Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates

Enzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still in demand. Here, we investigated and engineered the RhoPDEs from Salpingoeca rosetta, Choanoeca flexa and three other protists. All the RhoPDEs (fused with a cytosolic N-terminal YFP tag) can be expressed in Xenopus oocytes, except the AsRhoPDE that lacks the retinal-binding lysine residue in the last (8th) transmembrane helix. An N296K mutation of YFP::AsRhoPDE enabled its expression in oocytes, but this mutant still has no cGMP hydrolysis activity. Among the RhoPDEs tested, SrRhoPDE, CfRhoPDE1, 4 and MrRhoPDE exhibited light-enhanced cGMP hydrolysis activity. Engineering SrRhoPDE, we obtained two single point mutants, L623F and E657Q, in the C-terminal catalytic domain, which showed ~40 times decreased cGMP hydrolysis activity without affecting the light activation ratio. The molecular characterization and modification will aid in developing ideal light-regulated phosphodiesterase tools in the future.

Pre-metazoan origin of neuropeptide signalling

Neuropeptides are a diverse class of signalling molecules in metazoans. They occur in all animals with a nervous system and also in neuron-less placozoans. However, their origin has remained unclear because no neuropeptide shows deep homology across lineages and none have been found in sponges. Here, we identify two neuropeptide precursors, phoenixin and nesfatin, with broad evolutionary conservation. By database searches, sequence alignments and gene-structure comparisons we show that both precursors are present in bilaterians, cnidarians, ctenophores and sponges. We also found phoenixin and a secreted nesfatin precursor homolog in the choanoflagellate Salpingoeca rosetta. Phoenixin in particular, is highly conserved, including its cleavage sites, suggesting that prohormone processing occurs also in choanoflagellates. In addition, based on phyletic patterns and negative pharmacological assays we question the originally proposed GPR-173 (SREB3) as a phoenixin receptor. Our findings indicate that signalling by secreted neuropeptide homologs has pre-metazoan origins and thus evolved before neurons.

The History of Salpingoeca rosetta as a Model for Reconstructing Animal Origins

Choanoflagellates, the closest living relatives of animals, have the potential to reveal the genetic and cell biological foundations of complex multicellular development in animals. Here we describe the history of research on the choanoflagellate Salpingoeca rosetta. From its original isolation in 2000 to the establishment of CRISPR-mediated genome editing in 2020, S. rosetta provides an instructive case study in the establishment of a new model organism.

Genome editing in the choanoflagellate Salpingoeca rosetta v2

This protocol details the preparation and execution of CRISPR/Cas9 genome editing in S. rosetta. The protocol builds on a method to transfect macromolecules into S. rosetta for delivering a purified Cas9 ribonucleoprotein from Streptomyces pyogenes (SpCas9 RNP) into S. rosetta. Upon cleaving the S. rosetta genome at locations specified by the guide RNA (gRNA) of the SpCas9 RNP, S. rosetta can use DNA oligonucleotides as templates to repair the double-stranded break. Those repair templates can encode foreign sequences and mutations for editing the S. rosetta genome, so long as DNA oligonucleotides have >30 bases of sequence that is homologous to both sides of the Cas9 cleavage site.

Electroporation in the choanoflagellate Salpingoeca rosetta v1

This protocol details the electroporation of DNA plasmids into the choanoflagellate Salpingoeca rosetta. The protocol builds on a method to transfect macromolecules into S. rosetta via nucleofection.

Detection of horizontal gene transfer in the genome of the choanoflagellate Salpingoeca rosetta

Horizontal gene transfer (HGT), the movement of heritable materials between distantly related organisms, is crucial in eukaryotic evolution. However, the scale of HGT in choanoflagellates, the closest unicellular relatives of metazoans, and its possible roles in the evolution of animal multicellularity remains unexplored. We identified at least 175 candidate HGTs in the genome of the colonial choanoflagellate Salpingoeca rosetta using sequence-based tests. The majority of these were orthologous to genes in bacterial and microalgal lineages, yet displayed genomic features consistent with the rest of the S. rosetta genome—evidence of ancient acquisition events. Putative functions include enzymes involved in amino acid and carbohydrate metabolism, cell signaling, and the synthesis of extracellular matrix components. Functions of candidate HGTs may have contributed to the ability of choanoflagellates to assimilate novel metabolites, thereby supporting adaptation, survival in diverse ecological niches, and response to external cues that are possibly critical in the evolution of multicellularity in choanoflagellates.

Choanoflagellates and the ancestry of neurosecretory vesicles

Neurosecretory vesicles are highly specialized trafficking organelles that store neurotransmitters that are released at presynaptic nerve endings and are, therefore, important for animal cell–cell signalling. Despite considerable anatomical and functional diversity of neurons in animals, the protein composition of neurosecretory vesicles in bilaterians appears to be similar. This similarity points towards a common evolutionary origin. Moreover, many putative homologues of key neurosecretory vesicle proteins predate the origin of the first neurons, and some even the origin of the first animals. However, little is known about the molecular toolkit of these vesicles in non-bilaterian animals and their closest unicellular relatives, making inferences about the evolutionary origin of neurosecretory vesicles extremely difficult. By comparing 28 proteins of the core neurosecretory vesicle proteome in 13 different species, we demonstrate that most of the proteins are present in unicellular organisms. Surprisingly, we find that the vesicular membrane-associated soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein synaptobrevin is localized to the vesicle-rich apical and basal pole in the choanoflagellate Salpingoeca rosetta. Our 3D vesicle reconstructions reveal that the choanoflagellates S. rosetta and Monosiga brevicollis exhibit a polarized and diverse vesicular landscape reminiscent of the polarized organization of chemical synapses that secrete the content of neurosecretory vesicles into the synaptic cleft. This study sheds light on the ancestral molecular machinery of neurosecretory vesicles and provides a framework to understand the origin and evolution of secretory cells, synapses and neurons. This article is part of the theme issue ‘Basal cognition: multicellularity, neurons and the cognitive lens’.

Structural insights into the mechanism of rhodopsin phosphodiesterase

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.

Detection of Horizontal Gene Transfer in the Genome of the Choanoflagellate Salpingoeca rosetta

Horizontal gene transfer (HGT), the movement of heritable materials between distantly related organisms, is crucial in eukaryotic evolution. However, the scale of HGT in choanoflagellates, the closest unicellular relatives of metazoans, and its possible roles in the evolution of animal multicellularity remains unexplored. We identified 703 potential HGTs in the S. rosetta genome using sequence-based tests. The majority of which were orthologous to bacterial lineages, yet displayed genomic features consistent with the rest of the S. rosetta genome – evidence of ancient acquisition events. Putative functions include enzymes involved in amino acid and carbohydrate metabolism, cell signaling, the synthesis of extracellular matrix components, and the detection of bacterial compounds. Functions of candidate HGTs may have contributed to the ability of choanoflagellates to assimilate novel metabolites, thereby supporting adaptation, survival in diverse ecological niches, and response to external cues that are possibly critical in the evolution of multicellularity in choanoflagellates.

Genome editing enables reverse genetics of multicellular development in the choanoflagellate Salpingoeca rosetta

In a previous study, we established a forward genetic screen to identify genes required for multicellular development in the choanoflagellate, Salpingoeca rosetta (Levin et al., 2014). Yet, the paucity of reverse genetic tools for choanoflagellates has hampered direct tests of gene function and impeded the establishment of choanoflagellates as a model for reconstructing the origin of their closest living relatives, the animals. Here we establish CRISPR/Cas9-mediated genome editing in S. rosetta by engineering a selectable marker to enrich for edited cells. We then use genome editing to disrupt the coding sequence of a S. rosetta C-type lectin gene, rosetteless, and thereby demonstrate its necessity for multicellular rosette development. This work advances S. rosetta as a model system in which to investigate how genes identified from genetic screens and genomic surveys function in choanoflagellates and evolved as critical regulators of animal biology.