Cellular biology

DNAJC5, an HSP40 member, supports synaptic vesicle release and protein folding byactivating HSP70 ATPase activity. In humans, it localizes to presynaptic terminals and endomembrane compartments that are involved in protein trafficking. Mutations in DNAJC5 cause CLN4 disease, a rare adult-onset Batten disease. Dictyostelium discoideum, a model for neurodegenerative research, encodes a putative homolog of DNAJC5, Dnajc5 (DDB0306688), which remains uncharacterized. This study examined Dnajc5 localization and function in D. discodieum. Dnajc5 localized to the endoplasmic reticulum, cytoplasm and nucleolus under both growth and starvation conditions, suggesting a role in proteostasis. Unlike human DNAJC5, Dnajc5 was absent from endomembrane compartments and extracellularly during starvation. Protein quantification revealed increased levels during early development, peaking at the mound stage, and declining thereafter—paralleling gene expression. Immunoprecipitation of Dnajc5 showed no serine phosphorylation or ubiquitination, unlike human DNAJC5. These findings suggest functional differences despite a possible common role in proteostasis.

Author Keywords: actinomycin- D, CLN4, Dictyostelium discoiduem, DNAJC5, Immunoprecipitation, multicellular development

Crop losses due to pathogens, pests, and weeds account for 20–40% of global production, with fungal pathogens responsible for the most significant yield reductions and economic impact. The diseases caused by fungi spread through dormant spores, which protect its genetic material under adverse conditions. Dormancy is maintained until favorable germination conditions are met. Despite their importance in the fungal lifecycle, the molecular transitions from dormancy to germination remain poorly understood. The research presented uses the basidiomycete Ustilago maydis, the causal agent of Common Smut of Corn, to investigate fungal spore dormancy and germination. It aims to 1) identify the molecular transitions and stages of teliospore germination and 2) the roles of RNA helicases during teliospore germination. RNA-seq and respiration analyses were used to propose teliospore germination stages and a microdissection technique was developed for studying these stages. Transcriptomic analysis identified patterns of gene transcript level changes during germination, with GO term enrichment identifying genes involved in cell morphogenesis, metabolism, and RNA metabolism. Several RNA helicases were identified with potential roles during dormancy and germination. Previous work in the Saville Laboratory proposed that mRNAs are stored as dsRNA in dormant teliospores. I hypothesized that RNA helicases function to make these mRNAs available for translation upon germination. Forty-six RNA helicases were identified in U. maydis, and 28 RNA helicases were proposed to have roles in growth, pathogenesis, stress response, and teliospore dormancy and germination. The RNA helicases udbp3 and uded1 were selected for functional analysis by creating mutant strains. The results suggest that udbp3 negatively regulates osmotic stress response, potentially modulating stress-responsive genes during dormancy. The altered uded1 expression in mutant strains leads to slow and polarized growth and dsRNA formation. This suggests uded1 represses translation by stabilizing sense/antisense transcripts in dormant spores and then reactivates translation during germination. These findings increase our understanding of the molecular events during teliospore germination and offer insights into factors contributing to disease progression in fungal plant pathogens.

Author Keywords: gene expression, genome annotation, RNA helicases, RNA-seq, teliospore germination, Ustilago maydis

The neuronal ceroid lipofuscinoses (NCLs), collectively referred to as Batten disease, are a group of neurodegenerative diseases that affect all ages, primarily children. Batten disease is caused by mutations in 1 of the 13 ceroid lipofuscinosis neuronal (CLN) genes (CLN1-CLN8, CLN10-CLN14), each of which causes an NCL subtype when mutated. One of the NCL subtypes, CLN5 disease, is caused by mutations in the CLN5 gene. CLN5 is a soluble lysosomal protein that localizes to the endoplasmic reticulum (ER), the Golgi complex, the cytoplasm, and extracellularly. CLN5 has four putative molecular functions, including as a ceramide synthase, glycoside hydrolase, depalmitoylase, and bis(monoacylglycerol)phosphate synthase. CLN5 plays various roles within the cell, such as lipid metabolism, autophagy, and proteasome degradation. However, the function and the exact pathway in which CLN5 is involved are unclear. In addition, CLN5 is a secreted protein that, as shown via bioinformatics analysis, contains a signal peptide sequence. Furthermore, there are currently 70 CLN5 disease-causing mutations reported in the NCL mutation database. 12 CLN5 disease-causing mutations have been studied thus far in terms of their cellular impact, as well as the release of CLN5 to a certain extent. However, there is a lack of research into the functionality of the signal peptide in CLN5 and an in-depth analysis of the molecular impact of mutations in CLN5 disease. Consequently, this Ph.D. thesis focused on using comparative transcriptomics to reveal biological pathways affected by cln5-deficiency, revealing mechanisms that regulate the secretion of Cln5 and CtsD, and using Dictyostelium to gain insights into the molecular effects of mutations in CLN5 disease. Comparative transcriptomics reveal many differentially expressed genes that are linked to phenotypes observed in cln5-deficient cells and identified pathways affected in other CLN5 disease models, such as autophagy. Furthermore, novel findings, like affected expression of lysosomal enzymes and pathways, including secretion, are identified within the comparative transcriptomics analysis. Subsequently, this research also shows the secretory role of the signal peptide in Cln5 and CtsD. Finally, this Ph.D. thesis revealed that mutations in CLN5 disease affect the lysosomal biology and secretion of Cln5 and other lysosomal enzymes.

Author Keywords: Batten disease, CLN5, Dictyostelium discoideum, Enzymes, Lysosome, Secretion

Mutations in the CLN7 (MFSD8) gene, causes CLN7 disease, a subtype of neuronal ceroid lipofuscinosis. MFSD8 is a lysosomal transmembrane protein that transports chloride across membranes. Experimentation regarding Dictyostelium discoideum revealed that mfsd8 deficiency altered lysosomal enzyme activity. During starvation, the aggregation of mfsd8¬¬- cells was delayed, and cells formed more mounds that were smaller in size, phenotypes that were attributed to reduced cell-substrate adhesion and altered lysosomal enzymatic activities. This study examines the possible transcriptomic and secretomic basis for these phenotypes. This work generated new datasets for examining the effect of mfsd8 loss on the transcriptome and secretome. The validity of these datasets was supported by use of western blotting and RT-PCR along with a set of assays probing relevant biological processes. Together these results elucidate the biological mechanisms behind the observed phenotypes and lay the foundation for future studies to further study the cellular role of MFSD8.

Author Keywords: Battens disease, CLN7, Dictyostelium discoideum, MFSD8, Secretome, Transcriptome

Cytokinins (CKs) are a pervasive group of growth-promoting signaling molecules spanning every kingdom of life. Their roles are best known in plants, where they act as phytohormones controlling nearly all aspects of plant growth and development. CKs continue to be detected in new organisms, posing questions about their roles in such widespread forms of life. The research presented in this thesis, therefore, investigated CK dynamics in a non-plant system using the simple eukaryotic model, Dictyostelium discoideum. Prior to this thesis, CKs were established as key intercellular signals necessary for proper development of D. discoideum – specifically in the induction of sporulation and maintenance of spore dormancy. However, there were no documented roles of CKs prior to the late stages of multicellular development. Comprehensive mass spectrometric screening for CKs detected six novel CK forms during all stages of D. discoideum growth and development. Based on these findings, a model was proposed that mapped CK biosynthesis in D. discoideum. The CK profiles indicate that there are differing dominant CK forms during vegetative growth and early development compared to those detected during late multicellular development. This led to the hypothesis that CKs have different roles during the distinctive life cycle stages of D. discoideum. This hypothesis was tested by generating knockout and overexpression strains of the key, primary CK biosynthesis gene, iptA, to investigate potential expanded roles for CKs during growth and the early stages of D. discoideum development. iptA-deficiency resulted in cytokinesis defects and both iptA-deficiency and overexpression caused altered mitochondrial morphology, dysregulated TCA cycle and amino acid metabolism, as well as increased levels of the energy metabolite, AMP. These combined phenotypes were suggestive of mitochondrial-associated dysfunction in vegetative growth and provided the first evidence of expanded roles of CKs during the D. discoideum life cycle. This was the first metabolomics-based evidence of CKs influencing mitochondrial function in D. discoideum. Lastly, a key CK-activating enzyme was functionally characterized, DdLOG, and additional CK biosynthesis enzymes were identified for future examination. Together, the findings of this thesis provide insights into: CK biosynthesis in a non-plant associated model; new roles for CKs during the D. discoideum life cycle; and CK interactions with mitochondria. The methods established as part of this thesis can be used as a foundation for characterizing further CK biosynthesis enzymes and as a guide for detecting subtle sub-cellular phenotypes related to CK metabolism in D. discoideum and other CK-producing organisms.

Author Keywords: cytokinin biosynthesis, cytokinins, Dictyostelium discoideum, IptA, mass spectrometry, mitochondria

Giardia intestinalis is the causative agent of a diarrheal disease in mammals, but the mechanisms of disease pathogenesis are unclear. While proteins secreted by Giardia affect the host cells, the potential of hormone secretion has not been investigated to date. Cytokinins (CKs) are classified as phytohormones, but little is known about their role beyond plants. Mass spectrometry-based intracellular analysis revealed CKs typical of tRNA degradation, and extracellular analysis showed CK-riboside scavenging by Giardia with concurrent secretion of CK-free bases. Metabolomics profiling of culture supernatants showed similar trends where nucleosides were up taken, and nucleobases were secreted. The dynamics of amino acids, nucleosides and nucleobases were altered by CK-supplementation during encystation, along with inhibition of encystation. In summary, this is the first study to report CK synthesis and metabolism by Giardia along with the effects of CKs on the metabolite secretome of Giardia, while establishing a link between CK and nucleoside metabolism.

Author Keywords: Cytokinins, Giardia, mass spectrometry, metabolomics, parasite, secretome

The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are a family of fatal neurodegenerative disorders that primarily affect children. Several subtypes of NCLs have been reported, each being caused by a mutation in a distinct ceroid lipofuscinosis neuronal (CLN) gene; this results in aberrant lysosome function and the accumulation of lipoprotein aggregates (known as ceroid lipofuscin) within cells. Several innate cellular pathways exist to alleviate the stress caused by the buildup of aggregates. The endoplasmic reticulum (ER) is an essential organelle in this process because it is responsible for maintaining cellular homeostasis through protein production, quality control, and regulating several signalling pathways. The unfolded protein response (UPR) consists of several conserved pathways devoted to attenuating ER stress caused by an accumulation of misfolded proteins or aggregates; at the center of this stress response is GRP78, a molecular chaperone that binds to misfolded proteins to facilitate proper folding. The social amoeba Dictyostelium discoideum is an excellent model system for studying NCLs as it encodes more CLN-like proteins when compared to other classical model organisms (e.g., yeast, worm, fruit fly). In this study, D. discoideum was used to elucidate the effects of ER stress and build an understanding of how cells cope with increased stress. Beyond this, ER stress in D. discoideum models for CLN3 disease and CLN5 disease were evaluated. First and foremost, during the induction of ER stress by tunicamycin, there was an increase in intracellular and extracellular amounts of Grp78 accompanied by an increase in stress-related changes to the ER. Furthermore, models of CLN3 disease and CLN5 disease displayed increased amounts of Grp78 as well as a disrupted ER morphology. Interestingly, wildtype D. discoideum, AX3 cells, treated with tunicamycin displayed a similarly disrupted ER when compared to CLN models. Finally, when subjected to tunicamycin-induced ER stress, these NCL models displayed a trend towards increased Grp78 amounts, however, these cells appear to have a reduced sensitivity to tunicamycin-induced stress compared to wild-type cells. In summary, this study highlights D. discoideum as a model for studying ER stress through the conserved role of Grp78 in the stress response and concludes that an aberrant ER stress underlies the pathology of the NCLs.

Author Keywords: Batten disease, Dictyostelium discoideum, ER stress, GRP78, neuronal ceroid lipofuscinoses (NCLs)

The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are a family of inherited neurodegenerative lysosomal storage disorders. CLN7 disease is a subtype of NCL that is caused by mutations in the MFSD8 gene. MFSD8 encodes a lysosomal transmembrane protein that is predicted to play a role in transporting small substrates across membranes. However, little is known about its role and substrate specificity. Previous work identified an ortholog of human MFSD8 in the social amoeba Dictyostelium discoideum and reported its localization to endocytic compartments. In this study, the effects of mfsd8 loss during Dictyostelium growth and multicellular development were further characterized. Dictyostelium mfsd8- cells displayed increased rates of proliferation and pinocytosis in liquid media. During growth, loss of mfsd8 altered lysosomal enzymatic activities and reduced the intracellular and extracellular levels of autocrine proliferation repressor A. mfsd8- cells grown on a lawn of bacteria formed plaques in a shorter period of time compared to WT cells, providing additional support for the enhanced growth of mfsd8- cells. Upon starvation, the aggregation of mfsd8- cells was delayed, and mfsd8- cells formed more mounds that were smaller in size, which may be attributed to the reduced cell-substrate adhesion and altered lysosomal enzymatic activities observed for mfsd8- cells. Following aggregation, tipped mound formation was delayed, however, loss of mfsd8 did not affect the timing of slug/finger and fruiting body formation. Additionally, slug migration was reduced in mfsd8- cells. These aberrant phenotypes, excluding fruiting body formation, were effectively or partially rescued when Mfsd8-GFP was introduced into mfsd8- cells. Overall, these results show that Mfsd8 plays a role in regulating growth and developmental processes in Dictyostelium via lysosomal-associated functions.

Author Keywords: CLN7, Dictyostelium discoideum, Lysosomes, MFSD8, Neuronal Ceroid Lipofuscinoses

The emergence of fungal hybrid pathogens threatens sustainable crop production worldwide. To investigate hybridization, the related smut fungi, Ustilago maydis and Sporisorium reilianum, were selected because they infect a common host (Zea mays), can hybridize, and tools are available for their analysis. Hybrid dikaryons exhibited filamentous growth on plates but reduced virulence and limited colonization in Z. mays. Select virulence genes in the hybrid had similar transcript levels on plates and altered levels during infection of Z. mays relative to each parental dikaryon. Virulence genes were constitutively expressed in the hybrid to determine if its pathogenic development could be influenced. Little impact was observed in hybrids with increased expression of effectors known to modify host response and metabolism. However, increased expression of transcriptional regulators of stage specific pathogenic development increased the hybrid's capacity to induce symptoms. These results establish a base for investigating molecular aspects of fungal hybrid pathogen emergence.

Author Keywords: effectors, hybrid pathogenesis assays, Sporisorium reilianum, transcription factors, Ustilago maydis, virulence factors

Giardia intestinalis possesses four isotypes of cytochrome b5 (gCYTB-I-IV) that differ from their mammalian counterparts, suggesting different functions in this protozoan parasite. Although the recently discovered Giardia flavoenzyme, GiOR-1, reduces these cytochromes, its properties have not been thoroughly studied, owing to the difficulty in its expression. Here I describe successful conditions for expression of GiOR-1 using autoinduction. GiOR-1 is obtained with flavins bound as indicated by its UV-visible spectrum. Its ability to catalyze electron transfer from donors (NADH, NADPH) to acceptors (oxygen, ferricyanide, cytochrome c, gCYTB5-III) were studied in spectrophotometric rate assays. NADPH is the preferred electron donor, while cytochromes are the preferred electron acceptors. Interestingly, the His-tag used to purify gCYTB5-III decreases its reaction rate with GiOR-1, as an untagged version has slightly faster rates. These findings establish the appropriate conditions for further studies on GiOR-1, including the identification of endogenous electron acceptors.

Author Keywords: Autoinduction, Cytochrome b5, Cytochrome P450 oxidoreductase, Giardia intestinalis, GiOR-1, Polyhistidine tag