Bothrosome & EN

The Bothrosome and Endoplasmic Network

Background
One feature unique to the zoospore to vegetative cell transition in thraustochytrids (and other labyrinthulomycetes) is the production of the ectoplasmic network (EN) – branched extensions of the plasma membrane connected to the cell body by a unique organelle known as a bothrosome or sagenogenetosome – which is involved in the search for and attachment to food sources by vegetative cells (Hamamoto & Honda, 2019; Iwata & Honda, 2018). The EN seems functionally analogous to the rhizoids of chytrid fungi (perhaps the ancestral state of fungal hyphae, (Laundon et al., 2020)) or to animal filopodia. However, the EN likely represents an independent instance of the evolution of surface attachment and the identity, evolution, and functions of proteins in the EN and bothrosome remain to be revealed. Ultrastructural characterization of the EN by transmission electron microscopy reveals a wall-less, membrane-bound compartment that may contain ‘net elements’ described as vesicles or internal membrane cisternae, but no cytoplasmic organelles like ribosomes or mitochondria (Perkins, 1973). Actin may function in the delivery of cargo between the cell body and the distal regions of the EN (Fossier Marchan et al., 2018).

We combined time course proteomics and microscopy to characterize the transition from zoospore to a settled vegetative cell with an EN in Aurantiochytrium limacinum ATCC MYA-1381

Settlement and EN production of A. limacinum zoospores. High-definition time-lapse microscopy captures the progressive stages of A. limacinum zoospore settlement on glass-bottom petri dishes. 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. Black arrows indicate the emergence and progression of the EN. Growth in cell body size is noticeable at 2.5-3 hours, leading to cell division between 6-8 hours.
PCA plot shows the changing composition of cellular proteins as A. limacinum zoospores develop into settled vegetative cells. over the first eight hours of development. PC1 contains 62.3% of the variability, while PC2 contains 16.6%. Three replicates are shown for each time point. From time 0 (zoospores) to 4 hours post-settlement, the samples moved from left to right along PC1, suggesting that this axis is associated with changes over the first 4 hours of development from zoospore to vegetative cell. From time 0 to 2 hours post-settlement, the samples moved from top to bottom of PC2, then from 2 to 8 hours post-settlement they move back up PC2, suggesting that this axis may capture mainly transient changes associated more uniquely with settlement and early development of vegetative cells, which may include bothrosome development.