Building and applying high-throughput tools to uncover protist biology

Life has diversified into countless forms over the past several billion years (and continues to do so). During this time, certain groups of organisms have arisen that, for whatever reason, seem to possess exceptional levels of biological diversity. Perhaps the most impressive group — also one of the most neglected — are eukaryotes (organisms with cellular nuclei) that aren’t plants, animals, or fungi: the so-called protists.

We believe that, lurking among their ranks, protists possess biology that may transform our understanding of life and the evolutionary process. At least 200,000 protist species exist, spanning the majority of branches in the eukaryotic tree. Some are photosynthetic. Some hunt bacterial prey. Some live in the microbial world, others are the size of a grapefruit. Many form the bedrock of their ecosystems. Some may be crucial for generating climate solutions. The vast majority have not been characterized. Given their extensive variation and adaptability to laboratory conditions, protists may offer myriad opportunities to uncover novel problems — and potentially resolve long-standing questions — in cell biology.

We are building cost-effective tools for high-throughput characterization of protist biology. Our hope is that they will be useful for, and adopted by, the scientific community to help fill in our understanding of the evolutionary and molecular bases of diverse cellular traits.

Community Philosophy

We are committed to developing tools that are cheap, open, and easy to replicate. Given the sheer breadth of protist diversity, coupled with the size of datasets produced by modern imaging and omics, no single organization will be able to tackle the depth of protist biology alone. To this end, we are also constantly re-analyzing and integrating our results to identify minimal data amounts needed to conduct these experiments. Through these efforts, we hope to reduce overhead and empower community use, independent of funding level or institutional affiliation.

Goals

We are taking a multifaceted approach to tackling protist biology:

1. Generating methods for high-throughput measurement of cellular traits (phenotypes).

   We are working to create platforms and software for conducting rapid, label-free characterization of dozens to hundreds of cells in a single experiment. In conjunction, we are developing analysis pipelines for large motility, shape, and biochemistry datasets.

2. Mapping the macroevolutionary history of protists (genotypes).

   We are using phylogenetic inference to study evolutionary patterns across protist (and eukaryotic) taxa, with a special focus on the evolution of protein structure and cellular motility.

3. Creating tools for characterizing genetic diversity and dissecting microevolution (genotypes and phenotypes).

   Mechanistic studies hinge upon targeting candidate molecules and functions. By uncovering the relationship between genetic and phenotypic variation, association analyses (statistical links between phenotypes and individual genetic polymorphisms) are powerful tools for identifying such candidates. We are developing methods for rapidly measuring genetic diversity in protist populations (even within species whose genomes may not be sequenced or are of intractable complexity).

Progress

We want to be able to bring a novel protist species into the lab, rapidly phenotype it, compare the results phylogenetically, and mechanistically dissect its biology. Since genomic and molecular tools aren’t available for the vast majority of these species (and are costly and slow to produce) we aim to accomplish these goals using label- and sequence-free methods as much as possible.

To start, we are exploring imaging-based methods for high-throughput comparisons of unicellular traits. We have been building hardware to be able to observe and measure the phenotypes of many cells simultaneously. In parallel, we are developing quantitative tools for performing evolutionary comparisons of complex cellular phenotypes (such as motility). To work as we hope, we have had to figure out how to make these tools generalizable for the full breadth of protist sizes, shapes, and movement styles.

Tackling diverse motility types

To move, protists can swim, crawl, glide, and even walk. We aim to map the evolution of these motility types by combining computational tools from behavioral biology and comparative phylogenetics. A key first step here is deciding how to quantitatively represent different modes of cellular movement. While swimming, gliding, and walking all share features with other types of organismal movement (e.g. the swimming of protists and models such as zebrafish may be treated similarly), crawling can be difficult to model since it involves active changes to the cell’s shape.

To address this, we developed a computational framework for processing, representing, and comparing images of cells crawling. We found that we could capture the movement dynamics of diverse cell types in a single ‘movement space.’ Using this space, we were able to discover that crawling varies broadly across multiple dimensions. Furthermore, we developed a simple statistic that can measure the relationship between variation in cell shape and the types of movement that are generated. This work lays the foundation for identifying the mechanistic bases of cellular movement across large evolutionary distances.

Learn more about this tool and how to use it here:

What’s next?

Now that we have a computational framework for comparing movement, we are at work applying our tools to a diversity of protist species. We are excited to share the results of these efforts and all of the tools used to achieve them.

In the near future, we will be exploring methods for rapidly characterizing genetic diversity within and among protist species. We are generating the ability to genotype thousands of cells in a single experiment. We aim to intersect these results with high-throughput phenotyping in order to identify the genetic basis of cellular traits in novel protist species.

Longer-term, we hope to place this work in a larger macroevolutionary context in order to map the generation of eukaryotic cellular diversity.