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.

Insights into the origin of metazoan filopodia and microvilli.

Filopodia are fine actin-based cellular projections used for both environmental sensing and cell motility, and they are essential organelles for metazoan cells. In this study, we reconstruct the origin of metazoan filopodia and microvilli. We first report on the evolutionary assembly of the filopodial molecular toolkit and show that homologs of many metazoan filopodial components, including fascin and myosin X, were already present in the unicellular or colonial progenitors of metazoans. Furthermore, we find that the actin crosslinking protein fascin localizes to filopodia-like structures and microvilli in the choanoflagellate Salpingoeca rosetta. In addition, homologs of filopodial genes in the holozoan Capsaspora owczarzaki are upregulated in filopodia-bearing cells relative to those that lack them. Therefore, our findings suggest that proteins essential for metazoan filopodia and microvilli are functionally conserved in unicellular and colonial holozoans and that the last common ancestor of metazoans bore a complex and specific filopodial machinery.

Identification of a type I Ca2+/Mg2+-dependent endonuclease induced in maize cells exposed to camptothecin.

BACKGROUND: Camptothecin is a plant alkaloid that specifically binds topoisomerase I, inhibiting its activity and inducing double stranded breaks in DNA and activating the cell responses to DNA damage. RESULTS: Maize cultured cells were incubated in the presence of different concentrations of camptothecin. Camptothecin inhibits cultured cell growth, induces genomic DNA degradation, and induces a 32 kDa Ca2+/Mg2+-dependent nuclease activity. This nuclease, we called CaMNUC32, is inhibited by Zn2+ and by acid pH, it is mainly localized in the nucleus and it cleaves single- and double-stranded DNA, with a higher activity against single-stranded DNA. Two-dimensional electrophoresis combined with mass spectrometry suggests that CaMNUC32 is a member of the type I S1/P1 nuclease family. This type of nucleases are usually Zn2+-dependent but our results support previous indications that S1-type nucleases have a wide variety of enzyme activities, including Ca2+/Mg2+-dependent. CONCLUSIONS: We have identified and characterized CaMNUC32, a 32 kDa Ca2+/Mg2+-dependent nuclease of the S1/P1 family induced by the topoisomerase I inhibitor camptothecin in maize cultured cells.

Transcriptomic and proteomic profiling of maize embryos exposed to camptothecin.

BACKGROUND: Camptothecin is a plant alkaloid that specifically binds topoisomerase I, inhibiting its activity and inducing double stranded breaks in DNA, activating the cell responses to DNA damage and, in response to severe treatments, triggering cell death. RESULTS: Comparative transcriptomic and proteomic analyses of maize embryos that had been exposed to camptothecin were conducted. Under the conditions used in this study, camptothecin did not induce extensive degradation in the genomic DNA but induced the transcription of genes involved in DNA repair and repressed genes involved in cell division. Camptothecin also affected the accumulation of several proteins involved in the stress response and induced the activity of certain calcium-dependent nucleases. We also detected changes in the expression and accumulation of different genes and proteins involved in post-translational regulatory processes. CONCLUSIONS: This study identified several genes and proteins that participate in DNA damage responses in plants. Some of them may be involved in general responses to stress, but others are candidate genes for specific involvement in DNA repair. Our results open a number of new avenues for researching and improving plant resistance to DNA injury.

Integrin-Mediated Adhesion in the Unicellular Holozoan Capsaspora owczarzaki.

In animals, cell-matrix adhesions are essential for cell migration, tissue organization, and differentiation, which have central roles in embryonic development [1-6]. Integrins are the major cell surface adhesion receptors mediating cell-matrix adhesion in animals. They are heterodimeric transmembrane proteins that bind extracellular matrix (ECM) molecules on one side and connect to the actin cytoskeleton on the other [7]. Given the importance of integrin-mediated cell-matrix adhesion in development of multicellular animals, it is of interest to discover when and how this machinery arose during evolution. Comparative genomic analyses have shown that core components of the integrin adhesome pre-date the emergence of animals [8-11]; however, whether it mediates cell adhesion in non-metazoan taxa remains unknown. Here, we investigate cell-substrate adhesion in Capsaspora owczarzaki, the closest unicellular relative of animals with the most complete integrin adhesome [11, 12]. Previous work described that the life cycle of C. owczarzaki (hereafter, Capsaspora) includes three distinct life stages: adherent; cystic; and aggregative [13]. Using an adhesion assay, we show that, during the adherent life stage, C. owczarzaki adheres to surfaces using actin-dependent filopodia. We show that integrin ß2 and its associated protein vinculin localize as distinct patches in the filopodia. We also demonstrate that substrate adhesion and integrin localization are enhanced by mammalian fibronectin. Finally, using a specific antibody for integrin ß2, we inhibited cell adhesion to a fibronectin-coated surface. Our results suggest that adhesion to the substrate in C. owczarzaki is mediated by integrins. We thus propose that integrin-mediated adhesion pre-dates the emergence of animals.

The Capsaspora genome reveals a complex unicellular prehistory of animals.

To reconstruct the evolutionary origin of multicellular animals from their unicellular ancestors, the genome sequences of diverse unicellular relatives are essential. However, only the genome of the choanoflagellate Monosiga brevicollis has been reported to date. Here we completely sequence the genome of the filasterean Capsaspora owczarzaki, the closest known unicellular relative of metazoans besides choanoflagellates. Analyses of this genome alter our understanding of the molecular complexity of metazoans' unicellular ancestors showing that they had a richer repertoire of proteins involved in cell adhesion and transcriptional regulation than previously inferred only with the choanoflagellate genome. Some of these proteins were secondarily lost in choanoflagellates. In contrast, most intercellular signalling systems controlling development evolved later concomitant with the emergence of the first metazoans. We propose that the acquisition of these metazoan-specific developmental systems and the co-option of pre-existing genes drove the evolutionary transition from unicellular protists to metazoans.

Genetic, molecular and cellular approaches to the analysis of maize embryo development.

The development of embryo structures in plants is essential for the formation of the adult plant organs. In cereals, this process has distinct features which have attracted attention from different points of view. Differential gene expression analyses have been used in order to identify genes useful as molecular markers of certain physiological, molecular or developmental processes. Several maize mutants affected in embryo development have been isolated, but only a fraction of them have been characterized at the molecular level. Molecular markers can be useful in the characterization of embryo defective mutants. Here, we describe the different techniques used in the identification of molecular marker genes for embryo development. We describe in more detail some groups of genes coding for cell wall proteins. We also describe the application of these molecular markers in the characterization of some embryo mutants.