Keith Gull

Visualization of detyrosination along single microtubules reveals novel mechanisms of assembly during cytoskeletal duplication in trypanosomes

We have been able to use immunogold labeling with monoclonal antibodies specific for tyrosinated alpha-tubulin to define new microtubule assembly within the T. brucei pellicular cytoskeleton. Using this approach, we have been able to visualize and define the detyrosination gradient along single microtubules in vivo. New microtubules are seen to invade the cytoskeletal array early in the cell cycle between old microtubules. In post-mitotic cells, a unique form of microtubule assembly occurs, with very short microtubules being intercalated in the array. We propose that these are nucleated by lateral interaction with the MAPs on existing adjacent microtubules. This construction pattern suggests a templated morphogenesis of microtubule arrays with semi-conservative distribution to the daughter cells.

Use of monoclonal antibodies to analyse the expression of a multi‐tubulin family

We have used a panel of monoclonal antibodies in a study of the expression of multiple tubulins in Physarum polycephalum. Three anti‐β‐tubulin monoclonal antibodies, DM 1B, DM3B3 and KMX‐1 all reacted with the β1‐tubulin isotypes expressed in both myxamoebae and plasmodia. However, these antibodies showed a spectrum of reduced reactivity with the plasmodial β2‐tubulin isotype ‐ the competence of recognition of this isotype was graded DM1B > KMX‐1 > DM3B3. The anti‐α‐tubulin monoclonal antibody, YOL defined the full complement of Physarum α‐tubulin isotypes, whilst the anti‐α‐tubulin monoclonal antibody, KMP‐1 showed a remarkably high degree of isotype specificity. KMP‐1 recognises all of the myxamoebal α1‐tubulin isotypes but only recognises 3 out of the 4 α1‐tubulin isotypes expressed in the plasmodium (which normally focus in the same 2D gel spot). KMP‐1 does not recognise the plasmodial specific α2‐tubulin isotype. This monoclonal antibody reveals a new level of complexity amongst the tubulin isotypes expressed in Physarum and suggests that monoclonal antibodies are valuable probes for individual members of multi‐tubulin families.

Evidence that griseofulvin binds to a microtubule associated protein.

We have recently shown that the anti-mitotic drug, griseofulvin, inhibits assembly of brain microtubules in vitro [l] . Similar data have been presented by Weber et al. [2] . Our earlier experiments indicated that griseofulvin inhibits microtubule assembly by preventing the association between tubulin and the microtubule associated proteins (MAPS) which normally co-purify with tubulin [3] and promote assembly of purified tubulin dimer [4] . We now present evidence that griseofulvin binds specifically to MAPS and not to tubulin dimer at concentrations of griseofulvin which inhibits microtubule assembly.

Microtubular organization visualized by immunofluorescence microscopy during erythrocytic schizogony in Plasmodium falciparum and investigation of post-translational modifications of parasite tubulin.

We describe a novel procedure for the immunofluorescent investigation of Plasmodium falciparum. This has allowed us to visualize clearly microtubular structures and their changing conformation through the erythrocytic cell-cycle, to the stage of cytodifferentiation leading to merozoite release. The images of spindle development we observed, together with an analysis of nuclear body numbers in large numbers of parasites, indicate that there is an apparent asynchrony in chromosomal multiplication within a single parasite. Using antibodies specific for post-translational modification of alpha-tubulin, we also demonstrate that the C-terminal tyrosine-containing epitope of P. falciparum alpha-tubulin I is similar to that of other organisms. Lysine-40 in the same molecule, a target for highly specific in vivo acetylation in some organisms, is unmodified in the blood stages we examined here. After in vitro acetylation of this residue, however, the epitope to which it contributes was recognized by antibody, showing that the conformation of this part of the molecule is also conserved, despite a lack of primary sequence homology immediately downstream of the target lysine residue.

A comparison of the interaction of anthelmintic benzimidazoles with tubulin isolated from mammalian tissue and the parasitic nematode Ascaridia galli

Colchicine and a range of anthelmintic benzimidazoles inhibited the in vitro polymerization of tubulin purified from the parasitic nematode Ascaridia galli. In most cases, this inhibition was more pronounced than that detected when these drugs were incubated with tubulin purified from mammalian tissue. In particular, oxfendazole and thiabendazole had virtually no effect on mammalian tubulin assembly whereas they were both good inhibitors of nematode tubulin polymerization. Electron microscopic examinations revealed no morphological differences between microtubules from either nematode or mammalian tissues polymerized in the presence or absence of drug, though the length and number of microtubules was reduced in the drug-incubated samples. These results show that the benzimidazole group of anthelmintics interacts specifically with nematode tubulin and that their selectivity, at least in part, is a direct consequence of such interaction.

A comparison of tubulins from mammalian brain and Physarumpolycephalum using SDS—polyacrylamide gel electrophoresis and peptide mapping

IBM isolated from rn~rn~~ brain consists of two equimdar protein species designated (Y- and /?-tubulins. Conventionally cw-tubulin is the slower, and P-tubulin the faster migrating species during sodium dodecyl sulphate-polyacrylamide gel electrophoresis [ 11. Tubulin has been considered to be a highly conserved protein ]2,3]; however, we now show differences between tubulins from mammalian brain and myxamoebae of the slime mould Physarum poZycephaZum from which microtubule proteins have been purified by an assembly procedure [4]. Evidence is baaed on differences in migration of these tubulins on SDS- and urea-SDS-PAGE, and peptide mapping, and demonstrates that Physarum tubulin consists of an ol-tubulin which is similar to brain /3-tubulin, and a P-tubulin which is dissimilar to both brain subunits. In addition, the analogous proteins from Physarum and brain demonstrate micro heterogeneity during urea-SDS-PAGE. The migration of the Physarum P-tubulin is altered during urea-SDS-PAGE such as to cause the order of migration of the myxamoebal tubulins to be reversed with respect to that during SDS-PAGE. In addition the importance of the source of SDS in resolving tubu~ subunit proteins during PAGE is highlighted. Abbreviations: EGTA, ethylene glycol-bis@aminoethyl ether) tetra acetic acid; PAGE, polyac~~rn~e gel electrophoresis; PfPES,pi~r~~e-~~~is(2~th~e~~pho~c acid); SDS, sodium dodecyl sulphate

In vitro assembly of microtubule proteins from myxamoebae of Physarum polycephalum

Abstract Microtubule protein of >95% purity has been isolated by self-assembly from concentrated cell extracts of myxamoebae of Physarum polycephalum . Ninety-eight percent of the amoebal microtubule protein was tubulin. Both a and β subunits of amoebal tubulin were different from neurotubulin α and β subunits, but very similar to those of Tetrahymena ciliary tubulin. The non-tubulin components, which co-purified with tubulin through three assembly cycles, were essential to microtubule formation and contained several polypeptides including some of apparent molecular weights 49000, 57000 and 59000. Purified amoebal microtubule protein formed microtubules on warming in the absence of glycerol which were cold- and Ca 2+ -labile. In vitro, microtubule assembly was inhibited by vinblastine, benzimidazole derivatives and griseofulvin, but not by 10 −4 M colchicine. Amoebal tubulin had a much lower affinity than neurotubulin for colchicine.

Ultrastructure of the septum inCandida albicans

The ultrastructure of the septum in the mycelial phase of the dimorphic fungusCandida albicans has been studied both in thin sections of fixed material and in shadow casts of the chemically purified chitinous wall layer. The septum has a 25-nm central micropore which would not allow the passage of nuclei or mitochondria, thus delimiting these organelles within the mycelial compartments without preventing cytoplasmic continuity.

Inhibition by griseofulvin of microtubule assembly in vitro

The antimitotic drug, griseofulvin, has been shown to arrest cell division at a point close to metaphase in a variety of cells [1]. The microtubules in griseofulvinblocked HeLa cells appear to have a normal morphology and orientation within the spindle [2] and it has been suggested that griseofulvin inhibits some aspect of microtubule function rather than microtubule assembly [3]. Using a crude extract of brain, Wilson and Bryan [1 ] were unable to inhibit in vitro microtubule assembly, as measured by electron microscopy, with griseofulvin at a concentration known to inhibit mitosis (60/aM). We report here that griseofulvin at concentration between 20 and 200/IM does inhibit in vitro assembly of purified microtubule protein from brain as measured by turbidimetry, electron microscopy and a sedimentation method. Inhibition of assembly by griseofulvin is distinctly different from inhibition by colchicine. Disc gel electrophoretic analysis indicates that microtubule protein polymerized in the presence of griseofulvin is depleted in the high molecular weight (HMW) components which normally copurify with tubulin [4,5].

A Correlation between in Vivo and in Vitro Effects of the Microtubule Inhibitors Colchicine, Parbendazole and Nocodazole on Myxamoebae of Physarum polycephalum

The effects of the microtubule inhibitors colchicine, parbendazole and nocodazole on the growth of myxamoebae of Physarum polycephalum were closely paralleled by the effects of these drugs on the assembly in vitro of purified amoebal microtubule protein. Colchicine at 100 microM did not inhibit amoebal growth and did not inhibit formation, or depolymerization, of amoebal microtubules. The benzimidazole carbamate derivatives nocodazole and parbendazole were very effective in both inhibiting growth and inhibiting the assembly in vitro of amoebal microtubule protein. Parbendazole was the most effective.