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Abstract Although an established model organism, Tetrahymena thermophila remains comparatively inaccessible to high throughput screens, and alternative bioinformatic approaches still rely on unconnected datasets and outdated algorithms. Here, we report a new approach to consolidating RNA-seq and microarray data based on a systematic exploration of parameters and computational controls, enabling us to infer functional gene associations from their co-expression patterns. To illustrate the power of this approach, we took advantage of new data regarding a previously studied pathway, the biogenesis of a secretory organelle called the mucocyst. Our untargeted clustering approach recovered over 80% of the genes that were previously verified to play a role in mucocyst biogenesis. Furthermore, we tested four new genes that we predicted to be mucocyst-associated based on their co-expression and found that knocking out each of them results in mucocyst secretion defects. We also found that our approach succeeds in clustering genes associated with several other cellular pathways that we evaluated based on prior literature. We present the Tetrahymena Gene Network Explorer (TGNE) as an interactive tool for genetic hypothesis generation and functional annotation in this organism and as a framework for building similar tools for other systems. 

ABSTRACT The contractile vacuole complex (CVC) is a dynamic and morphologically complex membrane organelle, comprising a large vesicle (bladder) linked with a tubular reticulum (spongiome). CVCs provide key osmoregulatory roles across diverse eukaryotic lineages, but probing the mechanisms underlying their structure and function is hampered by the limited tools available for in vivo analysis. In the experimentally tractable ciliate Tetrahymena thermophila, we describe four proteins that, as endogenously tagged constructs, localize specifically to distinct CVC zones. The DOPEY homolog Dop1p and the CORVET subunit Vps8Dp localize both to the bladder and spongiome but with different local distributions that are sensitive to osmotic perturbation, whereas the lipid scramblase Scr7p colocalizes with Vps8Dp. The H+-ATPase subunit Vma4 is spongiome specific. The live imaging permitted by these probes revealed dynamics at multiple scales including rapid exchange of CVC-localized and soluble protein pools versus lateral diffusion in the spongiome, spongiome extension and branching, and CVC formation during mitosis. Although the association with DOP1 and VPS8D implicate the CVC in endosomal trafficking, both the bladder and spongiome might be isolated from bulk endocytic input.