Proteome remodeling in the zoospore-to-vegetative cell transition of the stramenopile Aurantiochytrium limacinum reveals candidate ectoplasmic network proteins

Abstract

Thraustochytrids are marine protists of ecological and biotechnological importance. Like many other eukaryotes, their life cycle includes a critical transition from a flagellated, swimming zoospore dispersal stage to a settled, surface-attached, growing vegetative cell. Unlike other eukaryotes, the settling vegetative cells of thraustochytrids (and their labyrinthulomycete relatives) attach to surfaces by producing a unique structure known as the ectoplasmic network, and its associated connection to the cytoplasm, the bothrosome. We conducted time-course proteomics and microscopy to study this transition in the model thraustochytrid Aurantiochytrium limacinum ATCC MYA-1381. We identified 623 proteins significantly differentially expressed between zoospores and samples collected 2, 4, 6, and 8 hours after settlement. Analysis of the differentially expressed proteins revealed broad cellular changes during the transition from zoospore to vegetative cell, including shifts in motility, signaling, and metabolism. A relative enrichment of proteasomal and ribosomal components in the zoospores suggests these proteins are stockpiled, priming the zoospore for rapid protein turnover upon settlement. Flagellar proteins were strongly downregulated upon settlement, coinciding with loss of motility. Environmental sensing systems, such as channelrhodopsins, declined post-settlement. The proteomic changes also suggest that zoospores rely on catabolism of stored lipids by beta-oxidation, whereas settled vegetative cells shift towards anabolic metabolism, including gluconeogenesis (growth media contained glycerol), and the biosynthesis of membrane lipids, amino acids, and nucleic acids. A search for proteins which were upregulated during vegetative cell settlement, and which were phylogenetically divergent in thraustochytrids, yielded a list of potential ectoplasmic network or bothrosome candidates, including potential homologs of micronemal adhesins and membrane-trafficking proteins. Our findings illuminate a critical life-history transition in A. limacinum, and identify targets for understanding the evolutionary origins and functions of unique labyrinthulomycete structures.

Fig 1. Time-resolved high-definition imaging of A. limacinum reveals coordinated ectoplasmic network (EN) development and cell division following zoospore settlement.

Key events include: initial zoospore motility, flagellar attachment (~90 min post-settlement), visible EN development (4-6 hours), and robust EN network formation by 8 hours. Arrows point out the emergence, extension, and connection of some strands of the EN. Growth in cell body size is noticeable at 2.5-3 hours, leading to cell division between 6-8 hours. Black scale bar at lower left indicates 10 µm. The contrast and brightness of these images was adjusted to increase the visibility of the EN; original un-adjusted images are available in S1 File.

Fig 2. Principal Component Analysis reveals major axes of protein expression variation during settlement and early vegetative growth.

PC1 (x-axis, 62.2% of variance) represents developmental changes from zoospores to vegetative cells over the initial 4 hours. PC2 (y-axis, 16.6% of variance) reflects transient changes associated with settlement and early vegetative cell development, particularly from 0 to 2 hours post-settlement, including loss of flagellar proteins and EN/bothrosome development. Data points are color-coded by time point, highlighting consistency across replicates.

Fig 3. Temporal expression patterns of some predicted flagellar proteins during the transition from free-swimming zoospore to non-motile vegetative cell.

The figure displays the log-centered intensity of two predicted alpha-tubulin proteins (A0A6S8DQS1 and A146288) and the putative mastigoneme protein A117061 across five time points: T0, T2, T4, T6, and T8. ‘Rep.’ indicates independent biological replicates. The reduction in expression levels over time suggests a decline in structures associated with motility, consistent with the observed loss of flagella and related proteins in the transition.

Fig 4. Heatmap visualization of four temporal expression clusters of 623 significant proteins revealing dynamic changes across the settlement time course.

Proteins were clustered using k-means and Pearson distance across time points T0, T2, T4, T6, T8 and replicates _1, _2, and _3. Colors represent log2-centered intensities (i.e., average log2 fold-change scaled protein-wise). Gene clusters C1-C4 are indicated at left.

Fig 5. Functional classification of differentially expressed proteins during the transition from zoospores to vegetative cells in A. limacinum.

Bars show the total number of significantly upregulated (red, right-facing) and downregulated (blue, left-facing) proteins (at any time point) relative to zoospores (time 0) for each selected KOG class or ko group. Unfilled triangles in the first column at right indicate functional groups significantly overrepresented (upward triangle) or underrepresented (downward triangle) among all differentially expressed proteins (Fisher’s Exact Test with 5% false discovery rate). Filled triangles in the last column indicate functional groups overrepresented among upregulated (C3 and C4, upward triangle) or downregulated (C1 and C2, downward triangle) genes in vegetative cells in comparison to zoospores. The comparisons shown here represent a curated subset of the full enrichment analysis shown in S6 Fig.