Cell differentiation controls iron assimilation in a choanoflagellate

Abstract

Marine microeukaryotes have evolved diverse cellular features that link their life histories to surrounding environments. How those dynamic life histories intersect with the ecological functions of microeukaryotes remains a frontier to understand their roles in essential biogeochemical cycles^1,2. Choanoflagellates, phagotrophs that cycle nutrients through filter feeding, provide models to explore this intersection, for many choanoflagellate species transition between life history stages by differentiating into distinct cell types^3-6. Here we report that cell differentiation in the marine choanoflagellate Salpingoeca rosetta endows one of its cell types with the ability to utilize insoluble ferric colloids for improved growth through the expression of a cytochrome b561 iron reductase (cytb561a). This gene is an ortholog of the mammalian duodenal cytochrome b561 (DCYTB) that reduces ferric cations prior to their uptake in gut epithelia⁷ and is part of an iron utilization toolkit that choanoflagellates and their closest living relatives, the animals, inherited from a last common eukaryotic ancestor. In a database of oceanic metagenomes^8,9, the abundance of cytb561a transcripts from choanoflagellates positively correlates with upwellings, which are a major source of ferric colloids in marine environments¹⁰. As this predominant form of iron^11,12 is largely inaccessible to cell-walled microbes^13,14, choanoflagellates and other phagotrophic eukaryotes may serve critical ecological roles by first acquiring ferric colloids through phagocytosis and then cycling this essential nutrient through iron utilization pathways^13-15. These findings provide insight into the ecological roles choanoflagellates perform and inform reconstructions of early animal evolution where functionally distinct cell types became an integrated whole at the origin of animal multicellularity^16-22.

Figure 1:. Thecates from the choanoflagellate S. rosetta are a distinct cell type.

(A) S. rosetta differentiates into morphologically distinct cell types. This schematic shows four of the cell types from S. rosetta that were stably cultured to produce transcriptome profiles. All of these cell types display the common choanoflagellate cell architecture in which an apical flagellum is encircled by a collar of actin-filled microvilli (indicated on the slow swimmer) that enables choanoflagellates to phagocytose bacteria and particulate matter. When nutrients are abundant, slow swimmers (green) respond to bacterial and algal cues to develop into multicellular rosettes (blue) that form by serial cell division. Cultures of slow swimmers also form chains of cells through serial cell divisions, but those chains are easily disrupted by mechanical force. Under starvation, slow swimmers and rosettes become fast swimmers (grey), which have a reduced cell body and collar with a longer flagellum. Under sustained nutrient deprivation, fast swimmers differentiate into thecates (red), a type of cell that adheres to substrates by constructing an extracellular apparatus called a theca. Thecates still proliferate in low nutrient conditions by dividing into swimmers that build their own theca. By taking the supernatant of thecate cultures in high nutrient conditions, thecates can then differentiate into slow swimmers. (B) The transcriptome profile of thecates stands apart from all other cell types. A principal component analysis of triplicate RNA-seq profiles from each cell type (panel A) shows that the 86% of the variance between samples is attributed to the thecate transcriptome profile. All other cell types cluster closely together.

Figure 2:. Thecates utilize ferric colloids through the expression of an iron reductase, cytb561a.

(A) Thecates highly express an iron reductase. The log₂ transformed ratio of transcript abundances (counts) from thecates and slow swimmers (x-axis) plotted against q-values (y-axis) highlights transcriptome changes in each cell type.. Among the genes with transcript abundances that reliably (q < 0.01) changed more than two-fold in thecates (red) compared to swimmers (green), PTSG_09715 is in the 98^th percentile. This gene, which is annotated as an iron reductase, encodes a single domain: cytochrome b561 (Pfam 03188). (B) A method to swap iron sources that support choanoflagellate growth. In preparation for iron limitation assays, S. rosetta was passaged in low-nutrient media and depleted of iron. Cells were then washed multiple times and inoculated with iron-limited feeder bacteria. At the same time, ferric EDTA (Fe³⁺•EDTA) or ferric colloids (Fe³⁺) were provided. Cultures grew for 48 hours at 27°C before assaying cytb561a expression (Fig. 2C and S2) and growth (Figs. 2D and S4) (C) cytb561a expression is part of the thecate regulon, and not determined by external iron conditions. The expression of cytb561a was monitored by RT-qPCR in cultures of slow swimmers (green) or thecates (red) that were grown with either ferric EDTA or ferric colloids. The expression of cytb561a was normalized to cofilin (PTSG_01554), a eukaryotic gene that displays high, consistent expression across all S. rosetta cell types. Independent triplicates were performed and P-values were calculated from a two-way ANOVA. (D) Thecates require cytb561a for increased proliferation with ferric colloids. To account for differences in growth between slow swimmers (green) and thecates (red), the cell density of cultures grown with ferric colloids was normalized to cultures grown with ferric EDTA. With this metric, a ratio greater than one indicates that the cell type displays increased growth with ferric colloids; whereas, a ratio less than one indicates the converse. A premature termination sequence introduced at position 151 in cytb561a with CRISPR/Cas9 genome editing produced a mutant allele (cytb561a^PTS) with a ten-fold reduction in cytb561a mRNA levels and stop codons that would truncate the protein translated from this transcript (Fig. S2 and Table 2). Unlike the wild-type strain (circles), thecate cells with cytb561a^PTS displayed no improved growth with ferric colloids (triangles). (E) Thecates ingest ferric colloids through phagocytosis. Time courses of wild-type thecates incubated with fluorescently labeled ferric colloids (see methods on ferric colloid labeling). Fluorescent ferric colloid particles were tracked (magenta outline) and observed being ingested and internalized through phagocytosis. The zero time point indicates initial contact with the ferric colloid particle.

Figure 3:. Iron acquisition pathways in choanoflagellates and animals evolved from a toolkit widely conserved in eukaryotes.

(A) Distribution of iron acquisition proteins across diverse eukaryotes. Iron acquisition proteins characterized in model eukaryotes were compiled into a list to search a curated database of proteomes predicted from the genomes and/or transcriptomes of diverse eukaryotes. The iron acquisition proteins are sorted into these categories from the top to bottom rows: transporters, reductases, oxidases, iron storage, and sharing/binding. Each column indicates a single species, and major eukaryotic lineages are denoted by color. The number of unique protein hits identified in each species is shown in greyscale. (B) Cell-type expression of iron acquisition genes identified S. rosetta cell types. Genes are denoted by unique identifiers for S. rosetta genes and gene names that were given in this work based on their homology with animals. Expression values are averaged triplicate values of transcripts per million (TPM) from cell type transcriptomes (Fig. 1). Of all genes and paralogs, cytb561a exhibits the most striking differential regulation. (C) The localization of iron acquisition proteins in thecates. An ALFA epitope was engineered into endogenous genetic loci at the carboxy terminus of the protein coding sequences. Although this tag was engineered into all iron acquisition genes (except the vacuolar transporters), only the proteins displayed in this panel were visible in western blots (Fig. S7). Immunofluorescent staining of ALFA-tags, tubulin and phalloidin indicate the localization of tagged protein relative to cytoskeletal markers of cellular architecture. Images of individual cells are representative of more than three independent staining experiments in which six or more images were captured of fields containing more than ten cells. Scale bar is 5 μm. (D) A putative iron acquisition pathway in S. rosetta. Iron is internalized by phagocytic vesicles derived at the base of the collar. Inside, Cytb561a reduces iron for transport into the cytosol by Dmt1. Iron is then assimilated by the cell. To maintain iron homeostasis, Fpn exports iron out of the cell, and Heph aids in maintaining a favorable concentration gradient for export.

Figure 4:. Cytb561 paralogs possess distinct biochemical properties and appear at different oceanic locations.

(A) Phylogeny of iron reductases reveals Cytb561a is an ortholog of animal DCYTB. Each paralog of S. rosetta falls into distinct clades of cytochrome b561. Circles on each branch are proportional to bootstrap values greater than sixty. Scale bar indicates the average number of substitutions per site. Branch colors correspond to clades as shown in the legend: choanoflagellata (choano.), tereto. Teretosporea (tereto.), amoeba (amoeb.), rhizari (rhiz.), rhodophyta (rhodo.), chromista (chr.), haptophyta (hapt.), metamonoda (metam.) (B) Predicted structures of S. rosetta Cytb561 paralogs show differences in dimerization and substrate-binding interfaces. Alphafold (v3) predictions show Cytb561a as the only paralog forming homodimers. Predicted electrostatic surfaces Cytb561 paralogs show different charge distributions around the substrate binding pocket (arrows). Models are angled 45° for viewing the lumenal surface where iron binds. (C) cytb561a transcript abundance correlates with ocean upwelling velocities. Maps (left panels) show the genomic (grey) and transcript (blue) abundance of each paralog. Plots show genomic (middle panels) and transcript (right panels) abundance on the y-axis and average upwelling velocities on the x-axis. Note, the x-axis for cytb561c transcript abundance is scaled differently than for cytb561a and cytb561b. r indicates Pearson correlation and P-values were calculated from a t-test.