Biology subjects

Turkewitz, A. P.

Publications and source records attributed to Turkewitz, A. P..

6 recordsLinked to original sources

bioRxiv2025

A V-ATPase complex, required for biogenesis of a lysosome-related organelle (LRO) in Tetrahymena thermophila, disassembles during LRO maturation

Lysosome-related organelles (LROs) are a heterogeneous family of organelles found in many cell types, whose significant similarities to lysosomes include their acidification by vacuolar-type ATPases (V-ATPases). However, some organelles with hallmarks of LROs are nonetheless non-acidic. Here we investigate this poorly understood phenomenon using the ciliate Tetrahymena thermophila, which have prominent secretory LROs called mucocysts. Using three different approaches, we show that mature mucocysts, poised for exocytosis, are non-acidic. Nonetheless, a specific V-ATPase a-subunit is targeted to mucocysts and is indispensable for mucocyst biogenesis and secretion. Consistent with this, immature mucocysts in the cytoplasm appear to be transiently acidified. The stage specificity of acidification can be explained by our finding that several canonical V-ATPase subunits are present in the immature, but not mature, mucocysts. Our data argue that a mucocyst-specific V-ATPase complex is targeted to newly-forming mucocysts and subsequently disassembles at a late stage in Tetrahymena LRO maturation, and that this depends on the AP-3 trafficking adaptor.

cell biology↗

bioRxiv2024

Inferring gene-pathway associations from consolidated transcriptome datasets: an interactive gene network explorer for Tetrahymena thermophila

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to high throughput screens, and alternative bioinformatic approaches still rely on unconnected datasets and outdated algorithms.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. Key PointsO_LIur approach integrates nearly 20-year-old microarray and contemporary RNA-seq datasets. C_LIO_LIrigorously compare co-expression clustering parametrization by way of computational controls. C_LIO_LICo-expression clustering identifies known and novel functionally associated genes in Tetrahymena. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/627356v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1dbe655org.highwire.dtl.DTLVardef@1ef09aaorg.highwire.dtl.DTLVardef@63b670org.highwire.dtl.DTLVardef@5e9209_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2022

An apical membrane complex controls rhoptry exocytosis and invasion in Toxoplasma

Apicomplexan parasites possess secretory organelles called rhoptries that undergo regulated exocytosis upon contact with the host. This process is essential for the parasitic lifestyle of these pathogens and relies on an exocytic machinery sharing structural features and molecular components with free-living ciliates. Here, we performed a Tetrahymena-based transcriptomic screen to uncover novel exocytic factors in Ciliata and Apicomplexa. We identified membrane-bound proteins, named CRMPs, forming part of a large complex essential for rhoptry secretion and invasion in Toxoplasma. In contrast to previously described rhoptry exocytic factors, TgCRMPs are not required for the assembly of the rhoptry secretion machinery and only transiently associated with the exocytic site - prior to invasion. CRMPs and their partners contain putative host cell-binding domains, and CRMPa shares similarity to GPCR proteins. We propose that the CRMP complex acts as host-molecular sensor to ensure that rhoptry exocytosis occurs when the parasite contacts the host cell.

cell biology↗

bioRxiv2020

ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block exit of the secretory cargo from the endoplasmic reticulum (ER), and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescent secretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory protein ESCargo (Erv29/Surf4-dependent Secretory Cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid transport out of the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used with many model organisms.

cell biology↗

bioRxiv2017

An evolutionary switch in the specificity of an endosomal CORVET tether underlies formation of regulated secretory vesicles in the ciliate Tetrahymena thermophila

In the endocytic pathway of animals, two related complexes, called CORVET (Class C Core Vacuole/Endosome Transport) and HOPS (Homotypic fusion and protein sorting), act as both tethers and fusion factors for early and late endosomes, respectively. Mutations in CORVET or HOPS lead to trafficking defects and contribute to human disease including immune dysfunction. HOPS and CORVET are conserved throughout eukaryotes but remarkably, in the ciliate Tetrahymena thermophila, the HOPS-specific subunits are absent while CORVET-specific subunits have proliferated. VPS8 (Vacuolar Protein Sorting), a CORVET subunit, expanded to 6 paralogs in Tetrahymena. This expansion correlated with loss of HOPS within a ciliate subgroup including the Oligohymenophorea, which contains Tetrahymena. As uncovered via forward genetics, a single VPS8 paralog in Tetrahymena (VPS8A) is required to synthesize prominent secretory granules called mucocysts. More specifically,{triangleup} vps8a cells fail to deliver a subset of cargo proteins to developing mucocysts, instead accumulating that cargo in vesicles also bearing the mucocyst sorting receptor, Sor4p. Surprisingly, although this transport step relies on CORVET, it does not appear to involve early endosomes. Instead, Vps8a associates with the late endosomal/lysosomal marker Rab7, indicating target specificity switching occurred in CORVET subunits during the evolution of ciliates. Mucocysts belong to a markedly diverse and understudied class of protist secretory organelles called extrusomes. Our results underscore that biogenesis of mucocysts depends on endolysosomal trafficking, revealing parallels with invasive organelles in apicomplexan parasites and suggesting that a wide array of secretory adaptations in protists, like in animals, depend on mechanisms related to lysosome biogenesis.\n\nAbbreviations

cell biology↗

bioRxiv2017

The Co-regulation Data Harvester for Tetrahymenathermophila: automated high-throughput geneannotation and functional inference in a microbialeukaryote

Identifying co-regulated genes can provide a useful approach for defining pathway-specific machinery in an organism. To be efficient, this approach relies on thorough genome annotation, which is not available for most organisms with sequenced genomes. Studies in Tetrahymena thermophila, the most experimentally accessible ciliate, have generated a rich transcriptomic database covering many well-defined physiological states. Genes that are involved in the same pathway show significant co-regulation, and screens based on gene co-regulation have identified novel factors in specific pathways, for example in membrane trafficking. However, a limitation has been the relatively sparse annotation of the Tetrahymena genome, making it impractical to approach genome-wide analyses. We have therefore developed an efficient approach to analyze both co-regulation and gene annotation, called the Co-regulation Data Harvester (CDH). The CDH automates identification of co-regulated genes by accessing the Tetrahymena transcriptome database, determines their orthologs in other organisms via reciprocal BLAST searches, and collates the annotations of those orthologs' functions. Inferences drawn from the CDH reproduce and expand upon experimental findings in Tetrahymena. The CDH, which is freely available, represents a powerful new tool for analyzing cell biological pathways in Tetrahymena. Moreover, to the extent that genes and pathways are conserved between organisms, the inferences obtained via the CDH should be relevant, and can be explored, in many other systems.

bioinformatics↗