Acetylated a-Tubulin in Physarum: Immunological Characterization of the Isotype and Its Usage in Particular Microtubular Organelles

We have used monoclonal antibodies specific for acetylated and unacetylated a-tubulin to characterize the acetylated a-tubulin isotype of Physarum polycephalum, its expression in the life cycle, and its localization in particular microtubular organelles. We have used the monoclonal antibody 6-11B-1 (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) as the probe for acetylated ct-tubulin and have provided a biochemical characterization of the monoclonal antibody KMP-1 as a probe for unacetylated tubulin in Physarum. Concomitant use of these two probes has allowed us to characterize the acetylated a-tubulin of Physarum as the a3 isotype. We have detected this acetylated a3 tubulin isotype in both the flagellate and in the myxameba, but not in the plasmodium. In the flagellate, acetylated tubulin is present in both the flagellar axonemes and in an extensive array of cytoplasmic microtubules. The extensive arrangement of acetylated cytoplasmic microtubules and the flagellar axonemes are elaborated during the myxameba-flagellate transformation. In the myxameba, acetylated tubulin is not present in the cytoplasmic microtubules nor in the mitotic spindle microtubules, but is associated with the two centrioles of this cell. These findings, taken together with the apparent absence of acetylated a-tubulin in the ephemeral microtubules of the plasmodium suggest a natural correspondence between the presence of acetylated a-tubulin and microtubule organelles that are intrinsically stable or cross-linked. I N many organisms there is now excellent evidence for the existence of multi-gene families encoding both ctand [3-tubulin. The organization and number of the tubulin genes varies between organisms and it is clear that in many cases there is a complex pattern of differential expression that leads to the appearance of different tubulin isotypes in particular cell types or tissues (6, 25). There is also good evidence that expression of these tubulin multi-gene families can lead to the presence of multiple aor 13-tubulin polypeptides within individual cells. However, at least in the case of ¢t-tubulin, this is not the only method by which an individual cell can provide itself with a variety of tubulin isotypes. Two well-defined, posttranslational modifications have been described that produce alternative isotypes of a-tubulin. The ct-tubulin polypeptide can undergo both acetylation and detyrosination; in both cases the posttranslational modification appears reversible to produce, respectively, the original unacetylated and tyrosinated form of the polypeptide (1, 15-17, 30). The posttranslational acetylation of a-tubulin was discovered in the unicellular green alga Chlamydomonas reinhardtii. In this and other organisms such as trypanosomes (20, 28, 29), the major ct-tubulin detected in the flagellum possesses unique electrophoretic coordinates on a two-dimensional gel. This particular a-tubulin isotype (ct3) is formed via a posttranslational event from the tt-tubulin (al) encoded by the tubulin mRNA and the modification appears to occur either immediately before or immediately after the inclusion of a tubulin polypeptide into the microtubule. In Chlamydomonas, this posttranslational modification has been shown to involve the acetylation of the e-amino group of a lysine residue in the a-tubulin polypeptide (17). More recently, the enzymatic activity responsible for the acetylation reaction has been identified and characterized in isolated flagella of Chlamydomonas (10). The a-tubulin acetylase has been shown to have high specificity for a-tubulin, however the Chlamydomonas enzyme will acetylate the a-tubulin of both Chlamydomonas and mammalian brain. An important development in understanding the cellular distribution of acetylated tubulin has come with the recent description of a monoclonal antibody that specifically recognizes this form of ct-tubulin (24). The antibody was raised against the tubulin from the axonemes of sea urchin sperm flagella but has been shown to recognize acetylated a-tubulin in cilia and flagella from a variety of organisms. The antigen was not found in the soluble, cytoplasmic pool tubulin, confirming the earlier view of the time of production of the acetylated a-tubulin isotype during microtubule construction in vivo (15, 20). Modification of a-tubulin by acetylation leads to the production of a novel tubulin isotype, the true © The Rockefeller University Press, 0021-9525/87/01/41/9 $1.00 The Journal of Cell Biology, Volume 104, January 1987 41-49 41 on A uust 7, 2017 jcb.rress.org D ow nladed fom function of which is, at present, unknown. In this context we have sought to ascertain whether the acetylated tubulin isotype is present in all cells in the life cycle of an organism and, if present, whether it functions within all of the microtubular organelles constructed by an individual cell. Knowledge of the precise natural distribution of acetylated tubulin is likely to be crucial in understanding how a cell controls its production and usage. We have chosen to use for these experiments the slime mould Physarumpolycephalum since this organism is known to possess distinct cell types that assemble microtubular organelles of precise arrangement and different function. Physarum myxamebas are single cells in which microtubules are present as components of the cytoplasmic cytoskeleton, centrioles, and the open mitotic spindle. The myxameba can undergo a reversible transformation into a nonproliferating flagellate that possesses polymerized tubulin in cytoplasmic microtubules, basal bodies, and the flagellar axonemes. Under appropriate conditions, a myxameba can also transform into a plasmodium. This is a macroscopic, single cell that does not possess cytoplasmic microtubules; tubulin is only polymerized during mitosis in the microtubules of the intranuclear mitotic spindle. The various cell types of the Physarum life cycle also express different • tubulin isotypes, some being the products of different genes and at least one being formed by a posttranslational modification (5, 9, 27). We have used two monoclonal antibodies that have enabled us to detect the acetylated and unacetylated isotypes of tz-tubulin in Physarum. We have been able to show that the Q3 tubulin isotype, produced via a posttranslational modification, is acetylated. This acetylated ct3 tubulin isotype is a major isotype in the flagellate, but is also present in the myxameba. We have been unable to detect acetylated tubulin in the plasmodial phase of the life cycle. In those cell types that possess acetylated tubulin there is a very precise distribution of the modified tubulin in particular microtubular organelles. In the flagellate, acetylated tubulin is present in both cytoplasmic microtubules as well as those of the flagellum axoneme. In the myxamebal cell, the acetylated tubulin appears to be associated with the two centrioles, whilst the adjacent cytoplasmic microtubules or mitotic spindle microtubules are unacetylated. These findings, taken together with the apparent absence of acetylated tubulin in the ephemeral microtubules of the plasmodium suggest a correspondence between the presence of acetylated ¢t-tubulin and microtubule organelles that are intrinsically stable or cross-linked. Materials and Methods Physarum polycephalum Cultures The strains of Physarum polycephalum myxamebas used in this study were CLd, CLd-AXE, and LU 352. LU 352 is a recently isolated strain of amebas which has inherited the ability to grow in liquid media from the mutant strain CLd-AXE (19) but unlike CLd-AXE has the ability to transform into flagellates when transferred to non-nutrient liquid conditions (7). The strain LU 352 was a gift from Dr. J. Dee (University of Leicester, Leicester, England). LU 352 and CLd-AXE were grown at 26*C in a liquid semidefined medium according to the method of McCullough and Dee (18), modified to include 1% (wt/vol) bacteriological peptone. CLd was grown on Escherichia coli lawns on liver infusion agar at 25°C. Physarum polyeephalum strain CLd x LU 862 was used for microand macroplasmodia studies. Plasmodial growth conditions were as described previously (26, 27). Monoclonal Antibodies The two main monoclonal antibodies used throughout this study were 6-UB-1 and KMP-1.6-11B-1 is a mouse monoclonal of the IgG type. It was raised against sea urchin sperm axonemes and reacts with acetylated a-tubulin from a variety of species (24). This antibody was kindly donated by Dr. G. Piperno (The Rockefeller University, New York). KMP-1 is a mouse monoclonal of the IgM type. It was raised against Physarum myxamebal tubulin (3). Other general anti-ct-tubulin monoclonal antibodies used were YOL1/34 and YLI/2 (13), which were gifts from Dr. J. Kilmartin (Medical Research Council, Cambridge) and DM1A (4), which was a gift from Dr. S. Blose (Cold Spring Harbor Laboratories, Cold Spring Harbor, NY). KMX-1 was used as a general anti-I]-tubulin monoclonal (3). Protein Purification Microtubule protein was purified from Physarum microplasmodial strain CLd x LU862 and myxamebal strain CLd-AXE essentially according to the method described by Roobol et al. (27). Chemical Acetylation Myxamebal microtubule protein (1.5 nag; 5 mg/ml) was dialyzed against a half-saturated aqueous solution of sodium acetate at 4°C, then modified by six additions at 5-rain intervals, each of 1 lal acetic anhydride (21). Protein and reactants were separated on a G25 column equilibrated with 50 mM Na phosphate, pH 7.0. The protein-containing eluate was made 1 mM in hydroxylamine and kept at 4°C for 30 rain, dialyzed against water at 4°C for 24 h, then lyophilized. A second 1.5-rng sample of the microtubule protein (the experimental control) was treated identically except that acetic anhydride was