon 1996, Marc 1997, Vaughn and Harper 1998). It has been suggested that it is involved in microtubule (Mt) nucleation, since it is located at the centrosome of animal cells (Stearns et al. 1991, Zheng et al. 1991) and at the spindle pole body (B. Oakley et al. 1990, Horio et al. 1991) and the spitzenk6rper (McDaniel and Roberson 1998) of

* Correspondence and reprints: Department of Botany, Faculty of Biology, University of Athens, GR-15784 Athens, Greece.

fungi. In animal cells, especially, discrete multiprotein ring complexes, consisting, among other components, of ?-tubulin and named y-somes, have been proposed to exist at the centrosome and to act as a template for Mt initiation and formation (Stearns and Kirschner 1994, Moritz eta!. 1995, Zheng et al. 1995).

The presence of 7-tubulin in higher plants (see Vaughn and Harper 1998) would shed light on the sites and mechanisms of Mt nucleation in cells which lack a discrete centrosome. 7-Tubulin is found in dividing cells of various flowering plants (Liu et al. 1993, 1994, 1995) and in developing guard cells (McDonald et al. 1993), as well as in spermatogenous fern cells (Hoffman et al. 1994).

However, apart from being a component of the centrosome of animal cells, 7-tubulin is also reported to be present along the Mts (Lajoie-Mazenc et al. 1994, 1996; Moudjou et al. 1996). In plant cells, as well, besides its Mt minus-end specificity (Li and Joshi 1995), y-tubulin is also located along the Mts (Hoffman et al. 1994, Joshi and Palevitz 1996). This pattern of distribution suggests that the role(s) of y-tubulin may be far more complicated and diverse than initially sup- posed (Vaughn and Harper 1998). Especially for plant cells, several proposals about a function alternative to Mt nucleation have been made (see Joshi and Palevitz 1996, Balczon 1996, Vaughn and Harper 1998).

Taking into account the formerly mentioned ambi- guity, we trace the distribution of y-tubulin throughout vegetative cell division in the liverworts Marchantia paleacea and Lunularia cruciata, the fern Adiantum capillus-veneris, and the angiosperm Vigna sinensis.

180 E. Panteris et al.: Gamma-tubulin localization in dividing higher-plant cells

D a t a conce rn ing y- tubul in d i s t r ibu t ion in b ryophytes

and ferns are l imi ted and, especially for vegeta t ive

cells, no i n fo rma t ion exists. Moreover , the possible

i n v o l v e m e n t of y- tubul in in Mt in i t i a t ion and/or orga-

n iza t ion in f lowering p lants is no t well established.

Besides n o r m a l cells, we examine the presence and

d is t r ibu t ion of y - tubul in in Mt-free, oryzal in-affected

cells, as well as in colchic ine- t rea ted cells con ta in ing

t ubu l i n paracrystals (Apos to lakos et al. 1990). The

possible re la t ionship of y - tubul in local izat ion to Mt

o rgan iza t ion is discussed.

Material and methods

Plant material

Thalli of the liverworts Marchantia paleacea and Lunularia cruciata as well as Adiantum capillus-veneris plants were collected from several sites around Athens, and cultures were kept in the labora- tory. Vigna sinensis seedlings were grown on cotton moistened with fresh water. Treatments with 10 gM oryzalin and 2mM colchicine were performed at room temperature as mentioned elsewhere (Apostolakos et al. 1990, Panteris et al. 1992). Oryzalin treatments lasted 6~48 h. In certain cases, a 6 h oryzalin treatment was followed by a recovery period of 10 h in fresh water. Colchicine treatment lasted 12 h.

Immunofluorescence microscopy

Young thalli of the liverworts and root tips of A. capillus-veneris and V. sinensis were fixed and prepared for immunofluorescence microscopy as described elsewhere (Panteris et al. 1991, Aposto- lakos and Galatis 1992). Incubations with the antibodies were carried out overnight at room temperature or for 1 h at 37 ~ In most cases, the material was first incubated with a mixture of anti- c~- (developed in rat, YOL 1/34; Seralab) and anti-y-tubulin antibody (developed in rabbit against Dictyostelium discoideum y-tubulin; Euteneuer et al. 1998) overnight, and afterwards with a mixture of ftuorescein isothiocyanate(FITC)-anti-rat (Sigma) and tetramethyl- rhodamine isothiocyanate(TRITC)-anti-rabbit (Sigma) at 37 ~ The anti-y-tubulin antibody was diluted 1 : 5 or 1 : 10 in phosphate- buffered saline (PBS) containing 1% bovine serum albumin. The same buffer was used for the dilution of all the antibodies applied. Anti-~-tubulin (developed in mouse; Amersham) was used, alterna- tively to anti-c~-tubulin, in certain cases, followed by FITC-anti- mouse (Sigma), while in others both anti-a- and anti-[%tubulin antibodies preceded the anti-y-tubulin antibody incubation. Control experiments omitting the incubation with anti-y-tubulin antibody were also performed.

After incubation with antibodies, the specimens were stained with Hoechst 33258 (Sigma) and mounted with a PBS-glycerol mixture containing 0.1% p-phenylendiamine (Sigma). They were examined under a Zeiss Axioplan epifluorescence microscope and photographed on Kodak T-Max 400 film, rated at ISO 1600 and developed in Kodak T-Max developer.

aminoethyl ether)-N,N,N',N'-tetraacetic acid, 0.2mM EDTA, 150mM KC1, 0.4M sucrose, 1% [w/v] polyvinylpyrrolidone 25, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, pH 6.9) and centrifuged at 9000 rpm (SLA1500 rotor) for 15 rain at 4 ~ The supernatant was again centrifuged at 31,000 rpm (70Ti rotor) for 45 min at 4 ~ Proteins in the supernatant were precipitated with ammonium sulfate (70% saturation) and pelleted at 9000 rpm (SS34 rotor) for 20 min at 4 ~ pellet was resuspended in 1 volume of homogenization buffer and mixed with sodium dodecyl sulfate (SDS) loading buffer. Proteins were separated by SDS elec- trophoresis on a 12.5% polyacrylamide gel (acrylamide-to-bisacry- lamide ratio 200 : 1). Proteins were transferred to a nitrocellulose membrane by semidry electroblotting (Kyhse-Andersen 1984). Blocking and antibody incubations were carried out as described elsewhere (Wieczorek et al. 1991). Alkaline phosphatase-conjugated anti-rabbit immunoglobulin G (Sigma, Deisenhofen, Federal Republic of Germany) was used as secondary antibody, and bands were visualized by the color reaction of nitroblue-tetrazolium chloride and bromo-chloro-indolyl phosphate (Biomol, Hamburg, Federal Republic of Germany).

Results

General remarks

Ant i -D ic tyos t e l i um y- tubul in does no t b ind to a - or

[3-tubulin, ne i the r of Dic tyos te l ium discoideum

( E u t e n e u e r et al. 1998) nor pig b ra in (R. Gr/~f unpub l .

obs.). Nevertheless , specificity of this polyc lonal anti-

body for y- tubul in in p lan t cells was conf i rmed by

i m m u n o b l o t s taining. As expected in V. sinensis cyto-

solic extracts the purif ied an t i se rum label led a single

b a n d at a molecu la r mass of approx imate ly 50 k D a

(Fig. 1), which could be clearly dis t inguished f rom a

cons iderab ly slower migra t ing b a n d s ta ined by the [3-

tubul in-specif ic m o n o c l o n a l an t ibody WA3 (data no t

shown). In immunof luo re scence microscopy, anti-y-

t ubu l i n s ta in ing is punc t ua t e and diffuse in all the

p lan t mater ia l used. The signal is more in tense in the

Immunoblotting

7-day-old seedlings of V. sinensis grown in dark were homogenized with 0.75 volumes of buffer (100 mM piperazine-N,N'-bis(2-ethane- sulfonic acid), 5raM MgSO4, 5mM ethylene glycol-bis([~-

Fig. 1. Immunoblot of a V. sinensis cytosolic extract with anti-y- tubulin (1). The relative molecular mass of standard proteins stained with Ponceau S (2) is indicated on the right

E. Panteris et al.: Gamma-tubulin localization in dividing higher-plant cells 181

Figs. 2-8. Preprophase (Figs. 2-4, 7, and 8), prophase (Fig. 5), and prometaphase (Fig. 6) cells of liverworts (Figs. 2-6) and A. capillus-veneris (Figs. 7 and 8). A Microtubules. B y-Tubulin. C DNA staining, xl000

Fig, 2. Anti-c~-tubulin staining is normal (A) in this control cell, in which anti-y-tubulin incubation is omitted. No signal is visible through the TRITC filter (B)

Fig. 3. An incomplete PPB is present at the right side (arrowhead in A) of this early preprophase cell (see nucleus after DNA staining in Fig. 4B inset), y-Tubulin is also present at the same site (arrowhead in B). Perinuclear Mts (A) are discernible and y-tubulin follows their pattern (B)

Fig. 4A, B. Same cell as in Fig. 3, but at median plane of focus. Intense Mt fluorescence is present at the poles of the perinuclear Mt array (arrowheads in A), where y-tubulin is also prominent (arrowheads in B). Note that y-tubulin is not restricted to the pole area but extends along the whole perinuclear Mt array

Fig. 5. Prophase cell in which y-tubulin (B) resembles the pattern of the perinuclear Mts (A)

Fig, 6A, B. Prometaphase cell in which the chromosomes are just released from the nuclear envelope (inset, after DNA staining). The forming Mt spindle (A) is resembled by the distribution of y-tubulin (B)

Fig. 7. ?-Tubulin (arrowhead in B) is present on the PPB (arrowhead in A) in this preprophase cell

Fig. 8. Median optical section of preprophase cell, in which y-tubutin (B) follows accurately the perinuclear Mts (A). y-Tubulin (arrowhead in !1) is also present at the PPB profile (arrowhead in A). Note that both Mts (A) and anti-7-tubulin staining (B) follow the protrusions of the nucleus (C)


liverworts, less intense in A. capillus-veneris, and rather weak in V. sinensis. Control specimens in all the plant material have no signal at all (Fig. 2). No difference in the pattern and intensity of immunostaining is observed, whether anti-7-tubulin antibody is applied before, after, or simultaneously with anti-a-tubulin and/or anti-13-tubulin antibodies. In specimens where both anti-a-tubulin and anti-13-tubulin antibodies are applied before anti-7-tubulin antibody, no difference is observed either.

Preprophase-prophase cells

Anti-y-tubulin immunostaining is more or less prominent along the preprophase band (PPB), in both early and late stages of organization (Figs. 3, 7, and 8). In the liverwort preprophase cells, where incomplete PPBs are present and the PPB does not follow the matura- tion stages, which are observed in higher plants (Apos- tolakos and Galatis 1985, 1992), anti-y-tubulin staining resembles the cell-specific Mt pattern (Fig. 3). Anti-y- tubulin staining is also detected on the perinuclear Mts, in both the preprophase and the prophase cells studied (Figs. 4, 5, and 8). It extends along the whole Mt arrays, at the PPB as well as the perinuclear Mts, although it is more intense at the preprophase- prophase spindle poles (Figs. 4, 5, 7, and 8). In A. capillus-veneris cells, where the preprophase nucleus protrudes towards the poles and the PPB, anti-y- tubulin follows the Mt pattern at the protrusions (Fig. 8). Especially in the liverworts, which display discrete polar organizers (Fowke and Pickett-Heaps 1978; Apostolakos and Galatis 1985, 1992; Brown and Lemmon 1990), two specific loci of intense anti- y-tubulin staining are observed on the poles of the preprophase-prophase spindle (Figs. 4 and 5). How- ever, even in this stage anti-y-tubulin staining is ex- tended along the whole preprophase-prophase spindle (Figs. 4 and 5). In prometaphase cells, anti-y-tubulin staining follows the pattern of the organizing metaphase spindle (Fig. 6).

Metaphase-anaphase cells

Anti-7-tubulin immunostaining can be observed all along the kinetochore Mt bundles, in metaphase cells of all the plants examined (Figs. 9, 11, and 13). 1,_ Tubulin is distributed all over the Mts and no special preference for the spindle poles and/or the kinetochores can be observed. During anaphase, while the

Figs. 9 and 10, Metaphase (Fig. 9) and anaphase (Fig. 10) cells of liverworts. The pattern of spindle Mts (A) is resembled by y-tubulin (B), 7-Tubulin is not restricted to the pole area but extends all along the spindle Mts (compare B with A). 7-Tubulin is also present together with interzonal Mts (arrowheads in Fig. 10) during anaphase. Insets Chromosomes after DNA staining, xl000

two daughter chromosome groups separate (Figs. 10 B inset, 12C, and 14C), anti-y-tubulin staining follows the Mt pattern (Figs. 10,12, and 14). Short kinetochore Mt bundles are stained by anti-y-tubulin all over their length (Figs. 10 and 14). In cells where interzonal Mts are present, they are lined by y-tubulin (Figs. 10, 12, and 14).

Telophase-cyrokinesis cells

In all the telophase-cytokinesis cells examined, anti-y- tubulin staining follows with accuracy the pattern of the phragmoplast Mts. At early stages of cytokinesis, where phragmoplast Mts are confined between the two daughter nuclei (Figs. 15A and 16A), anti-y- tubulin staining is prominent at exactly the same site (Figs. 15B and 16B). In cells, which are at a more advanced cytokinetic stage, where the phragmoplast Mts are restricted to the edges of the cell plate (Figs. 17A and 18A), anti-y-tubulin staining follows the same pattern (Figs. 17B and 18B). In all cases, y- tubulin covers the whole Mt length, showing no special preference for the Mt end either proximal or distal to the cell plate (Figs. 15-18). In late-cytokinetic and postcytokinetic cells of V. sinensis, exhibiting perinuclear Mt organization (Fig. 19 A), anti-y-tubulin stain-

E. Panteris et al.: Gamma-tubulin localization in dividin I hilher-Ilant cells 183

Figs. 11-14. Metaphase (Figs. 11 and 13) and anaphase (Figs. 12 and 14) cells of A. capillus-veneris (Figs. 11 and 12) and V. sinensis (Figs. 13 and 14). A Microtubules. B Anti-7-tubulin immunostaining. C DNA staining, xl000

Fig. 11. Anti-y-tubulin staining follows the Mts along the whole spindle in this cell (compare B with A)

I~g. 12. Intense anti-7-tubulin fluorescence (B) is observed on the interzonal Mts (A) of this anaphase (C) cell

Fig. 13. Anti-y-tubulin staining (B) is almost identical to the spindle Mts (A), from the poles to the kinetochores

Fig. 14. Anaphase cell (C) with short kinetochore Mt bundles and interzonal Mts (arrowhead in A). Anti-7-tubulin staining (B) follows the Mt pattern both at the former and the latter arrays (arrowhead in B)

ing is coincident with the per inuclear Mts (Fig. 19 B). In pos tcytokinet ic ceils of A. capillus-veneris, as well as in l iverwort cells of the same stage, which exhibit intense Mt f luorescence on the whole daughte r wall surface (Fig. 2 0 A ) (see also Panteris et al. 199], Apos - to lakos and Galatis 1992), anti-?-tubulin staining is also intense at the same site (Fig. 20B) .

Treatments with anti-Mr drugs

Adiantum capillus-veneris cells t rea ted with oryzalin for 6 b contain no Mts at all (Fig. 21 A). In such cells, no anti-y-tubulin staining can be observed (Fig. 21B). In cells recover ing f rom oryzal in t rea tment , anti-?- tubulin staining is located at the reappear ing Mt arrays. For example in the cell depic ted in Fig. 22, ?- tubulin (Fig. 22B) is localized along the whole reappear ing k ine tochore Mt bundles (Fig. 22A) . A p a r t f rom the reappear ing Mt arrays, no o ther site of the cell displays anti-?-tubulin staining (Fig. 22B).

Cells of L. cruciata t rea ted with oryzal in for 12 48 h contain no Mts at all (Fig. 23 A) or, in certain cases, some f ragmented disorganizing Mts still persist (Fig. 24A) . Anti-7-tubulin staining is present exclusively on these degrading Mt arrays (Fig. 24B) , while it is absent f rom Mt-free cells or cell sites (Figs. 23 B and 24B) . It should be no ted that anti-y-tubulin staining is also absent f rom the spindle poles of Mt- free p rophase ceils (Fig. 23). It is no t ewor thy that, in the p rep rophase -p rophase cell in Fig. 24, y-tubulin (Fig. 24B) is localized only at the site of the pole where Mt remnants persist (Fig. 24A) , while it is absent f rom the other.

Vigna sinensis cells t rea ted with colchicine include a re t iculum of tubulin paracrystals but no Mts at all (Figs. 2 5 A and 2 6 A ) (see also Apos to l akos et al. 1990). Anti-7-tubulin staining follows m o r e or less accurately the pat terns of these paracrystals (Figs. 25B and 26B) , being absent f rom any o ther cell site.


Figs. 15-20. Telophase cytokinetic (Figs. 15-18) and postcytokinetic (Figs. 19 and 20) cells of L. cruciata (Fig. 15), A. capiUus-veneris (Figs. 16, 17, and 20) and V. sinensis (Figs. 18 and 19). A Microtubules. B y-Tubulin, xl000

Figs. 15 and 16. The phragmoplast (A) is accurately followed by the distribution of y-tubulin (B) in these early cytokinetic cells

Fig. 17A, B. Cell at an advanced stage of cytokinesis. A Phragmoplast Mts are restricted at the border of the developing cell plate. B y- Tubulin exhibits the same pattern of distribution

Fig. 18. A Phragmoplast Mts from the margin of the cell plate of a broken cell at advanced cytokinesis. B y-Tubulin resembles with accuracy the pattern of the Mts

Fig. 19. A Post-cytokinetic cell showing many Mts around the daughter nuclei. B y-Tubulin distribution is also perinuclear

Fig. 20. A The daughter wall is lined by many Mts (arrowhead) in this cell. B Intense y-tubulin fluorescence is present at the same cell site (arrowhead)

Discu ss ion

The overal l y- tubulin dis t r ibut ion in all the ma te r i a l examined , e i ther n o r m a l or expe r imen ta l ly man ipu -

la ted, can be s u m m a r i z e d as follows. (a) 7-Tubutin is p r e sen t a long the M t a r rays of every cell division stage.

N o special p r e f e r ence for poss ible Mt minus ends can be observed . (b) y-Tubulin is absen t f r o m any cell site

devoid of Mts, in b o t h n o r m a l and oryza l in - t rea ted cells. (c) y-Tubulin is exclusively p re sen t in ~[3- tubul in paracrys ta l s in co lch ic ine- t rea ted cells.

The un i fo rm lining of all Mt a r rays with y-tubulin,

wi thout any p re fe rence at all for Mt minus ends, is an

obse rva t ion difficult to in terpre t . The poss ib le role of y- tubul in in Mt nuc lea t ion is es tabl i shed by its special locat ion at the mic ro tubu le -o rgan iz ing centers

( M T O C ) of an imal and fungal cells (see In t roduc t ion) . In p lan t cells, however , y- tubulin has been r epea t ed ly

shown to localize a long the Mts ins tead of be ing res t r ic ted to any special cell site(s) (Liu et al. 1993,

1994; H o f f m a n et al. 1994). I ts poss ib le role in Mt nucleat ion, based on its p r e f e r ence for the Mt minus


Figs. 21-26. Cells of A. capillus-veneris (Figs. 21 and 22), L. cruciata (Figs. 23 and 24), and V. sinensis (Figs. 25 and 26), treated with oryzalin (Figs. 21-24) or colchicine (Figs. 25 and 26). A c~/[3-Tubulin. B y-Tubulin. C DNA staining, xi000

Fig. 21. A Mts are totally absent from this cell, treated for 6 h with oryzalin. B No y-tubulin is present either; inset chromosomes after DNA staining

Fig. 22. Mitotic cell treated for 6 h with oryzalin, then left to recover for 10 h. A tripartite spindle is formed by the reappearing Mts (A). The same pattern is followed by y-tubulin (B); inset chromosomes after DNA staining

Fig. 23. Prophase (C) cell with no Mrs at all (A) after 24 h of oryzalin treatment. No y-tubulin signal can be observed either (B)

Fig. 24A, B. Preprophase-prophase cell treated with oryzalin for 12 h. A The remnants of Mts are limited to one pole of the perinuclear array (arrowhead). B y-Tubulin is exclusively present at the same cell site (arrowhead). C Nucleus after DNA staining

Figs. 25 and 26. A c~/13-Tubulin paracrystals form reticuli in these cells treated with colchicine for 12 h. B y-Tubulin resembles the patterns of the paracrystals

end (Li and Joshi 1995), is cha l l enged , s ince no struc-

tu res s imi lar to the y-somes (S tea rns and K i r s c h n e r

1994, M o r i t z e t al. 1995, Z h e n g et al. 1995) have b e e n

o b s e r v e d in p l an t cells. In our ma te r i a l , as well, the

h ighe r f luorescence in tens i ty of an t i -y- tubul in s ta in ing

at ce r ta in cell sites, such as the po les of the

p r e p r o p h a s e - p r o p h a s e sp ind le in l iverwor t s (Figs. 4 B

and 5 B), c anno t be d i rec t ly a t t r i b u t e d to a pu t a t i ve

M T O C act iv i ty p r o p o s e d for these si tes ( F o w k e and

P i c k e t t - H e a p s 1978; S t ee r 1984; A p o s t o l a k o s and

186 E, Panteris et al.: Gamma-tubulin localization in dividing higher-plant cells

Galatis 1985,1992; Brown and Lemmon 1990,1992). It may just be the result of clustering and converging of many Mts at these sites. This is further supported by the absence of anti-7-tubulin staining at these sites after oryzalin treatment. In Mt arrays where no Mt convergence occurs, such as the phragmoplast, anti- 7-tubulin staining intensity is uniform all along the Mts (see Figs. 15-18).

In addition, the role of 7-tubulin in animal cells is also doubted, since it is also found along the spindle Mts and not only at the centrosome (Lajoie-Mazenc et al. 1994, 1996; Moudjou et al. 1996; Heald et al. 1997). Especially in metaphase HeLa cells, 7-tubulin is present close to the kinetochore end of kinetochore Mt bundles (see Lajoie-Mazenc et al. 1994: fig. 5). Although there are several interpretations for the distribution of 7-tubulin along plant Mts (see Marc 1997, Vaughn and Harper 1998), clearly, a role in Mt nucleation cannot be assigned to 7-tubulin.

The most obvious difference in 7-tubulin distribution along Mts between this and previous studies in other plant cells (see Introduction) is that in the present study y-tubulin is not absent from the plus end of the Mts. This difference is not easy to understand. The anti-y-tubulin antibody applied here has been developed against Dictyostelium y-tubulin (Euteneuer et al. 1998) and, although plants and slime molds are evolutionary distant, it shows clear reaction with a single band around 50 kDa in extracts of V. sinensis by immunoblotting (Fig. 1), confirming its specificity for 7-tubulin in plant cells as well. Besides, in cells of brown algae it localizes at the centrosome (D. Karyophyllis, Department of Botany, Faculty of Biology, University of Athens, pers. commun.). An explanation could be that several different y-tubulins are present along Mts, which are recognized by the polyclonal anti-y-tubulin antibody applied in the present study. It is possible that the anti-y-tubulin antibodies applied by other authors do not stain the plus- end-specific y-tubulin isotype. This view, although highly speculative, can also be supported by the difference in anti-y-tubulin staining intensity between the plant taxa examined in the present study. Because of the evolutionary divergence between 7-tubulins of various species (Sobel and Snyder 1995, Euteneuer et al. 1998), every specific anti-y-tubulin antibody may show different affinity to different plant y-tubulin isotypes.

y-Tubulin distribution, in all the plant material examined in this study, cannot also be correlated to

any MTOC-relevant structure. Its total absence from Mt-free cells supports that it is an Mt-specific and not an MTOC-specific protein. Besides, in cells recovering from anti-Mt drug treatment, anti-y-tubulin staining appears only when the Mts reappear and does not precede their organization. Similar observations have been made recently in plant cells treated with several anti-Mt drugs (Binarova et al. 1998). In that study, ?- tubulin seems to have some sort of special relationship with the kinetochores. Whether there is a possible role in modulation and/or stabilization of kinetochore-Mt interactions (Binarova et al. 1998) or not, the fact is that y-tubulin is present together with the Mts at the kinetochores and is restricted neither there nor to the spindle poles. These observations favor the view that y-tubulin may play a certain stabilizing role along the Mts, rather than promote Mt initiation (Hoffman et al. 1994, Joshi and Palevitz 1996). It could well be that, apart from the a$-tubulin dimers, some dimers or even trimers containing y-tubulin are present along the Mt trunk, taking part in its stabilization and/or facilitating its reformation (Vaughn and Harper 1998).

The most novel observation of the present study is the presence of y-tubulin in colchicine-induced paracrystals. Such paracrystals have been observed in various higher-plant cells after colchicine treatment and have been proved to consist of, or at least to contain, cz- and ~-tubulin (Apostolakos et al. 1990, Karagiannidou et al. 1995). The presence of y-tubulin in these paracrystals shows that this protein is related to a- and ~3-tubulin in a more general way and not only in functional structures such as the Mts. As a con- clusion, 7-tubulin may have in plant as well as in animal cells a role far more complicated than that of a unique Mt initiator, and further research is required to understand its possible involvement in the various cell phenomena.

Acknowledgment

We ~harzk George Komis for his va[ualzle hellz in preparing samples for immunoblotling,

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Gamma-tubulin colocalizes with microtubule arrays and tubulin paracrystals in dividing vegetative cells of higher plants