Transfection of Capsaspora owczarzaki, a close unicellular relative of animals

How animals emerged from their unicellular ancestor remains a major evolutionary question. New genome data from the closest unicellular relatives of animals have provided important insights into the evolution of animal multicellularity. We know that the unicellular ancestor of animals had an unexpectedly complex genetic repertoire, including many genes that are key to animal development and multicellularity. Thus, assessing the function of these genes among unicellular relatives of animals is key to understanding how they were co-opted at the onset of the Metazoa. However, such analyses have been hampered by the lack of genetic tools. Progress has been made in choanoflagellates and teretosporeans, two of the three lineages closely related to animals, whereas no tools are yet available for functional analysis in the third lineage: the filastereans. Importantly, filastereans have a striking repertoire of genes involved in transcriptional regulation and other developmental processes. Here, we describe a reliable transfection method for the filasterean Capsaspora owczarzaki. We also provide a set of constructs for visualising subcellular structures in live cells. These tools convert Capsaspora into a unique experimentally tractable organism to use to investigate the origin and evolution of animal multicellularity.Fil: Parra Acero, Helena. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; EspañaFil: Ros Rocher, Núria. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; EspañaFil: Perez Posada, Alberto. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; EspañaFil: Kożyczkowska, Aleksandra. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; EspañaFil: Sánchez Pons, Núria. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; EspañaFil: Nakata, Azusa. Prefectural University of Hiroshima; JapónFil: Suga, Hiroshi. Prefectural University of Hiroshima; JapónFil: Najle, Sebastián Rodrigo. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Ruiz Trillo, Iñaki. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; España. Institució Catalana de Recerca i Estudis Avancats; España. Universidad de Barcelona; Españ

Emerging of unconventional model organism - Corallochytrium limacisporum - to study origin of animal multicellularity

Trabajo presentado en el 2nd IBE PhD Symposium, celebrado online el 4 y 5 de febrero de 2021.The Holozoa clade emerges as an important group for comparative cell biology analyses among eukaryotes. In addition to a well-studied Metazoa group, it includes four different unicellular lineages (Choanoflagellata, Filasterea, Ichthyosporea and Corallochytrea). Interestingly, these unicellular lineages are highly heterogeneous in their ecological distribution and have diverse developmental modes, cell morphologies, and life stages. Importantly, some species in each of the unicellular holozoan clades can transiently form multicellular structures resembling those in animals. The best way to understand in depth their biology, in particular their complex life cycles is by performing functional analysis. To do so we need to have genetic tools available in each of the four lineages.So far, genetic tools have been recently established in all of those holozoan lineages, except for Corallochytrea, a clade that occupy a key phylogenetic position as sister-group to Ichthyosporea. To have the complete approach for comparative cell biology among this relevant eukaryotic group, we need to develop genetic tools, ideally stable transfection, in the remaining lineage of Corallochytrea.Corallochytrea includes two taxa described so far, Corallochytrium limacisporumand Syssomonas multiformis. C. limacisporumis an understudied marine free-living walled saprotroph, that in addition to its key phylogenetic position, has features that make it relevant to be developed as a model organism: a peculiar and still uncharacterized life cycle. C. limacisporumhas also a well-annotated genome which contains several conserved homolog genes with animals. It grows fast and it can be cultured in axenic conditions, in both, liquid and solid medium facilitating the isolation of clonal lines.We here report a setof genetic tools that allow stable transfection in C. limacisporum. We have also developed a battery of cassettes tagging key cellular components, such as nucleus, plasma membrane, cytoplasm and actin filaments, that can serve for a better understanding of life cycle of C. limacisporum. Using nuclear labelling we have already gained some insights into particularities of C. limacisporum cell biology. We discovered that C. limacisporumis bi-nucleate for the majority of its life cycle. Interestingly, unlike most studied eukaryotes, the nuclear division is decoupled from the cellular division. We could also identify that C. limacisporumcan go through multinucleate stage, hence the life cycle is non-linear. Progress and the potential implications of our research will be presented and further discussed.Peer reviewe

Stable transfection in protist Corallochytrium limacisporum identifies novel cellular features among unicellular animals relatives

The evolutionary path from protists to multicellular animals remains a mystery. Recent work on the genomes of several unicellular relatives of animals has shaped our understanding of the genetic changes that may have occurred in this transition.1, 2, 3 However, the specific cellular modifications that took place to accommodate these changes remain unclear. To address this, we need to compare metazoan cells with those of their extant relatives, which are choanoflagellates, filastereans, ichthyosporeans, and corallochytreans/pluriformeans. Interestingly, these lineages display a range of developmental patterns potentially homologous to animal ones. Genetic tools have already been established in three of those lineages.4, 5, 6, 7 However, there are no genetic tools available for Corallochytrea. We here report the development of stable transfection in the corallochytrean Corallochytrium limacisporum. Using these tools, we discern previously unknown biological features of C. limacisporum. In particular, we identify two different paths for cell division—binary fission and coenocytic growth—that reveal a non-linear life cycle. Additionally, we found that C. limacisporum is binucleate for most of its life cycle, and that, contrary to what happens in most eukaryotes, nuclear division is decoupled from cellular division. Moreover, its actin cytoskeleton shares characteristics with both fungal and animal cells. The establishment of these tools in C. limacisporum fills an important gap in the unicellular relatives of animals, opening up new avenues of research to elucidate the specific cellular changes that occurred in the evolution of animals.This work was funded by the Gordon and Betty Moore Foundation’s Marine Microbiology Initiative for establishing Emerging Model Systems Grant Number: 4973.01.Peer reviewe