The architecture of cell differentiation in choanoflagellates and sponge choanocytes

Collar cells are ancient animal cell types which are conserved across the animal kingdom and their closest relatives, the choanoflagellates. However, little is known about their ancestry, their subcellular architecture, or how they differentiate. The choanoflagellate Salpingoeca rosetta expresses genes necessary for animal multicellularity and development and can alternate between unicellular and multicellular states making it a powerful model to investigate the origin of animal multicellularity and mechanisms underlying cell differentiation. To compare the subcellular architecture of solitary collar cells in S. rosetta with that of multicellular 'rosettes' and collar cells in sponges, we reconstructed entire cells in 3D through transmission electron microscopy on serial ultrathin sections. Structural analysis of our 3D reconstructions revealed important differences between single and colonial choanoflagellate cells, with colonial cells exhibiting a more amoeboid morphology consistent with relatively high levels of macropinocytotic activity. Comparison of multiple reconstructed rosette colonies highlighted the variable nature of cell sizes, cell-cell contact networks and colony arrangement. Importantly, we uncovered the presence of elongated cells in some rosette colonies that likely represent a distinct and differentiated cell type. Intercellular bridges within choanoflagellate colonies displayed a variety of morphologies and connected some, but not all, neighbouring cells. Reconstruction of sponge choanocytes revealed both ultrastructural commonalities and differences in comparison to choanoflagellates. Choanocytes and colonial choanoflagellates are typified by high amoeboid cell activity. In both, the number of microvilli and volumetric proportion of the Golgi apparatus are comparable, whereas choanocytes devote less of their cell volume to the nucleus and mitochondria than choanoflagellates and more of their volume to food vacuoles. Together, our comparative reconstructions uncover the architecture of cell differentiation in choanoflagellates and sponge choanocytes and constitute an important step in reconstructing the cell biology of the last common ancestor of the animal kingdom

Towards a quantitative understanding of chytrid cellular development

The current understanding of fungal developmental biology is almost entirely derived from dikaryan hyphae and yeast, neglecting the diversity and prevalence of other major fungal lineages. The ‘chytrids’ (phylum Chytridiomycota) are a predominantly unicellular group of fungi pervasive throughout aquatic environments. As prominent saprotrophs, parasites, and pathogens, chytrids are integral to biogeochemical cycling in aquatic ecosystems. Additionally, they retain ancestral cellular characteristics present in the last common ancestor of branching (i.e hyphal and rhizoidal) fungi, making chytrids powerful models to study the evolution of fungal-specific innovations. Despite the evolutionary and ecological importance of chytrids, their basic cell biology and development remains poorly resolved. This fundamental gap must be closed if a proper appreciation for chytrids is to be achieved. To address this, this thesis aimed to present a quantitative picture of chytrid development and identify shifts in biology across the life cycle. Using Rhizoclosmatium globosum as a model species for chytrid biology, this thesis set out to 1) establish an experimental toolkit for chytrid developmental biology, 2) identify the cellular and molecular drivers of the chytrid life cycle, and 3) quantify the development of the rhizoid, all of which were achieved. The combination of 3D electron microscopy reconstructions and transcriptomic profiling achieved a holistic developmental atlas for the chytrid life cycle shedding light on lipid metabolism, vacuolisation, and zoospore development in R. globosum, and revealing the chytrid apophysis to be a functionally delineated structure governed by intracellular trafficking. Live-cell confocal microscopy and reconstruction of developing rhizoids demonstrated that rhizoid growth was analogous to hyphal morphogenesis, adaptive to resource availability, and capable of spatiotemporal functional differentiation. Overall, this thesis achieved a quantitative characterisation of chytrid development, uncovered previously hidden cellular complexities important for ecological and evolutionary chytrid biology, and will provide a solid foundation for future investigations into chytrid biology

Chytrid rhizoid morphogenesis resembles hyphal development in multicellular fungi and is adaptive to resource availability

Key to the ecological prominence of fungi is their distinctive cell biology, our understanding of which has been principally based on dikaryan hyphal and yeast forms. The early-diverging Chytridiomycota (chytrids) are ecologically important and a significant component of fungal diversity, yet their cell biology remains poorly understood. Unlike dikaryan hyphae, chytrids typically attach to substrates and feed osmotrophically via anucleate rhizoids. The evolution of fungal hyphae appears to have occurred from rhizoid-bearing lineages and it has been hypothesized that a rhizoid-like structure was the precursor to multicellular hyphae. Here, we show in a unicellular chytrid, Rhizoclosmatium globosum, that rhizoid development exhibits striking similarities with dikaryan hyphae and is adaptive to resource availability. Rhizoid morphogenesis exhibits analogous patterns to hyphal growth and is controlled by β-glucan-dependent cell wall synthesis and actin polymerization. Chytrid rhizoids growing from individual cells also demonstrate adaptive morphological plasticity in response to resource availability, developing a searching phenotype when carbon starved and spatial differentiation when interacting with particulate organic matter. We demonstrate that the adaptive cell biology and associated developmental plasticity considered characteristic of hyphal fungi are shared more widely across the Kingdom Fungi and therefore could be conserved from their most recent common ancestor

Healthy herds in the phytoplankton: the benefit of selective parasitism

The impact of selective predation of weaker individuals on the general health of prey populations is well-established in animal ecology. Analogous processes have not been considered at microbial scales despite the ubiquity of microbe-microbe interactions, such as parasitism. Here we present insights into the biotic interactions between a widespread marine thraustochytrid and a diatom from the ecologically important genus Chaetoceros. Physiological experiments show the thraustochytrid targets senescent diatom cells in a similar way to selective animal predation on weaker prey individuals. This physiology-selective targeting of ‘unhealthy’ cells appears to improve the overall health (i.e., increased photosynthetic quantum yield) of the diatom population without impacting density, providing support for ‘healthy herd’ dynamics in a protist–protist interaction, a phenomenon typically associated with animal predators and their prey. Thus, our study suggests caution against the assumption that protist–protist parasitism is always detrimental to the host population and highlights the complexity of microbial interaction

Structural diversity of supercoiled DNA

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA min icircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function

Patient Time Requirements for Anticoagulation Therapy with Warfarin

Most patients receiving warfarin are man- aged in outpatient office settings or anticoagulation clinics that require frequent visits for monitoring

A large colonial choanoflagellate from Mono Lake harbors live bacteria

As the closest living relatives of animals, choanoflagellates offer insights into the ancestry of animal cell physiology. Here, we report the isolation and characterization of a colonial choanoflagellate from Mono Lake, California. The choanoflagellate forms large spherical colonies that are an order of magnitude larger than those formed by the closely related choanoflagellate Salpingoeca rosetta. In cultures maintained in the laboratory, the lumen of the spherical colony is filled with a branched network of extracellular matrix and colonized by bacteria, including diverse Gammaproteobacteria and Alphaproteobacteria. We propose to erect Barroeca monosierra gen. nov., sp. nov. Hake, Burkhardt, Richter, and King to accommodate this extremophile choanoflagellate. The physical association between bacteria and B. monosierra in culture presents a new experimental model for investigating interactions among bacteria and eukaryotes. Future work will investigate the nature of these interactions in wild populations and the mechanisms underpinning the colonization of B. monosierra spheres by bacteria.publishedVersio

A cellular and molecular atlas reveals the basis of chytrid development

The chytrids (phylum Chytridiomycota) are a major fungal lineage of ecological and evolutionary importance. Despite their importance, many fundamental aspects of chytrid developmental and cell biology remain poorly understood. To address these knowledge gaps, we combined quantitative volume electron microscopy and comparative transcriptome profiling to create an ‘atlas’ of the cellular and molecular basis of the chytrid life cycle, using the model chytrid Rhizoclosmatium globosum. From our developmental atlas, we describe the transition from the transcriptionally inactive free-swimming zoospore to the more biologically complex germling, and show that lipid processing is multifaceted and dynamic throughout the life cycle. We demonstrate that the chytrid apophysis is a compartmentalised site of high intracellular trafficking, linking the feeding/attaching rhizoids to the reproductive zoosporangium, and constituting division of labour in the chytrid cell plan. We provide evidence that during zoosporogenesis, zoospores display amoeboid morphologies and exhibit endocytotic cargo transport from the interstitial maternal cytoplasm. Taken together, our results reveal insights into chytrid developmental biology and provide a basis for future investigations into non-dikaryan fungal cell biology