Examining the Function of Protein Acyltransferase via the DHHC Domain of the PAZ5 Protein in the Organism Dictyostelium discoideum

Protein S-palmitoylation plays a crucial role in many biological systems. S-palmitoylation involves the post-translational attachment of palmitate to a cysteine residue through a reversible thioester linkage. S-Palmitoylation is used to modify both integral and membrane proteins, many of which are involved in intracellular trafficking, membrane localization, and signal transduction pathways. Intracellular palmitoylation is mediated by a family of protein acyltransferases (PATs). PAT mutations are associated with neurological diseases and cancer progression. Proteins in the PAT family are defined by the presence of a 51-amino acid cysteine-rich domain (CRD), which contains a highly conserved aspartate-histidine-histidine-cysteine (DHHC) motif. The structure and function of DHHC proteins are still under investigation due to the difficulty in purifying eukaryotic multi-pass transmembrane proteins. In the model eukaryotic organism Dictyostelium discoideum, fourteen DHHC proteins have been discovered, labeled PAZ1-14. Previous work has investigated the localization of these proteins during growth and differentiation, with attention paid towards their effect on palmitoylation of the Gα2 subunit of the G protein. The PAZ5 protein was chosen for further investigation as cells transformed with a knockout PAZ5 presented a unique malformed phenotype in the fruiting body stage of the D. discoideum lifecycle. The fruiting body would collapse as fewer cells differentiated into stalk cells; the cell lines would return to normal functioning after rescue with the wildtype. It was also previously shown that mutations to the DHHC motif of PAZ5 induced the same malformed phenotype as the knockout, indicating a crucial role for PAZ5 in D. discoideum development. Using site-directed mutagenesis, seventeen independent mutations to PAZ5 were created, both in the DHHC-CRD and other conserved motifs. These were ligated into a fluorescent-tagged extrachromosomal expression plasmid (pTX-GFP) and transformed into D. discoideum. They were analyzed for fluorescent localization and the previously seen fruiting body malformations. Unforeseen low transformation efficiency prevented the full use of all seventeen mutants. The mutants that were able to be analyzed produced a phenotype dissimilar to both the wildtype and knockout strains; moreover, fluorescent localization of PAZ5 was unable to be discerned. It was determined that the plasmid experienced a loss of function, though this was not apparent until after the results were collected. Consequently, it is unknown to what extent the observations are experimentally valid. The work can be salvaged by re-ligating the mutant PAZ5 proteins into a new fluorescent expression plasmid. Further investigation into the DHHC-CRD of PAZ5 would provide an important eukaryotic analog for PAT research

The role of space, dispersal and active movement in fungal community assembly

Most of the theory of community ecology has been developed studying the unitary organisms. Therefore, the applicability of established theory to modular organisms remains unclear. Here we present theoretical developments that allow the community ecology of modular organisms to be firmly embedded within the established community ecology frameworks of modern coexistence theory and movement ecology. Within modular organisms, our primary focus is on filamentous fungi. The interplay of space and movement of organisms is critical for community assembly and species coexistence. Several research areas such as metacommunity theory, modern coexistence theory, and movement ecology aim to describe this interplay for animals and plants. These disciplines have assembled theoretical knowledge about the persistence and dynamics of biological diversity that is intended to be universally applicable to living systems. Applying theoretical concepts largely developed for unitary macro-organisms to filamentous fungi is challenging given their modular, network-like body structure. Here, we reviewed relevant knowledge from modern coexistence theory, movement ecology, and fungal ecology and developed two concepts that enable the application of established community ecology to filamentous fungi. We named these concepts unit of community interactions (UCI) and active movement of fungi. The first concept provides an operational definition of individual and population that is central to modern coexistence theory, but is problematic for clonal/modular life forms. This concept is introduced in the first chapter of this thesis along with modern coexistence theory applied to fungal systems. In the second chapter, we introduce the concept of active movement in fungi, demonstrating how the framework of movement ecology can be applied to filamentous fungi at all relevant spatial scales. We show that in modular organisms, physiological and morphological movements have a coupled ecological function and can thus influence community assembly via processes predicted by movement ecology. We further demonstrate this in the third chapter, where we describe the development of an agent-based model of hyphal dispersal in micro-structured environments and provide an initial evaluation of the model

THE UNIQUE PHYLOGENETIC DISTRIBUTION OF VAULT PARTICLES REVEALS ITS FUNCTIONAL ROLES

Ph.DDOCTOR OF PHILOSOPH

Cancer across the tree of life: cooperation and cheating in multicellularity

Multicellularity is characterized by cooperation among cells for the development, maintenance and reproduction of the multicellular organism. Cancer can be viewed as cheating within this cooperative multicellular system. Complex multicellularity, and the cooperation underlying it, has evolved independently multiple times. We review the existing literature on cancer and cancer-like phenomena across life, not only focusing on complex multicellularity but also reviewing cancer-like phenomena across the tree of life more broadly. We find that cancer is characterized by a breakdown of the central features of cooperation that characterize multicellularity, including cheating in proliferation inhibition, cell death, division of labour, resource allocation and extracellular environment maintenance (which we term the five foundations of multicellularity). Cheating on division of labour, exhibited by a lack of differentiation and disorganized cell masses, has been observed in all forms of multicellularity. This suggests that deregulation of differentiation is a fundamental and universal aspect of carcinogenesis that may be underappreciated in cancer biology. Understanding cancer as a breakdown of multicellular cooperation provides novel insights into cancer hallmarks and suggests a set of assays and biomarkers that can be applied across species and characterize the fundamental requirements for generating a cancer

Individual and collective dynamics of chemotaxing cells

The study of the dynamics of interacting self-propelled entities is a growing area of physics research. This dissertation investigates individual and collective motion of the eukaryote Dictyostelium discoideum, a system amenable to signal manipulation, mathematical modeling, and quantitative analysis. In the wild, Dictyostelium survive adverse conditions through collective behaviors caused by secreting and responding to chemical signals. We explore this collective behavior on size scales ranging from subcellular biochemistry up to dynamics of thousands of communicating cells. To study how individual cells respond to multiple signals, we perform stability analysis on a previously-developed computational model of signal sensing. Polarized cells are linearly stable to perturbations, with a least stable region at about 60 degrees off the polarization axis. This finding is confirmed through simulations of the model response to additional chemical signals. The off-axis sensitivity suggests a mechanism for previously observed zig-zag motion of real cells randomly migrating or chemotaxing in a linear gradient. Moving up in scale, we experimentally investigate the rules of cell motion and interaction in the context of thousands of cells. Migrating Dictyostelium discoideum cells communicate by sensing and secreting directional signals, and we find that this process leads to an initial signal having an increased spatial range of an order of magnitude. While this process steers cells, measurements indicate that intrinsic cell motility remains unaffected. Additionally, migration of individual cells is unaffected by changing cell-surface adhesion energy by nine orders of magnitude, showing that individual motility is a robust process. In contrast, we find that collective dynamics depend on cell-surface adhesion, with greater adhesion causing cells to form smaller collective structures. Overall, this work suggests that the underlying migration ability of individual Dictyostelium cells operates largely independent of environmental conditions. Our gradient-sensing model shows that polarized cells are stable to small perturbations, and our experiments demonstrate that the motility apparatus is robust to considerable changes in cell-surface adhesion or complex signaling fields. However, we find that environmental factors can dramatically affect the collective behavior of cells, emphasizing that the laws governing cell-cell interaction can change migration patterns without altering intrinsic cell motility

Preliminaries for distributed natural computing inspired by the slime mold Physarum Polycephalum

This doctoral thesis aims towards distributed natural computing inspired by the slime mold Physarum polycephalum. The vein networks formed by this organism presumably support efficient transport of protoplasmic fluid. Devising models which capture the natural efficiency of the organism and form a suitable basis for the development of natural computing algorithms is an interesting and challenging goal. We start working towards this goal by designing and executing wet-lab experi- ments geared towards producing a large number of images of the vein networks of P. polycephalum. Next, we turn the depicted vein networks into graphs using our own custom software called Nefi. This enables a detailed numerical study, yielding a catalogue of characterizing observables spanning a wide array of different graph properties. To share our results and data, i.e. raw experimental data, graphs and analysis results, we introduce a dedicated repository revolving around slime mold data, the Smgr. The purpose of this repository is to promote data reuse and to foster a practice of increased data sharing. Finally we present a model based on interacting electronic circuits including current controlled voltage sources, which mimics the emergent flow patterns observed in live P. polycephalum. The model is simple, distributed and robust to changes in the underlying network topology. Thus it constitutes a promising basis for the development of distributed natural computing algorithms.Diese Dissertation dient als Vorarbeit für den Entwurf von verteilten Algorithmen, inspiriert durch den Schleimpilz Physarum polycephalum. Es wird vermutet, dass die Venen-Netze dieses Organismus den effizienten Transport von protoplasmischer Flüssigkeit ermöglichen. Die Herleitung von Modellen, welche sowohl die natürliche Effizienz des Organismus widerspiegeln, als auch eine geeignete Basis für den Entwurf von Algorithmen bieten, gilt weiterhin als schwierig. Wir nähern uns diesem Ziel mittels Laborversuchen zur Produktion von zahlreichen Abbildungen von Venen-Netzwerken. Weiters führen wir die abgebildeten Netze in Graphen über. Hierfür verwenden wir unsere eigene Software, genannt Nefi. Diese ermöglicht eine numerische Studie der Graphen, welche einen Katalog von charakteristischen Grapheigenschaften liefert. Um die gewonnenen Erkenntnisse und Daten zu teilen, führen wir ein spezialisiertes Daten-Repository ein, genannt Smgr. Hiermit begünstigen wir die Wiederverwendung von Daten und fördern das Teilen derselben. Abschließend präsentieren wir ein Modell, basierend auf elektrischen Elementen, insbesondere stromabhängigen Spannungsquellen, welches die Flüsse von P. poly- cephalum nachahmt. Das Modell ist simpel, verteilt und robust gegenüber topolo- gischen änderungen. Aus diesen Gründen stellt es eine vielversprechende Basis für den Entwurf von verteilten Algorithmen dar

Regulation of dynamic cyclic nucleotide signalling in social amoebas

Over the past years, much information has been gathered about the importance of the individual key components that control cAMP signalling in eukaryotes and prokaryotes. In mammalians, several different classes of ACs produce cAMP and much is known about their mechanism of stimulation and function. In contrast, much less is known about the enzymes that control degradation, the cAMP-PDEs and their regulatory mechanism. Nonetheless, it has become evident that cAMP is degraded by many isoforms of PDEs and that PDEs play a crucial role in controlling dynamic cAMP signalling. Throughout my PhD I have been interested in several aspects of cAMP signalling in the model organism Dictyostelium discoideum with particular emphasis on the roles of the phosphodiesterases in dynamic cAMP signalling. PdsA is the most critical enzyme in the control of aggregation and post-aggregative morphogenesis. It is controlled by three promoters; the vegetative, aggregative and the late promoter, which direct expression during growth, aggregation and late development, respectively. Despite its known crucial role in multicellular development not much is known about the regulation of PdsA or its expression after aggregation. In chapter 2, I present studies on the patterns of the aggregative and late promoter activity throughout development, the signals that regulate promoter activity and the promoter sequences that mediate regulation by these signals. D. discoideum and related species use extracellular cAMP as chemoattractant for aggregation, but most other species use other chemoattractants than cAMP. In chapter 3, I identified orthologues of the PdsA gene throughout the Dictyostelid phylogeny and studied their expression during development. Additionally, I show that one of them encodes a fully functional PDE. Cyclic AMP waves propagate throughout the Dictyostelium slug. This is somewhat enigmatic since ACA, the enzyme considered to be responsible for pulsatile cAMP signalling, is only expressed at the slug anterior. ACG is expressed at the posterior region of the slug, where it was shown to be responsible for induction of prespore differentiation. In chapter 4, I describe experiments showing that similar to ACA, ACG is transiently activated by stimulation of cAMP receptors. This could explain why cAMP waves can propagate in slug posteriors. In Dictyostelium, cGMP is associated with chemotaxis and plays a major role in myosin II regulation during cytoskeletal reorganisation. In the chapter 5, I describe experiments showing that modulation of the expression of the PdeD gene, a cGMPstimulated cGMP-PDE, has a pronounced effect on the expression of early genes, necessitating re-evaluation of the role of cGMP in Dictyostelium chemotaxis.UBL - phd migration 201

Application of optical coherence tomography in investigating cell migration

Chemotaxis and cell migration are important processes for life, involved in organism development and homeostasis and implicated in a number of disease states. Dictyostelium discoideum, an amoeba, is a useful model for investigation of chemotaxis and development, due to its ability to undergo chemotactic aggregation and development upon starvation. Although cell migration has been well described on planar transparent surfaces, it is uncertain how well these conditions replicate the natural environment of a cell. However, attempts to better replicate these environments generally make use of opaque substrates and 3D matrices, in which it is more challenging to image cell migration. Protocols were developed to enable optical coherence tomography, a 3D structural imaging technique which requires no sample processing or staining, to be successfully employed in imaging Dictyostelium cell migration in time-lapse on non-transparent substrata and within an agarose gel. I compared the effects of two substrates, a nitrocellulose filter and a polystyrene Petri dish on aggregating cells and found differences in speed but not persistence. Extension of this to include cells within agarose revealed that these cells exhibited less directed migration, but their velocity was unaffected. I showed that cells lacking myosin II failed to complete development within an agarose gel and had significantly reduced velocity and directional migration when compared to their parent strain. Furthermore, the velocities of cells migrating within agarose gel were bimodally distributed, potentially indicating two distinct cell populations, fast and slow, and fast movement was shown to be largely myosin II dependent. Great potential therefore exists for cell-substrate and cell-matrix interactions to affect the migration character of cells, even those, such as Dictyostelium, which do not form strong focal adhesions. Moreover a properly ordered cytoskeleton is implicated in enabling cells to effectively utilise different modes of cell motility

Signal Transduction Systems in the Myxococcus xanthus Developmental Program

Myxococcus xanthus serves as a prokaryotic model organism for the regulation of complex social behaviors. During all aspects of its life cycle M. xanthus favors multicellular behavior, including a developmental program in which the population is segregated into at least three distinct cell fates (sporulation inside multicellular fruiting bodies, peripheral rods and cell lysis). Neither the evolutionary advantage of producing these distinct cell fates, nor the mechanism by which cell fate segregating is induced are fully understood. However, MrpC, a major developmental trans-criptional regulator, is a good candidate for the regulation of cell fate segregation. MrpC accumulation is controlled by multiple distinct signaling systems including the (orphan) histidine protein kinases (HPKs) Esp, TodK, Red and Hpk30. A strain lacking three of the pathways (Δesp Δred ΔtodK) massively over-accumulates MrpC and displays a striking phenotype in which all cells appear to sporulate inappropriately rapidly, producing lawns of spores. In this thesis research, I first report how M. xanthus benefits from production of spores inside of fruiting bodies. I next address how fruiting body formation and cell fate segregation can be controlled. To do so, I characterized the mechanisms by which Red, TodK and Hpk30 could control MrpC accumulation. We have previously observed that a strain lacking Esp, Red and TodK signaling systems is deficient in the formation of organized fruiting bodies and essentially produces lawns of spores. By taking advantage of this mutant strain, I addressed the role of cell fate segregation in dispersal and environmental resistance of M. xanthus fruiting bodies. I showed that loss of fruiting body morphology leads to enhanced dispersal by the vector Drosophila melanogaster. However, this comes at the expense of environmental resistance as could be demonstrated by the impact of UV exposure on mutant and wild type fruiting bodies as well as wild type single spores. To clarify how signaling systems may converge to regulate MrpC accumulation, I confirmed a putative connection between the Red signaling system and the Ser/Thr kinase cascade thought to control MrpC activity by phosphorylation. To start to identify possible mechanisms by which TodK and Hpk30 could affect MrpC accumulation, I carried out a detailed characterization of their respective signal flows. TodK functions as a bifunctional histidine protein kinase/phosphatase, whose activity is likely modulated by the two N-terminal PAS-domains. Hpk30 characterization revealed kinase activity as the signal output and that this activity is modulated by its two receiver domains as well as a hypothetical protein, MXAN_4466. Together, these data suggest a model in which separate signaling systems converge to regulate MrpC accumulation in distinct cell types leading to segregation of cells into either peripheral rods outside or spores inside fruiting bodies. Altering the spatial and/or temporal accumulation of MrpC within cells in the developing population is used to adapt fruiting body morphology to specific environmental conditions in which either dispersal or long-term resistance might enhance M. xanthus survival