Our lab is deeply interested in evolutionary cell biology


Eukaryotic cells possess an endomembrane network of organelles that underlie secretion and uptake, as well as many other cellular functions.  Many detailed studies over the past several decades have established the important principle that many mechanisms underlying the endomembrane network are broadly conserved.  A limitation of these studies is that they have been pursued chiefly in a group of relatively closely-related organisms, namely animals and fungi.  By studying membrane trafficking in a ciliate, which is very distantly related from the lineage including both animals and fungi, we aim to provide a better perspective on the relative contributions of conservation vs innovation in the evolution of the endomembrane network.  This idea pervades all of our work, and is discussed in several reviews (Elde, Long, and Turkewitz, 2007Bowman, Cowan, and Turkewitz, 2009Briguglio and Turkewitz, 2014Guerrier et al., 2016). Summaries of our different interests are below:

Formation of regulated secretory vesicles in Tetrahymena

Tetrahymena release proteins via stimulus-coupled exocytosis, from densely packed vesicles called mucocysts.  Mucocysts are an example of a wide class of specialized secretory compartments in protists, which have been called extrusomes.  In general, remarkably little is known about the mechanisms underlying extrusome biogenesis, or even whether extrusomes are truly a single class of organelles.  The best-studied extrusomes are several organelles in the parasitic Apicomplexan parasites, and the secretory vesicles in ciliates.


Work in our laboratory established that mucocysts contain two chief classes of cargo proteins. One class of proteins undergoes obligatory proteolytic processing in a process of mucocyst maturation, and we identified the proteases responsible.  Most importantly, we are finding that the two classes of proteins appear to take different routes to reach the mucocysts.  One class of proteins depends for its targeting on its tendency to form large aggregates.  The second class of proteins, and also at least some of the proteases, depend on a pathway that resembles receptor-dependent sorting to lysosomes.  Based on this work, we believe that mucocysts, and perhaps a wide set of extrusomes, should be considered lysosome-related organelles (LROs).  After the initial discovery of the key receptor, we identified a set of other proteins consistent with the LRO hypothesis, including the AP-3 adaptor complex and an endosomal syntaxin.


Further reading:



The dual pathways of proteins to mucocysts may represent an innovative solution to assembling these highly complex vesicles.  Mucocysts function as macromolecular springs during exocytosis, undergoing irreversible expansion upon exposure to extracellular calcium.  Yet these springs are assembled during mucocyst maturation in the secretory pathway, where multiple compartments are rich in calcium. By harnessing lysosome-related machinery, Tetrahymena may precisely control the access of mucocyst-associated proteases to their substrates and thus regulate spring assembly in a compartment-specific fashion.


Further reading:


The role of endosomal tethers in compartmental specificity

Another protein that is required for mucocyst biogenesis is an endosomal tether.  Such tethers are believed to act as one level of control for specifying appropriate fusion partners, in membrane trafficking.  In eukaryotes, two related endosomal tethering complexes, each composed of 6 subunits, have been described, called HOPS and CORVET.  Remarkably, Tetrahymena and its close relatives have lost one of these complexes, while some genes encoding subunits of the remaining complexes have expanded into small gene families.  Our informatic and functional analysis suggest that, by this expansion, Tetrahymena expresses a set of tethers that have been tailored for individual pathways.  We are now focused on understanding how individual complexes achieved their specificity. This project is a collaboration with Joel Dacks’ group, at the University of Alberta Edmonton.

Developing forward genetics in Tetrahymena

Tetrahymena has long been established for classical genetic analysis, but various technical obstacles have prevented researchers from identifying the precise genetic lesions in Tetrahymena mutants.  These hurdles can now be overcome by taking advantage of whole genome sequencing.  We previously isolated and characterized Tetrahymena mutants in mucocyst formation and exocytosis.  We are now developing approaches to rapidly and efficiently identify the precise genetic defects in these mutants.

Further reading:

Transcriptional profiling to dissect pathways of membrane trafficking

Thanks to work by a set of Tetrahymena-focused researchers, and particularly Wei Miao and colleagues in Wuhan, China, there is online access to the transcriptional profiles of most Tetrahymena genes, with mRNA at different stages of culture growth, starvation, and mating (Tetrahymena Functional Genomics Database).   By using these data, we realized that many genes associated with mucocyst formation were co-regulated, and that moreover co-regulation itself could be used as a screen to identify novel mucocyst-associated factors.  To further develop this approach, we developed an automated method, called the Co-regulation Data Harvester, to identify and annotate co-regulated genes.  The program is accessible via the Tetrahymena Genome Database.

Further reading:

Mapping the pathways of membrane traffic in Tetrahymena

Thanks to a rich history of microscopy-based studies, the morphological complexity of ciliate cells has been obvious for many decades.  However, the significance of many structures is difficult or impossible to assess in the absence of molecular markers.  To begin to address this, we catalogued the family of Rab GTPases in Tetrahymena.  Rabs function as molecular switches that can control traffic to and from compartments, and are thus key determinants of specificity. Consistent with previous ideas about the complexity of ciliate cells,  Tetrahymena turned out to have roughly as many Rabs as are encoded in the human genome, and five times as many as are present in budding yeast.  We determined the subcellular localization of a large majority of the Tetrahymena Rabs, to generate a set of cell biological markers for this organism. We also asked whether the Rabs in animals and ciliates had similar evolutionary trajectories, leading to an estimate of the relative roles of conservation vs innovation in a variety of pathways.

Further reading:

In a similar fashion, we examined the role of dynamin GTPases in this organism. In these studies, we uncovered an example of a novel dynamin playing a conserved role (i.e., convergent evolution of dynamins in ciliates and animals), as well as a recently-evolved dynamin that took on an essential role in establishing nuclear dimorphism in Tetrahymena.

Further reading:

Aspects of mucocyst biogenesis

A set of Rab videos (visit Bright et al. for details and our Gallery for more videos):


  • RabD14 localizes at membranes that appear associated with the contractile vacuole
  • RabD15 localizes in relatively large vesicles at the periphery of a subset of phagosomes
  • RabD20 localizes to phagosomes fated to fuse at the cytoproct
  • Rabs D32 and 11B localize to the oral apparatus and mobile cytoplasmic puncta
  • RabD39 localizes to the nascent oral apparatus and to the primary and secondary cortical meridians
  • Rab4A localizes to the oral apparatus, primary meridian, and the contractile vacuole pore
  • Rabs 6B, 6C, and 6D localize to Golgi apparati at the cell cortex
  • Rab7 localizes to late endosomes/lysosomes, as judged by co-localization with LysoTracker
  • RabD3 localizes to puncta on cytoplasmic microtubules, which appear to correspond to vesicles moving from the cytoproct (where old phagosomes fuse with the plasma membrane) to the oral apparatus, where phagosomes arise.

Collaborations with other Tetrahymena labs


Our lab is also involved in stimulating and rewarding collaborations with other Tetrahymena laboratories: