Search

Exocytic membrane traffic can be visualized using regulatable fluorescent secretory cargoes. In cells from diverse eukaryotes, a red fluorescent fusion protein termed ESCargo can be trapped as aggregates in the endoplasmic reticulum and then released by ligand addition to create a wave of transport through the secretory pathway. This tool should have broad utility.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

N6-methyldeoxyadenosine (6mA or m6dA) was shown more than 40 years ago in simple eukaryotes. Recent studies revealed the presence of 6mA in more prevalent eukaryotes, even in vertebrates. However, functional characterizations have been limited. We use Tetrahymena thermophilaas a model organism to examine the effects of 6mA on nucleosome positioning. Independent methods reveal the enrichment of 6mA near and after transcription start sites with a periodic pattern and anti-correlation relationship with the positions of nucleosomes. The distribution pattern can be recapitulated by in vitro nucleosome assembly on native Tetrahymenagenomic DNA but not on DNA without 6mA. Model DNA containing artificially installed 6mA resists nucleosome assembling compared to unmodified DNA in vitro. Computational simulation indicates that 6mA increases dsDNA rigidity, which disfavors nucleosome wrapping. Knockout of a potential 6mA methyltransferase leads to a transcriptome-wide change of gene expression. These findings uncover a mechanism by which DNA 6mA assists to shape the nucleosome positioning and potentially affects gene expression.

Lysosome-related organelles (LROs) are secretory organelles formed by convergence between secretory and endosomal trafficking pathways. In Tetrahymena, secretory vesicles that resemble dense core granules are a new class of LROs whose synthesis depends on a conserved syntaxin required for heterotypic fusion and AP-3 for maturation.

Identifying co-regulated genes provides a useful approach for defining pathway-specific machinery in an organism. To be efficient, this approach relies on thorough genome annotation, a process much slower than genome sequencing per se. Tetrahymena thermophila, a unicellular eukaryote, has been a useful model organism and has a fully sequenced but sparsely annotated genome. One important resource for studying this organism has been an online transcriptomic database. We have developed an automated approach to gene annotation in the context of transcriptome data in T. thermophila, called the Co-regulation Data Harvester (CDH). Beginning with a gene of interest, the CDH identifies co-regulated genes by accessing the Tetrahymenatranscriptome database. It then identifies their closely related genes (orthologs) in other organisms by using reciprocal BLAST searches. Finally, it collates the annotations of those orthologs’ functions, which provides the user with information to help predict the cellular role of the initial query. 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 viathe CDH should be relevant, and can be explored, in many other systems.

ABSTRACTIn many organisms, sophisticated mechanisms facilitate release of peptides in response to extracellular stimuli. In the ciliate Tetrahymena thermophila, efficient peptide secretion depends on specialized vesicles called mucocysts that contain dense crystalline cores that expand rapidly during exocytosis. Core assembly depends of endoproteolytic cleavage of mucocyst proproteins by an aspartyl protease, cathepsin 3 (CTH3). Here, we show that a second enzyme identified by expression profiling, Cth4p, is also required for processing of proGrl proteins and for assembly of functional mucocysts. Cth4p is a cysteine cathepsin that localizes partially to endolysosomal structures and appears to act downstream of, and may be activated by, Cth3p. Disruption of CTH4results in cells (?cth4) that show aberrant trimming of Grl proproteins, as well as grossly aberrant mucocyst exocytosis. Surprisingly, ?cth4cells succeed in assembling crystalline mucocyst cores. However, those cores do not undergo normal directional expansion during exocytosis, and they thus fail to efficiently extrude from the cells. We could phenocopy the ?cth4defects by mutating conserved catalytic residues, indicating that the in vivofunction of Cth4p is enzymatic. Our results indicate that as for canonical proteins packaged in animal secretory granules, the maturation of mucocyst proproteins involves sequential processing steps. The ?cth4defects uncouple, in an unanticipated way, the assembly of mucocyst cores and their subsequent expansion and thereby reveal a previously unsuspected aspect of polypeptide secretion in ciliates.

In animal cells, the assembly of dense cores in secretory granules is controlled by proteolytic processing of proproteins. The same phenomenon occurs in the ciliate Tetrahymena thermophila, but the proteases involved appear to be highly unrelated, suggesting that similar regulatory mechanisms have different molecular origins.

Biologists have long recognized that some single-celled organisms show striking morphological and behavioral complexity, and details of the genetic underpinnings can be mined from the trove of newly-sequenced genomes. Ciliates, among which Tetrahymena thermophilaand Paramecium tetraureliahave received most attention, provide clear examples of a lineage in which, as in animal cells, the core pathways of membrane traffic have undergone dramatic expansion and elaboration to facilitate multiple modes of exocytosis and endocytosis. Recent surveys of the Rab GTPases in T. thermophila, including analysis of a large set of GFP-tagged copies, provide a new set of compartmental markers for this lineage, as well as striking views of membrane dynamics in these cells. In addition, phylogenetic analysis of the Tetrahymena Rabs suggests that different eukaryotic lineages may have independently evolved some functionally similar pathways.

ABSTRACTDense core granules (DCGs) in Tetrahymena thermophilacontain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal ß/?-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRLgenes, ?GRT1?GRT2cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in ?GRT1?GRT2cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in TetrahymenaDCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from ?GRT1?GRT2cells appear less adhesive than those from the wild type.

Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal β/-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, GRT1 GRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in GRT1 GRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from GRT1 GRT2 cells appear less adhesive than those from the wild type.

In some cells, the polypeptides stored in dense core secretory granules condense as ordered arrays. In ciliates such as Tetrahymena thermophila, the resulting crystals function as projectiles, expanding upon exocytosis. Isolation of granule contents previously defined five Granule lattice (Grl) proteins as abundant core constituents, whereas a functional screen identified a sixth family member. We have now expanded this screen to identify the nonredundant components required for projectile assembly. The results, further supported by gene disruption experiments, indicate that six Grlproteins define the core structure. Both in vivo and in vitro data indicate that core assembly begins in the endoplasmic reticulum with formation of specific hetero-oligomeric Grl proprotein complexes. Four additional GRL-like genes were found in the T. thermophilagenome. Grl2p and Grl6p are targeted to granules, but the transcripts are present at low levels and neither is essential for core assembly. The ΔGRL6cells nonetheless showed a subtle change in granule morphology and a marked reduction in granule accumulation. Epistasis analysis suggests this results from accelerated loss of ΔGRL6granules, rather than from decreased synthesis. Our results not only provide insight into the organization of Grl-based granule cores but also imply that the functions of Grl proteins extend beyond core assembly.

Regulated exocytosis of dense core secretory granules releases biologically active proteins in a stimulus-dependent fashion. The packaging of the cargo within newly forming granules involves a transition: soluble polypeptides condense to form water-insoluble aggregates that constitute the granule cores. Following exocytosis, the cores generally disassemble to diffuse in the cell environment. The ciliates Tetrahymena thermophila and Paramecium tetraurelia have been advanced as genetically manipulatable systems for studying exocytosis via dense core granules. However, all of the known granule proteins in these organisms condense to form the architectural units of lattices that are insoluble both before and after exocytosis. Using an approach designed to detect new granule proteins, we have now identified Igr1p (induced during granule regeneration). By structural criteria, it is unrelated to the previously characterized lattice-forming proteins. It is distinct in that it is capable of dissociating from the insoluble lattice following secretion and therefore represents the first diffusible protein identified in ciliate granules.

ABSTRACTRegulated exocytosis of dense core secretory granules releases biologically active proteins in a stimulus-dependent fashion. The packaging of the cargo within newly forming granules involves a transition: soluble polypeptides condense to form water-insoluble aggregates that constitute the granule cores. Following exocytosis, the cores generally disassemble to diffuse in the cell environment. The ciliates Tetrahymena thermophilaand Paramecium tetraureliahave been advanced as genetically manipulatable systems for studying exocytosis via dense core granules. However, all of the known granule proteins in these organisms condense to form the architectural units of lattices that are insoluble both before and after exocytosis. Using an approach designed to detect new granule proteins, we have now identified Igr1p (induced during granule regeneration). By structural criteria, it is unrelated to the previously characterized lattice-forming proteins. It is distinct in that it is capable of dissociating from the insoluble lattice following secretion and therefore represents the first diffusible protein identified in ciliate granules.

Formation and discharge of dense-core secretory vesicles depend on controlled rearrangement of the core proteins during their assembly and dispersal. The ciliate Tetrahymena thermophilaoffers a simple system in which the mechanisms may be studied. Here we show that most of the core consists of a set of polypeptides derived proteolytically from five precursors. These share little overall amino acid identity but are nonetheless predicted to have structural similarity. In addition, sites of proteolytic processing are notably conserved and suggest that specific endoproteases as well as carboxypeptidase are involved in core maturation. In vitro binding studies and sequence analysis suggest that the polypeptides bind calcium in vivo. Core assembly and postexocytic dispersal are compartment-specific events. Two likely regulatory factors are proteolytic processing and exposure to calcium. We asked whether these might directly influence the conformations of core proteins. Results using an in vitro chymotrypsin accessibility assay suggest that these factors can induce sequential structural rearrangements. Such progressive changes in polypeptide folding may underlie the mechanisms of assembly and of rapid postexocytic release. The parallels between dense-core vesicles in different systems suggest that similar mechanisms are widespread in this class of organelles.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.