Bezbaruah, Supriya; (1999) Heterologous expression of the mammalian microtubule associated proteins (MAPs) tau, MAP2c and MAP4 in the fission yeast, Schizosaccharomyces pombe. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The mammalian microtubule associated proteins (MAPs) tau, MAP2c and MAP4 were subcloned and expressed in the fission yeast Schizosaccharomyces pombe using the thiamine repressible pREP1 vector. Tau, MAP2c and MAP4 have similar C-terminal microtubule binding domains, but unique N-terminal projection domains. At the start of this study, there were no known MAPs in S. pombe and therefore this appeared to be a unique in vivo system to both study the roles of the three mammalian MAPs and to further define the function of microtubules. All three MAPs inhibited growth of wild type cells at 36°C over a range of temperatures. However each MAP produced distinct phenotypes in fission yeast, indicating that their effect was specific for that MAP. Tau expression resulted in a weak phenotype of long, multiseptate or branched cells. The MAP2c-induced phenotype was stronger, and resulted in long cells with bulbous ends, whilst MAP4 expression produced bent or hammer shaped cells. The MAPs tau and MAP2c accelerated and slowed entry into mitosis, respectively, of G2-arrested cdc25.211 cells. Expression of tau and MAP2c in tea1 cells (which plays a role in directing the cell to grow along a perfectly opposed longitudinal axis) and tea2 cells (which codes for a kinesin) result in distorted shapes and loss of the original phenotype. Immunofluorescence studies showed that each MAP had a unique effect not only on the cell phenotype but also on microtubule organisation of the cell. Tau caused microtubule bundling and displacement towards the cell periphery, MAP2c resulted in short microtubules around the nucleus, whilst MAP4 caused total depolymerisation of interphase microtubules. Both tau and MAP2c appeared to bind to microtubules, but MAP4 was seen distributed throughout the cell. Similarly, MAP2c caused the formation of short microtubules in teal cells, but tau had very little effect. tea2 control cells have short microtubules, and tau expression resulted in even shorter microtubules near the nucleus. MAP2c expression in tea2, however, resulted in microtubule depolymerisation, and assymetrical distribution towards one end of the cell. Tau and MAP2c also appeared to rescue the combined sensitivity of fission yeast to the cold and the microtubule depolymerisation agent, thiabendazole (TBZ). This observation was used as a strategy to isolate possible S. pombe MAPs by expressing an S. pombe cDNA library subcloned in pREP. Eight clones were isolated, and sequence analysis of two of those clones revealed that they coded for fatty acid synthetase (lsd1+) and the ribosomal protein L19.

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