Tubulin post-translational modifications and the construction of microtubular organelles in

ROSEMARY SASSE and KEITH GULL* hiologicnl Laboratory, , Canterbury, Kent CT2 7NJ, UK

* Author for correspondence

Summary

We have used specific monoclonal antibodies to in the cell cycle. T. brucei therefore, represents a facilitate a study of acetylated and tyrosinated cell type with extremely active mechanisms for a'- in the (MT) arrays in the the post-translational modification of a-tubulin. Trypanosoma brucei cell. Acetylated a-tubulin is Our analyses of the timing of acquisition and not solely located in the stable microtubular modulation in relation to MT construction in T. arrays but is present even in the ephemeral brucei, suggest that acetylation and detyrosin- of the mitotic spindle. Moreover, ation of a'-tubulin are two independently regu- there is a uniform distribution of this isoform in lated post-translational modifications, that are all arrays. Studies of flagella complexes show that not uniquely associated with particular subsets of acetylation is concomitant with assembly of MTs. MTs of defined lability, position or function. Post- There is no subsequent major modulation in the assembly detyrosination of a-tubulin may pro- content of acetylated a'-tubulin in MTs. Con- vide a mechanism whereby the cell could discri- minate between new and old MTs, during con- versely, polymerizing flagellar MTs have a high struction of the cytoskeleton through the cell tyrosinated n-tubulin content, which is sub- cycle. However, we also suggest that continuation sequently reduced to a basal level at a discrete of detyrosination, allows the cell, at cell division, point in the cell cycle. The MTs of the intranu- to partition into daughter cells two equivalent sets clear mitotic spindle appear never to contain of cytoskeletal MTs. tyrosinated a-tubulin, suggesting that they are actually constructed of detyrosinated a'-tubulin or Key words: tubulin, acetylation, tyrosination, that detyrosination is extremely rapid at this time microtubules, Trypanosoma bnicei.

Introduction 1982) and the second involves the acetylation of a lysinc within the polypeptide chain (L'Hernault & Roscn- Tubulin isotypc diversity can be established in cells by baum, 1983, 1985a,b). Evidence that these events lead the expression of a heterogeneous multi-gene family to the production of distinct sub-populations of MTs (Cleveland & Sullivan, 1985). However, initial examin- within cells comes mainly from the use of monoclonal ation of the products of these genes suggests that they and polyclonal antibodies that can discriminate be- do not become located in specific microtubule (MT) arrays within the cell (Lewis et al. 1987; Lopata & tween the tubulin isoforms; tyrosinated and detyrosin- Cleveland, 1987). In contrast to this position, two ated (Glu) a'-tubulin and acetylated a'-tubulin (Gun- reversible post-translational events are now known that dersen et al. 1984; Thompson et al. 1984; Piperno & lead not only to the production of specific tubulin Fuller, 1985; LeDizet & Piperno, 1986; Diggins & isoforms, but ultimately to the production of hetero- Dove, 1987; Kreis, 1987; Piperno et al. 1987; Sassee/ geneous populations of microtubules (MTs) within one al. 1987; Wehland & Weber, 1987). cell. Both reversible post-translational modifications In cultured animal cells, while the majority of affect the a'-tubulin molecule. The first involves the interphase MTs appear to be tyrosinated removal of the carboxy-terminal tyrosine residue O'-tubulin-rich (Wehland et al. 1983; Gunderscn et al. (Barra et al. 1973; Agarana et al. 1980; Thompson, 1984), Glu ar-tubulin-rich MTs are confined to Journal of Cell Science 90, - (1988) Printed in Great Britain © The Company of Biologists Limited 1988 577 specialized arrays such as the centrioles, primary cilia their tubulin isoform composition. and midbodies, and some differentiated structures We have used the monoclonal antibody, 6-1 IB-1 such as axons (Gundersen & Bulinski, 1986a,6; Cam- (Piperno & Fuller, 1985), in order to provide an bray-Deakin & Burgoyne, 1987a,6; VVehland & Weber, immunological characterization of the acetylated 1987). A subpopulation of Glu O'-tubulin-rich MTs a'-tubulin isoform of T. brucei. Use of this antibody in does exist in the cytoplasm of some cells; however, immunofluorescence studies reveals that acetylated their number can vary between cell lines and even a'-tubulin is present throughout all of the MT arrays of between cells of the same cell line (Gundersen et al. this cell, even in the spindle MTs. Therefore, acety- 1984; Gundersen & Bulinski, 19866; Cambray-Deakin lated a'-tubulin is not solely located to the stable MT & Burgoyne, 19876; Kreis, 1987; Schulze et al. 1987; arrays. Studies of isolated flagella complexes show that Wchland & Weber, 1987). In many cases Glu MTs MTs become acetylated during their polymerization have been characterized as being more stable in terms phase and show no subsequent major modulations in of drug resistance, cold stability and turnover (Cam- the content of acetylated a'-tubulin. Conversely, stain- bray-Deakin & Burgoyne, 19876; Gundersen et al. ing with the tyrosinated cv-tubulin-specific antibody 1987; Kreis, 1987; Schulze et al. 1987; Wehland & YL1/2 (Kilmartin et al. 1982; Wehland et al. 1984), Weber, 1987). Acetylation of a'-tubulin was originally shows that these polymerizing MTs have a high characterized as a post-translational modification oc- tyrosinated a'-tubulin content, which is subsequently curring during formation of the flagellum in Chlamy- reduced to a basal level. This may provide the cell with domonas (L'Hernault & Rosenbaum, 1983, 1986a,6). a method for discriminating between new and old MTs Subsequently, it was detected in other microorganisms during the cell cycle, whilst also permitting the pro- and, via the use of monoclonal antibodies, in animal duction of equivalent daughter cells at cytokinesis. The cells (Thompson et al. 1984; Piperno & Fuller, 1985; MTs of the intranuclear mitotic spindle appear never to Diggins & Dove, 1987; LiDizet & Piperno, 1987; contain tyrosinated a'-tubulin, suggesting that they are Piperno et al. 1987; Sasse et al. 1987). In animal cells actually constructed of detyrosinated a'-tubulin or that the population of acetylated MTs has a distribution detyrosination is extremely rapid at this time in the cell pattern showing a remarkable similarity to that of Glu cycle. T. brucei therefore, represents a cell type with MTs (compare Gundersen et al. (1984) with Thomp- extremely active mechanisms for the post-translational son et al. (1984) and Piperno et al. (1987)). These modification of a'-tubulin. Our analyses of the timing relationships have recently been assessed directly by of acquisition and modulation in relation to MT comparing the stable, the acetylated and the detyrosin- construction in T. brucei suggests that acetylation and ated MTs within the same cell (Cambray-Deakin & detyrosination of a'-tubulin are two independently Burgoyne, 19876; Schulze et al. 1987). In some regulated post-translational modifications, that are not cultured animal cells these were found to be properties uniquely associated with particular subsets of MTs of of the same cytoplasmic MT sub-set; however, vari- defined lability, position or function. ations in this relationship were found in other cell types (Schulze et al. 1987). Materials and methods We have recently shown that both the acetylation and tyrosination post-translational modification cycles op- Trypanosomes erate in Ttypanosoma brucei (Stieger et al. 1984; Procyclic Trypanosoma brucei brucei, stock 427 were grown Schneider et al. 1987; Sherwin et al. 1987). The in tissue-culture flasks in SDM 79 medium (Brun & Schonen- construction of new MT arrays takes place at discrete berger, 1979). Cells were harvested during exponential times within the cell cycle and a number of morpho- growth, i.e. at approximately 5X 106 cells ml"'. logical markers permit identification of the position of individual cells within the cell cycle (Sherwin et al. Antibodies 1987; Sherwin & Gull, 1988). The MT arrays con- The antibodies used in this study are as follows: 6-11B-1, a structed during the cell cycle vary in their type, mouse monoclonal antibody raised against sea-urchin sperm stability and position. Two sets of stable MTs are axonemes and characterized as reacting specifically with constructed, subpellicular MTs and flagellar MTs, acetylated a'-tubulin (Piperno & Fuller, 1985); YLl/2, a rat both of which remain present thereafter even during monoclonal antibody raised against yeast tubulin and charac- mitosis. Later in the cell cycle ephemeral MTs are terized as reacting with tubulin carrying an aromatic amino acid at its carboxy terminus (Kilmartin et al. 1982; Wehland polymerized in the intranuclear mitotic spindle (Vick- et al. 1984); DM1 A, a general crossreacting anti-tr-tubulin erman & Preston, 1976). This system therefore now monoclonal antibody (Blose et al. 1984); KMX-1, a general provides an opportunity to establish the relationship crossreacting anti-/3-tubulin monoclonal antibody (Birkett et between construction of discrete MT arrays, the timing al. 1985); a"T12, a rabbit polyclonal antisera specific for of their acquisition of post-translationally modified detyrosinated a'-tubulin (Kreis, 1987). O"T12 was used as a forms of tubulin and the subsequent modulation of potential probe for detyrosinated tubulin in immunofluor-

578 R. Sasse and K. Gull escence studies but it gave no reaction with T. brucei cells. ation of the new and old flagella complexes. The staining was This lack of reactivity was most probably due to the non- the same regardless of the fixative used. conserved amino acid composition of trypanosome and ver- tebrate brain tubulin in the epitope region (Ponstingl et al. Two-dimensional gel electrophoresis and 1981; Kimmel et al. 1985). FITC-labelled second antibodies immiinoblotting were bought from Sigma. Two-dimensional electrophoresis was performed essentially according to the procedure of O'Farrell (1975) with the Immunofluorescence modifications described by Burlande/ al. (1983). Transfer of Single indirect immunofluorescence of whole T. bnicei cells proteins to nitrocellulose and immunostaining was carried was carried out either as described by Sherwin et al. (1987) out as described (Birkett et al. 1985). or, for the more detailed study of the stages of mitosis, the following protocol was used. Cells were harvested by low- speed centrifugation, resuspended in 3-7% formaldehyde in Results phosphate-buffered saline (PBS), pH7-4, for 5min, har- vested by centrifugation, resuspended in PBS, settled on Immunological characterization of the acetylated a- polylysine-coated slides, fixed again in cold methanol for 5 min and rehydrated in PBS for 5 min. The antibody tubulin isoform ofT. brucei incubations and washes were then carried out as previously The monoclonal antibody 6-11B-1, which has been described. After the last wash, 4,6-diamidino-2-phenylindole characterized as reacting specifically with cv-tubulin, (DAP1) l^gml"' in water was applied to the slides for 30s which is acetylated at lysine 40 (Piperno & Fuller, and the slides were washed in water. The cells were mounted 1985; LeDizet & Piperno, 1987), was used to character- 1 under coverslips using Moviol containing 1 mgml" p-phen- ize immunologically the acetylated cv-tubulin isoform ylenediamine as an antifade. Cells were fixed in solution to of T. brucei. Fig. 1A,B shows the tubulin regions of study mitosis, in order to stop flattening of the cells and two identical Western blots of total T. brucei proteins distortion of the positions of the nuclei, which can occur separated by two-dimensional gel electrophoresis. The when the cells are settled on coverslips before fixing. This, however, led to some parts of the cells being out of the focal blot in Fig. 1A was probed with the general cross- plane when focusing on the nuclear region. reacting anti-ar-tubulin monoclonal antibody DM1 A, Double YLl/2 indirect/KMX-1 direct immunofluor- and shows the distinctive a\/a1 tubulin isoform escence was carried out by following the above protocol, pattern previously characterized as representing the using YLl/2 as the first antibody and FITC-conjugated goat primary translation product (a'1-tubulin) and a post- anti-rat igG (whole molecule) as the second antibody. Then, translationally derived isoform (a'3-tubulin) before application of the DAPI, two further antibody incu- (Schneider et al. 1987). The blot depicted in Fig. IB bations were performed: first, the cells were incubated in probed with 6-11B-1, however, shows only the more 10% rat serum in PBS and washed in PBS; then the cells abundant acidic a'3-tubulin isoform, indicating that were incubated in rhodamine sulphonylchloride-conjugated only this isoform is acetylated. This is in agreement KMX-1 in PBS containing 1 % rat serum and washed in PBS. with our previous in vivo labelling experiments Rhodamine sulphonylchloride was conjugated to KMX-1 (Schneider et al. 1987). according to the method of Brandtzaeg (1973). Isolated flagella complexes were prepared for immunoflu- orescence as follows. Cells were harvested, resuspended in PBS and settled on polylysine-coated coverslips; cytoskel- etons of the cells were obtained by incubating the coverslips in MME buffer (lOmM-Mops, pH6-9, lmM-EGTA, 1 mM- MgSO4) containing 0-1% Triton X-100 on ice with three changes of buffer for 10 min; isolated flagella complexes were obtained from these cytoskeletons by incubating the cover- slips in MME buffer containing 1 M-NaCl and 0-1 % Triton X-100 on ice with three changes of buffer, until the subpelli- cular MTs were judged by phase-contrast microscopy to have been solubilized; the flagella complexes were washed in PBS, fixed in either cold methanol, cold acetone or 3-7% formal- dehyde in PBS for 10 min, then rehydrated in PBS. Antibody incubations were then carried as normally and the coverslips Fig. 1. Tubulin regions of two identical Western blots of were mounted as above. This protocol did not always lead to total T. brucei proteins separated by two-dimensional the complete solubilization of the subpellicular MTs and electrophoresis. A. Probed with the general anti-a'-tubulin indeed a group of subpellicular MTs attached to basal bodies monoclonal antibody DM1A showing the pattern of a\- forming the flagellar pocket (our own ultrastructural studies; and a'3-tubulin isoforms. B. Probed with the anti- Sherwin & Gull, 1988) appeared to be most resistant to the acetylated a'-tubulin monoclonal antibody 6-11B-1 showing treatment. These MTs did not, however, hamper visualiz- only the a'3-tubulin isoform.

Tubulin modifications and microtubule construction 579 Spatial localization of acetylated a-tubidin Our previous analysis of the cr-tubulin isoform content of the flagellar axoneme and cell body MTs by cell fractionation and two-dimensional gel electrophoresis has shown that the acetylated a^-tubulin isoform is present in both. However, the ratio of acetylated to unacetylated cv-tubulin differs between the two, the flagella containing almost exclusively acetylated cv-tubulin and the cell body MTs containing both unacetylated and acetylated a-tubulin (Schneider etal. 1987). These biochemical characterizations were unable to define whether local heterogeneities were present within MT arrays or indeed whether modu- lations of acetylated tf-tubulin occurred as MT mor- phogenesis progressed through the cell cycle. Having established the selectivity and specificity of the mono- clonal antibody 6-11B-1 for the acetylated a^-tubulin isoform in T. brucei we were then able to address these questions using an immunofluorescence approach. Indirect immunofluorescence using 6-11B-1 was car- ried out on an asynchronous culture of procyclic T. brucei. A typical set of micrographs of the acetylated O'-tubulin immunofluorescence image and the corre- sponding DAPI fluorescence image of the nuclear and kinetoplast DNA of the same cells are shown in Fig. 2A,B. From studying such micrographs, it can be seen that the acetylated tf-tubulin appears evenly distributed throughout the subpellicular MTs of all cells. Acetylated O"-tubulin is also present in the flagellar axonemes; however, this is more difficult to visualize in intact cells, since flagella are attached to the cell body along almost their entire length. The bright fluorescence from the cell body therefore obscures the image of the flagellum. An advantage of T. brucei is that the DAPI fluorescence image provides an indicator of the position of a cell in the cell cycle by assessment of the number and positions of kinetoplasts and nuclei (Shcrwin et al. 1987; Sherwin & Gull, 1988). Using this counter-staining technique it can be seen that the distribution of acetylated ce-tubulin is essentially the same at all stages (see Fig. 2 and Fig. 3A—C, which show individual cells at three discrete stages through the cell cycle as determined by their DAPI fluor- escence, Fig. 3G—I), indicating no gross modulation of the acetylated ar-tubulin isoform in subpellicular MTs through the cell cycle. Close inspection of cells at different depths of focus revealed a certain percentage of cells that had an extra very bright area or line of immunofluorescence showing through the cell body (arrowed at each end in a cell in Fig. 2A). When such cells were aligned with their DAPI fluorescence image, Fig. 2. 6-11B-1 indirect immunofluorescence (A) and they always possessed two kinetoplasts some distance DAPI fluorescence (B) micrographs of an asynchronous apart and the staining fell over the nuclear region, population of procyclic T. brucei cells. Acetylated indicating that the fluorescence image represented the O"-tubulin is evenly distributed throughout the subpellicular MTs, the flagellar axoneme of all cells, and in the mitotic spindle MTs showing through the subpellicular intranuclear spindle MTs (arrowed at each end) of a cell at staining (see below). Acetylated cr-tubulin is, there- mitosis. Bar, lOftm.

580 R. Sasse and K. Gull Fig. 3. Two panels of indirect immunofluorescence micrographs of individual cells at three discrete stages of the cell cycle, as determined by their DAPI fluorescence images (G-L). The distribution pattern of acetylated a'-tubulin as revealed by 6-11B-1 (A-C) is essentially the same at each stage. Thus, indicating that no major modulation in the presence of acetylated a'-tubulin occurs during the cell cycle. The distribution pattern of tyrosinated a'-tubulin as revealed by YLl/2 (D—F), however, changes during the cell cycle. Early in the cycle tyrosinated a'-tubulin is preferentially located in the posterior third of the cell and in the basal body, but not in the flagellum (D). Later in the cycle tyrosinated a'-tubulin is also detected in the new growing flagellum (arrowed in E). Tyrosinated a'-tubulin is lost from the new flagellum, by the time the cell is ready to undergo cytokinesis; however, it is retained in the basal body (F). Bar, 5 fim. fore, present not only in the subpellicular MTs and in clusions from the original biochemical descriptions of the flagellar axoneme, but also in the intranuclear acetylation of a'-tubulin in Chlamydomonas (L'Her- mitotic spindle. nault & Rosenbaum, 1985a). The important question, The results of our earlier biochemical study are therefore, is when do the a'-tubulin subunits in an consistent with the idea that acetylated tubulin is elongating MT become acetylated? Are they acetylated present mainly, if not totally, in the assembled MTs at the point of assembly or is there a lag between and is not present to any significant extent in the assembly and acetylation? We have been able to address unpolymerized pool tubulin (Schneider et al. 1987). this question in T. brucei by analysing the acetylation Initially, this assembled tubulin of the axoneme and state of the MTs of the new flagellum as it elongates subpellicular MTs appears to be the tyrosinated form throughout the cell cycle. The old flagellum alongside of tubulin and is subsequently modified to the detyro- acts as a reference point in these experiments. We are sinated form. Consequently, immunofluorescence de- able to attach cells to coverslips and remove the main tection of tyrosinated tubulin reveals it to have a bulk of the subpellicular MTs, leaving flagella com- restricted location within the MT arrays, and to be plexes attached to the coverslip. Since cells are present subject to a temporal modulation during the cell cycle. at all stages in the cell cycle, the resulting flagella Thus, tyrosinated tubulin appears to act as a marker for complexes represent all stages of growth of the new newly assembled MTs (Sherwin et al. 1987). There- flagellum. These flagella complexes were stained with fore, the lack of a restricted segregation pattern or the 6-11B-1 antibody, using indirect immunofluor- modulation of acetylated a'-tubulin, which we now escence microscopy. Fig. 4A-E shows the results of show is in contrast to the pattern observed for tyrosi- this experiment. It is clear from these experiments that nated a'-tubulin (compare Fig. 3A—C with 3D—F), the MTs of the new flagellum are acetylated during the suggests some level of independence of the modifi- elongation stage. Even very short flagella arc acetylated cation cycles. and careful comparison of these images with phase- The lack of the acetylated o\3-tubulin isoform in the contrast images of the same flagella showed that there unpolymerized tubulin pool fraction suggests that was no detectable zone of unacetylated a'-tubulin at the acetylation occurs around the time of assembly of the extending distal tip of the elongating flagellum. Also, MT arrays. These results * confirmed similar con- double staining with directly labelled anti-j3-tubulin

Tubulin modifications and microtubule construction 581 Fig. 4. Indirect imniunofluorescence micrographs of isolated flagellacomplexe s from individual cells ordered according to the length of the new flagellum. Complexes probed with 6-11B-1 (A-E) show that acetylated a-tubulin is present in the new flagellumfro m the time of assembly and its presence is not subsequently modulated. Probing with YLl/2 (F—J) showed that the tubulin of the new flagellumi s heavily tyrosinated, but that the tyrosine is subsequently removed selectively from the axoneme, but not the basal body, to reach the steady-state level of tyrosination characteristic of a uniflagellated cell (F). In each case, the distal tip of the new flagellum is indicated with an arrow and the distal tip of the old flagellum is marked with an arrowhead. In A and F the new basal body is marked with a small arrow. Bar, Sftm. antibody (KMX-1) and 6-11B-1 showed complete assembly and elongation. This is most dramatically coincidence of staining (results not shown). Thus, we illustrated by Fig. 4H, where a clear contrast is seen conclude from these results that, at least for the MTs of between the bright fluorescence of the new flagellum the flagellar axoneme, acetylation of tubulin subunits is and the low fluorescence of the old. The tyrosination coincident with assembly of these subunits. Further, level of flagellar MTs remains relatively high during examination of the old flagellum of the complexes elongation of the flagellum (shown by Fig. 4G-I), revealed in Fig. 4A-E indicates that there is no dra- although by the end of flagellum growth there appears matic modulation of the acetylation state of MTs with to have been a detectable amount of detyrosination. time. The intensity of staining with the 6-1 IB-1 However, even at these late stages the MTs of the new antibody remains relatively constant after the assembly flagellum are still more tyrosinated than those of the of flagellar MTs (Fig. 4A-E). old. This de-tyrosination process must then continue in We have used these isolated flagella complexes to the later stages of the cell cycle in order to reduce the provide further insight into the detyrosination of tyrosination state of the new flagellum down to that of O'-tubulin subunits in relation to construction of MT the old. This last point comes from examination of arrays. YLl/2 staining of isolated flagella complexes uniflagellated cells at the start of the cell cycle. Since confirmed our earlier general results obtained using these comprise a single population in respect of the whole cells. Cells at the start of a cell cycle possess a flagellum tyrosination state, it appears that diminution single flagellum (the old flagellum), which contains to a low level of tyrosination is achieved by the start of only low levels of tyrosinated tubulin, consequently it the new cell cycle. Use of the isolated complexes has stains very poorly with YLl/2 antibody (Fig. 4F). At a revealed an additional detail regarding the detyrosin- later stage in the cell cycle cells initiate the formation of ated old flagellum. The improved resolution of the a new flagellum alongside the old. The new flagellum present results, shows that although the old flagellum is showed intense fluorescence during the period of MT generally detyrosinated, the distal tip still shows some

582 R. Sasse and K. Gull YLl/2 staining. It may be that this staining is a detect the spindle despite the fluorescence of the reflection of a low level of tubulin turnover within a subpellicular MTs. Careful focussing close to the mature flagellar axoneme. centre of the cell revealed spindles of differing mor- Therefore, the newly assembled MTs of the flagellar phologies in an asynchronous cell population. We were axoneme show a high level of a-tubulin tyrosination. able to use counterstaining with DAPI to provide the This extent of tyrosination declines with time, a morphological markers required to order cells in terms process that apparently begins before the flagellum is of their relative positions within the mitotic phase of complete. By the end of the cell cycle the extent of the cell cycle. In this study cells were fixed in solution tyrosination of the old and new flagella is indistinguish- (see Materials and methods) before application of able. antibodies in order to stop flattening of cells and distortion of the nuclear position, which may occur The mitotic spindle of trypanosome cells when cells are allowed to settle on polylysine-coated Mitosis in trypanosomes is intranuclear and, although slides before fixing. many early workers clearly observed and described Representative ordered images obtained using mitotic forms seen by light microscopy (Hoare, 1972), KMX-1, a monoclonal antibody that detects /?-tubulin relatively few ultrastructural studies have been carried and therefore displays the whole population of spindle out. Much debate still exists regarding chromosome MTs, are shown in Fig. 5. In T. brucei nuclear division number, arrangement and spindle morphology (Vick- occurs after kinetoplast DNA replication and division. erman & Preston, 1970; Solari, 1980). Moreover, there Kinetoplast separation is initiated at 0-65 of the unit has been no immunofluorescence study of the trypano- cell cycle (Sherwin & Gull, 1988). Fig. 5A,G,M, some spindle. Our initial observations using anti- shows a cell just after this point in the cell cycle; the cell tubulin antibodies suggested that it was possible to has two kinetoplasts but does not yet possess a mitotic

M N

Fig. 5. KMX-1 indirect immunofluorescence (A-F), DAPI fluorescence (G-L) and phase-contrast (M—R) micrographs of individual cells ordered to show the morphological changes that occur during mitosis. A,G,M. A cell without a spindle just before mitosis. B,H,N. A cell at 'metaphase'. C,I,O and D,J,P. Cells undergoing 'anaphase A'. E,K,Q. Cells undergoing 'anaphase B'; and F,L,R, 'telophase'. Bar, 5[im.

Tubulin modifications and microtubide construction 583 spindle. Mitosis is initiated at 0-84 of the unit cell cycle (Sherwin & Gull, 1988) and a cell at this stage of the cell cycle is seen in Fig. 5B,H,N. Cells at this stage had oval nuclei with no clear nucleolus in their phase image (Fig. SN), and the immunofluorescence image showed an oval spindle with pointed ends (Fig. 5B). At this early stage the nuclear DNA, as detected by DAPI, showed a degree of congression along the spindle mid- line but was excluded from a central line running from pole to pole (Fig. 5H). Cells at the second stage of mitosis could be identified by their possession of slightly longer spindles with more rounded ends (Fig. 5C,D). At this stage the kinetoplast connected to the maternal flagellum had moved to the side of the posterior end of the nucleus (Fig. 51). The images seen in Fig. 5C,I and 5D,J appear to represent stages in the anaphase A movement of chromosomes to spindle poles. After this anaphase A movement the spindle elongates and immunofluorescence shows two bundles of MTs that appear to twist and cross in the middle (Fig. 5E). The DAPI image of cells at this stage showed that the nuclear DNA had been sequestered at the two poles and that the kinetoplasts had moved further towards the anterior end of the cell, with the one associated with the old flagellum lying near the centre of the nucleus (Fig. 5K). The final stage that could be identified is characterized by cells showing a single line of bright immunofluorescence that, when overlaid on the DAPI image, lies been two distinct areas of nuclear DNA (Fig. 5F,L). Cells containing two distinct nuclei with very clear nucleoli had lost the single line of spindle immunofluorescence.

Acetylated a-tubulin is present in the intranuclear mitotic spindle MTs Our above analysis of the mitotic spindle reveals a MT organelle that is constructed at around 0-84 of the unit cell cycle and has completely disappeared before cyto- kinesis is initiated. Thus, we decided to investigate the presence of post-translationally modified cv-tubulin in the MTs of this ephemeral organelle. Fig. 6. 6-11B-1 indirect immunofluorescence micrographs We used the monoclonal antibody 6-11B-1 to reveal of cells at four characterized stages of mitosis (A,D,G,J) as the distribution of acetylated ar-tubulin in the mitotic determined by their DAPI fluorescence (B,E,H,K) and spindle. Results of this analysis indicate that acetylated phase-contrast images (C,F,I,L), showing that acetylated cr-tubulin is present in the MTs of the intranuclear (T-tubulin is present in the intranuclear spindle at all of mitotic spindle at all stages of mitosis. Examples of the these stages. Bar, 5/.im. staining pattern are seen in Fig. 6A,D,G,J, illustrating the distribution of acetylated ar-tubulin at each of the tyrosinated cv-tubulin, may act as a marker for newly four characteristic stages of mitosis. Close examination assembled MTs, a phenomenon most clearly seen in of the overall pattern at this level of resolution shows the case of flagellum growth. We have shown above that that there is no major difference between the distri- the MTs of the new flagellum are both acetylated and bution of acetylated cv-tubulin and total tubulin. tyrosinated. In this context we now asked whether the acetylated MTs of the mitotic spindle were also tyrosi- YL112 does not stain spindle MTs nated and so detectable with the YLl/2 antibody. Data presented in our previous study (Sherwin et al. When YLl/2 immunofluorescence micrographs of ex- 1987) and above show that YLl/2, which stains ponentially growing T. brucei cells were studied, none

584 R. Sasse and K. Gull of the spindle structures identified using KMX-1 or yet in none of the cells can the spindle be detected using 6-11B-1 could be visualized, even though mitotic forms the YLl/2 antibody. In order to show conclusively that could be clearly identified from phase-contrast and such cells did indeed contain spindle MTs, we carried DAPI images and masking of the immunofluorescence out double-label experiments with YLl/2 and image due to subpellicular MT staining was negligible, KMX-1. When YLl/2 indirect/KMX-1 direct immu- as YL1/2 does not stain the majority of these. This nofluorescence was carried out the same result was result is illustrated in Fig. 7. These micrographs illus- evident, even with very early spindles that could be trate cells at the characteristic stages of mitosis, as easily identified with the directly labelled KMX-1 described above (see DAPI images, Fig. 5B,E,H,K), (Fig. 8). This lack of staining with YLl/2 was a totally consistent and reproducible result over many exper- iments. We searched many thousands of cells in asynchronous populations for cells showing all of the characteristics of the earliest stage of mitosis. Examin- ation of all of these cells never revealed a cell with a spindle that stained with YLl/2.

Discussion

In our previous study we provided biochemical evi- dence that the electrophoretically defined O'3-tubulin of T. brucei was produced by a post-translational acety- lation event (Schneider et al. 1987). Our present work with the 6-11B-1 monoclonal antibody now provides immunological confirmation of this conclusion. More- over, this result indicates that the acetylation site on T. brucei a'-tubulin is the same as that detected in other organisms. The epitope on a'-tubulin detected by 6- 11B-1 has recently been characterized as encompassing an aeetylated lysine at position 40 (LcDizet & Piperno, 1987). The known a'-tubulin sequence from trypano- somes has a lysine at position 40 (Kimmel et al. 1985). This present immunological characterization of aeety- lated a'-tubulin in T. brucei, together with our previous immunological characterization of tyrosinated a'-tubulin (Sherwin et al. 1987), has enabled us to address the nature of the relationship between both of these post-translational modifications and the construc- tion of different MT arrays within one cell. Use of the 6-11B-1 antibody showed that aeetylated a'-tubulin has a universal distribution throughout all the microtubular arrays of T. brucei. This is in marked contrast to the observed location of aeetylated a'-tubulin in many other organisms or types of cultured animal cells where the aeetylated a'-tubulin is restricted to subpopulations of MTs (Cambray-Deakin & Bur- goyne, 1987a,6; Diggins & Dove, 1987; LeDizet & Piperno, 1987; Piperno et al. 1987; Sasse et al. 1987; Schulze et al. 1987). The immunological detection of aeetylated a'-tubulin within the subpellicular and fla- gellar MTs is consistent with the generally observed Fig. 7. YLl/2 indirect immunofluorescence micrographs of individual cells (A,D,G,J) at the four characterized view that highly cross-linked MTs or MTs oper- stages of mitosis, as determined by their DAPI ationally defined as more stable do contain more fluorescence (B,E,H,K) and phase images (C,F,I,L), aeetylated a'-tubulin. The flagella axonemc and subpel- showing that tyrosinated a'-tubulin is not detectable in the licular MTs of T. brucei have been characterized as spindle at any stage. Bar, 5|Um. being highly cross-linked, more stable to MT depolym-

Tubulin modifications and microtubule construction 585 Fig. 8. Double labelling with YLl/2 (indirect) and KMX-1 (direct) of two individual cells at an early (A-D) and a later (E-H) stage of mitosis. YLl/2 immunofiuorescence visualized in the FITC channel (A,E) shows the distribution pattern of tyrosinated a'-tubulin with no staining of the spindle. Total /3-tubulin distribution observed in the rhodamine channel by KMX-1, shows the presence of a spindle in these identical cells (B,F). The DAPI fluorescence images are shown in (C,G) and the phase-contrast images in (D,H). Bar, 5fim. erization conditions and resistant to many MT depoly- becomes difficult to detect with YLl/2 staining (Sher- merizing drugs (Schneider et al. 1987). However, our win et al. 1987). Our present results show that this description of acctylated tubulin in the MTs of the detyrosination can also be visualized in the detergent- intranuclear spindle does not fit readily with this extracted, salt-treated axonemes associated with the general correlation of MT stability and the presence of isolated flagella complexes. In this context our findings acetylated tubulin. Spindle MTs in T. bntcei are both regarding the tyrosination state of the mitotic spindle ephemeral and susceptible to certain MT-depolymeriz- MTs are surprising. We have made a careful and ing drugs (K. Gull & C. R. Birkett, unpublished data). exhaustive study of this structure and have been Thus, consideration of the diverse MT arrays of a completely unable to detect tyrosinated a'-tubulin at single T. brucei cell reveals that there is no strict any stage of mitosis. The most straightforward expla- correlation between possession of acetylated a'-tubulin nation of these data is that the spindle MTs are and MT stability. Interestingly, a recent report that composed of detyrosinated tubulin. Unfortunately, we compared different mammalian cultured cells has pro- cannot test for this directly, since we have no efficient vided similar evidence regarding cytoplasmic MTs. probe for T. brucei detyrosinated a'-tubulin (see Ma- Although the stable sub-set of cytoplasmic MTs may terials and methods). There are, of course, a number of contain acctylated a'-tubulin in one cell type, this may alternative explanations that would account for this not be the case with another (Schulze et al. 1987). finding. It may be that we are not detecting the Thus, it appears that there is a spectrum of tubulin tyrosinated a'-tubulin because the YLl/2 monoclonal modification levels exhibited by cells. T. brucei appears antibody is not admitted into the nucleus, or that the to represent a cell type at an extreme end of the binding of YLl/2 is prevented by the epitope masking. acetylation spectrum, in that the MTs of both stable Several lines of evidence point against these alterna- and ephemeral organelles are modified. tives. Access to the nucleus and staining of the nuclear We have shown (Sherwin et al. 1987) that the spindle occurs with many other anti-tubulin mono- dctyrosination of a'-tubulin is another important post- clonal antibodies (including the KMX-1 used in translational modification operating in T. brucei. The double-staining protocols above). Moreover, YLl/2 analysis of YLl/2 staining of the new versus old itself can clearly detect MTs in the intranuclear flagclla in intact cells provides evidence for the relation- spindles of other microorganisms such as yeast (Kil- ship between detyrosination of

586 R. Sasse and K. Gull conditions with constant results. Further, we have used difference in the timing of acquisition of the modified two different protocols in order to produce detergent- tubulin in the MTs. Acetylated

Tubulin modifications and microtubule construction 587 provides the cell with an opportunity for discriminating BRUN, R. & SCHONENBERGER, M. (1979). Cultivation and between new and old MTs as evidenced by the in vitro cloning of procyclic culture forms of flagellum. This may have general significance in the Trypanosoma bnicei in a semi-defined medium. Acta tropica 36, 289-292. context of progression through the cell cycle. The subpellicular MTs of the T. brucei cortex do not BURLAND, T. G., GULL, K., SCHEDL, T., BOSTON, R. S. & DOVE, W. F. (1983). Cell type-dependent expression depolymerize during cell division. Consequently, if we of tubulins in Physarum. J. Cell Biol. 97, 1852-1859. consider a cell that has initiated a new cell cycle: it faces CAMBRAY-DEAKIN, M. A. & BURGOYNE, R. D. (1987a). the problem of initiating and elongating a new set of Posttranslational modifications of a'-tubulin: Acetylated subpellicular MTs, whilst both maintaining the orig- and detyrosinated forms in axons of the rat cerebellum. inal set and not allowing them to polymerize further. J. Cell Biol. 104, 1569-1574. Thus, at this formal level the cell must discriminate CAMBRAY-DEAKIN, M. A. & BURGOYNE, R. D. (19876). between a new MT and an old MT. As has been Acetylated and detyrosinated a'-tubulin are colocalized in suggested earlier, a post-polymerization event such as stable microtubules in rat meningeal fibroblasts. Cell dctyrosination could provide a mechanism whereby Motil. Cytoskel. 8, 284-297. discrimination is generated. However, we suggest also CLEVELAND, D. W. & SULLIVAN, K. F. (1985). Molecular that an important aspect is the cell-cycle context. biology and genetics of tubulin. A. Rev. Biochem. 54, 331-365. Dctyrosination over the cell cycle results in the pro- DIGGINS, M. A. & DOVE, W. F. (1987). Distribution of duction of equivalent daughter cells. If it is important acetylated alpha-tubulin in Physarum polvcephalum. J. to discriminate between MTs during a cell cycle, then Cell Biol. 104, 303-309. it is necessary to ensure a resetting of the system by the FORREST, G. L. & KLEVECZ, R. R. (1978). Tyrosyltubulin time of cell division. This may be of extreme import- ligase and colchicine binding activity in synchronized ance in cells like T. bnicei where the possibility exists of Chinese hamster cells. J. Cell Biol. 78, 441-449. conservative inheritance of cytoskeletal elements. GUNDERSEN, G. G. & BULINSKI, J. C. (1986a). Distribution of tyrosinated and nontyrosinated cv-tubulin We thank Drs Gianni Piperno, Steve Blose, John Kilmar- during mitosis. J. Cell Biol. 102, 1118-1126. tin and Thomas Kreis for their generous gifts of antibodies, GUNDERSEN, G. G. & BULINSKI, J. C. (19866). and Trevor Sherwin, Chris Birkett and our other colleagues Microtubule arrays in differentiated cells contain elevated in Canterbury for helpful discussions during the course of levels of a post-translationally modified form of tubulin. this work. The work was supported by grants to K. Gull from Eur.J. Cell Biol. 42, 288-294. the Science and Engineering Research Council, the Medical GUNDERSEN, G. G., KALNOSKI, M. H. & BULINSKI, J. C. Research Council and the UNDP/World Bank/WHO (1984). Distinct populations of microtubules: Special Programme for Research and Training in Tropical Tyrosinated and nontyrosinated alpha tubulin are Diseases. distributed differently in vivo. Cell 38, 779-789. GUNDERSEN, G. G., KHAWAJA, S. & BULINSKI, J. C. (1987). Postpolymerization detyrosination of a'-tubulin: References A mechanism for subcellular differentiation of microtubules. J. Cell Biol. 105, 251-264. ARGARANA, C. E., BARRA, H. S. & CAPUTTO, R. (1980). HOARE, C. A. (1972). 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588 R. Sasse and K. Gull functionally distinct microtubules. Cell 49, 539-548. and cytoskeletal modulations. Phil. Trans. R. Soc. Ix>nd. L'HERNAULT, S. W. & ROSENBAUM, J. L. (1983). B (in press). Chlanivdomonas cr-tubulin is posttranslationally modified SHERWIN, T., SCHNEIDER, A., SASSE, R., SEEBECK, T. & in the flagella during flagellar assembly. J. Cell Biol. 97, GULL, K. (1987). Distinct localization and cell cycle 258-263. dependence of COOH terminally tyrosinolated fl'-tubulin L'HERNAULT, S. W. & ROSENBAUM, J. L. (1985a). in the microtubules of Trypanosoma bnicei brucei. J. Reversal of the posttranslational modification on Cell Biol. 104, 439-446. Chlamydomonas flagellar ar-tubulin occurs during SOLARI, A. J. (1980). The 3-dimensional fine structure of flagellar resorption. jf. Cell Biol. 100, 457-562. the mitotic spindle in Trypanosoma cmzi. Clnvmosoma L'HERNAULT, S. W. & ROSENBAUM, J. L. (19856). 78, 239-255. Chlamydomonas cr-tubulin is posttranslationally STIEGER, J., WYLER, T. & SEEBECK, T. (1984). Partial modified by acetylation on the e-amino group of a lysine. purification and characterization of the microtubular 24, 473-478. protein from Trypanosoma brucei. J. biol. Chem. 259, LOPATA, M. A. & CLEVELAND, D. W. (1987). In vivo 4596-4602. microtubules are copolymers of available /3-tubulin THOMPSON, W. C. (1982). The cyclic isotypes: Localization of each of six vertebrate /J-tubulin tyrosination/detyrosination of alpha tubulin. Meth. Cell isotypes using polyclonal antibodies elicited by synthetic Biol. 24, 235-255. peptide antigens. J. Cell Biol. 105, 1707-1720. THOMPSON, W. C, ASAI, D. J. & CARNEY, D. I-I. (1984). O'FARRELL, P. H. (1975). High resolution two dimensional Heterogeneity among microtubules of the cytoplasmic electrophoresis of proteins. J. biol Chem. 250, microtubule complex detected by a monoclonal antibody 4007-4021. to alpha tubulin. J. Cell Biol. 98, 1017-1025. PIPERNO, G. & FULLER, M. T. (1985). Monoclonal VICKERMAN, K. & PRESTON, T. M. (1970). Spindle antibodies specific for an acetylated form of tf-tubulin microtubules in the dividing nuclei of trypanosomes. Jf. recognize the antigen in the cilia and flagella from a Cell Sci. 6, 365-383. variety of organisms. J. Cell Biol. 101, 2085-2094. VICKERMAN, K. & PRESTON, T. M. (1976). Comparative PIPERNO, G., LEDIZET, M. & CHANG, X.-J. (1987). cell biology of the kinetoplastid flagellates. In Biology of Microtubules containing acetylated O'-tubulin in the Kinetoplastida, vol. 1 (ed. W. C. A. Lumsden & D. mammalian cells in culture. J. Cell Biol. 104, 289-302. A. Evans), pp. 35-130. New York: Academic Press. PONST1NGL, H., KRAUHS, K., LITTLE, M. & KEMPF, T. WEBSTER, D. R., GUNDERSEN, G. G., BULINSKI, J. C. & (1981). Complete amino acid sequence of cv-tubulin from BORISY, G. G. (1987). Assembly and turnover of porcine brain. Proc. natn. Acad. Sci. U.SA. 78, detyrosinated tubulin in vivo.J. Cell Biol. 105, 265-267. 2757-2761. WEHLAND, J., SCHODER, H. C. & WEBER, K. (1984). SASSE, R., GLYN, M. C. P., BIRKETT, C. R. & GULL, K. Amino acid sequence requirements in the epitope (1987). Acetylated ar-tubulin in Physarum: recognized by the a"-tubulin-specific rat monoclonal Immunological characterization of the isotype and its antibody YLl/2. EMBOJ. 3, 1295-1300. usage in particular microtubular organelles. J. Cell Biol. WEHLAND, J. & WEBER, K. (1987). Turnover of the 104, 41-49. carboxy-terminal tyrosine of cv-tubulin and means of SCHNEIDER, A., SHERWIN, T., SASSE, R., RUSSELL, D. G., reaching elevated levels of detyrosination in living cells. GULL, K. & SEEBECK, T. (1987). Subpellicular and J. Cell Sci. 88, 185-203. flagellar microtubules of Trypanosoma bnicei brucei WEHLAND, J., WILLINGHAM, M. C. & SANDOVAL, 1. V. contain the same ar-tubulin isoforms. Jf. Cell Biol. 104, (1983). A rat monoclonal antibody reacting specifically 431-438. with the tyrosylated form of a"-tubulin. 1. Biochemical SCHULZE, E., ASA1, D. J., BULINSKI, J. C. & KlRSCHNER, characterization, effects on microtubule polymerization M. (1987). Posttranslational modifications and in vitro and microtubule polymerization and organization microtubule stability. J. Cell Biol. 105, 2167-2177. in vivo.J. Cell Biol. 97, 1467-1475. SHERWIN, T. & GULL, K. (1988). The division cycle of Trypanosoma brucei brucei: Timing of event markers (Received 23 March 1988-Accepted 19 April 19SS)

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