Sporulation competence in Physarum polycephalum CL

The central aim of this work was to explore the possible value of P. polycephalum CL as a model of eukaryotic differentiation. Initially it was hoped to obtain a method of reproducibly obtaining sporulation and later to analyse sporulation both biochemically and genetically. Microplasmodia of the CL strain were found to yield the highest sporulation frequencies when harvested at the end of exponential growth. Sporulation frequencies of microplasmodia harvested at other points in the growth cycle could be improved by allowing overnight growth, as a surface plasmodium, before exposure to starvation medium. The minimum length of starvation in the dark required before one light pulse would induce sporulation of all plasmodia was found to be 72 h. Plasmodia became committed to sporulation about 4 h after illumination. The time of commitment to sporulation was related to the time of illumination, and not the overall length of starvation, since altering the time of illumination also altered the time of commitment. The importance of DNA replication and mitosis during the process of sporulation was assessed by examining the effects of inhibitors of these events on starving plasmodia. Nocodazole, an inhibitor of microtubule assembly, prevented sporulation if added any time up to 48 h during starvation. It was assumed, therefore, that the last mitosis during starvation occurred at about 48 h. However, nocodazole might also have affected some other event, besides mitosis, which involved microtubules. The DNA synthesis inhibitor, hydroxyurea, prevented sporulation if added at any time up to 24 h during starvation. This suggested that the last DNA replication preceded the last event susceptible to inhibition by nocodazole by some 24 h. By pulse labelling with methyl-HJ thymidine during starvation, the periods of DNA synthesis during the 72 h starvation period were defined. Periods of DNA replication began at about 4 h, 15 h and 24 h during starvation, confirming that the last replication occurred at about 24 h and demonstrating that this was the third replication to occur during starvation. The patterns of DNA replication in a sporogenous and an asporogenous culture were compared in an attempt to further clarify the role of DNA replication during sporulation. In the asporogenous derivative, the third period of DNA synthesis, normally detected in the wild type strain, did not occur, yet the previous two rounds of DNA synthesis took place normally. The asporogenous strain was produced by continuous subculture of microplasmodia in broth medium. Before the strain became fully asporogenous, it showed a delay in response to light before it would sporulate. Thus the strain only sporulated after a light pulse at 96 h instead of the normal 72 h. There was, in this strain, a concomitant delay in the escape of the plasmodium from nocodazole inhibition of sporulation. Thus the final replication at about 24 h during starvation and the nocodazole sensitive event which followed some 24 h later were important for determining the condition of the plasmodium for response to light and advance to sporulation. Microplasmodial cultures were able to grow in the presence of both hydroxyurea and the protein synthesis inhibitor, cycloheximide, at concentrations which normally inhibited growth, if incubated in their presence for extended lengths of time. This may have been due to instability of the drugs at the incubation temperature or P. polyc6phalum may have been capable of breaking these drugs down. Similar results were obtained with nocodazole, but in addition micro-plasmodia also developed resistance to the drug. A method of isolating sporulation deficient mutants was developed and several such mutants were obtained. In a preliminary genetic analysis of sporulation a cross between sporogenous (Spo+) amoebae and asporogenous (Spo-) amoebae was made. Although the diploid Spo+/Spo- plasmodium sporulated, none of the progeny able to form plasmodia in clones (matAh) were able to do so. Sporulation capacity of Spo+/Spo- heterokaryons formed by fusion of plasmodia was also investigated, in these asporogeny was dominant. Although no definite results were obtained from the genetic aspect of this work it has provided a base for further genetical studies on the process of sporulation in P. polycephalum CL

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.European Research Council ERC2014-ADG669898 TARLOOPMinisterio de Economía y Competitividad BFU2016-75058-PJunta de Andalucía BIO123

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies

Endocytosis in filamentous fungi

Endocytosis is little understood in filamentous fungi. For some time it has been controversial as to whether endocytosis occurs in filamentous fungi. A comparative genomics analysis between Saccharomyces cerevisiae and 10 genomes of filamentous fungal species showed that filamentous fungi possess complex endocytic machineries. The use of the endocytic marker dye FM4-64, and various vesicle trafficking inhibitors revealed many similarities between endocytosis in the filamentous fungus Neurospora crassa, and endocytosis in budding yeast and mammalian cells. Actin polymerization was found to be crucial for endocytosis in N. crassa, and the microtubule cytoskeleton seemed to be necessary for long distance movement of putative early endosomes. Brefeldin A (BFA) blocked vesicular transport to the Spitzenkörper. Three putative endocytic proteins (WASP, clathrin light chain and Rab5) were labelled with fluorescent proteins in N. crassa. WASP-GFP was found to localise to motile, punctate structures in the plasma membrane just behind the hyphal apex in growing hyphae. This localisation changed to the hyphal apex when growth was temporarily arrested, indicating a possible role in endocytosis and polarized growth. Clathrin light chain-GFP was found to be concentrated in a region just behind the Spitzenkörper, which is consistent with there being a high concentration of clathrinmediated endocytosis in this region. Clathrin light chain-GFP also labelled putative Golgi and this labelling was found to be BFA sensitive, whereas BFA did not have a detectable effect on FM4-64 internalisation and organelle staining. GFP-Rab5 labelled putative early endosomes and decorated microtubules. Knock-outs of putative endocytic proteins in N. crassa, generated as part of the Neurospora genome consortium gene knock-out project, were analysed for defects in endocytosis. 14 out of 17 gene knock-outs were found to be ascospore lethal. The Rab5 knock-out was viable, but did not show a detectable effect on the endocytic internalisation of FM4-64 or its pattern of staining. However, it did exhibit a defect in sexual crossing

Spliceosomal intron and spliceosome evolution in Giardia lamblia and other diplomonads

Spliceosomal introns interrupt protein coding genes in all characterized eukaryotic nuclear genomes and are removed by a large RNA-protein complex termed the spliceosome. Diplomonads are diverse unicellular eukaryotes that display compact genomes with few spliceosomal introns. My thesis objectives were to explore spliceosomal intron and spliceosome diversity as well as RNA processing mechanisms in the diplomonads Giardia lamblia and Spironucleus spp. Surprisingly, G. lamblia was found to contain a proportionally large number of fragmented spliceosomal introns that are spliced in trans from separate pre-mRNA molecules. Next, both evolutionarily divergent and conventional spliceosomal small nuclear RNAs were identified in G. lamblia and Spironucleus spp. and an RNA 3ʹ end motif was determined to be involved in processing of both non-coding RNAs and trans-introns in G. lamblia. These findings shed light on spliceosome and spliceosomal intron evolution in eukaryotes undergoing severe genomic reduction and potentially complete loss of their spliceosomal introns.University of Lethbridge (SGS Graduate Fellowship), Natural Sciences and Engineering Research Council of Canada (NSERC) (Discovery Grant and Alexander Graham Bell Canada Graduate Scholarships)