Tubulin Post-Translational Modifications and the Construction of Microtubular Organelles in Trypanosoma Brucei
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Tubulin post-translational modifications and the construction of microtubular organelles in Trypanosoma brucei ROSEMARY SASSE and KEITH GULL* hiologicnl Laboratory, University of Kent, 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'-tubulin in the microtubule (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- microtubules 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