Research Focus of the Caspari lab

In this lab we study endosymbiotic organellogenesis, and specifically the role of protein import in this process. Endosymbiotic organellogenesis of mitochondria and chloroplasts drastically changed the biology of the emerging chimeric cells, enabling the development of complex life. Understanding how organellogenic processes can be replicated in the laboratory using synthetic biology would be an important step towards understanding fundamental aspects of the evolution of life as well as a powerful biotechnological tool for generating organisms with desired properties. To achieve this goal, three aspects need to be fulfilled: First, two organisms must be made to interact for mutual benefit (mutualism). Secondly, one (microbial) organism must be accommodated in the cells of the other, creating an endosymbiosis. Thirdly, the endosymbiosis must be transformed into full-fledged organellogenesis by transferring control over key reproductive and metabolic functions of the internal partner to the host - protein import is crucial for this step. In our work in this lab, all three aspects are investigated using the unicellular model green alga Chlamydomonas reinhardtii.

Organelle-host relations

To better understand the interplay between host cell and organelle, we investigate protein import in chloroplasts and opposing retrograde signalling in Chlamydomonas. Highly sequence-divergent N-terminal chloroplast transit peptides (cTPs) determine the import of cytosolically produced proteins. Research in the lab builds on the finding that cTPs consist of unstructured sequence elements upstream and downstream of a central amphipathic helix (Caspari et al. 2023) and that these sequence elements extend beyond the site where cTPs are cleaved from the transported protein (Caspari 2022). We use a genetic screen to test the role of cTP elements in protein targeting. To do this, we use leaky stop codon technology (Caspari 2020), which allows a combination of complementation and fluorescence microscopy. Similarly, we are interested in finding out whether the affinity of the transit peptides for the lipid composition of the target organelle membranes plays a role in determining the subcellular target site. In addition, we are investigating the role of H2O2 as a possible retrograde signal, which informs the nucleus which types of proteins are required in response to light stress.

A Venus fluorescent protein is targeted into different parts of the cell: Without a peptide, Venus accumulates in the cytoplasm. The Rubisco activase cTP generates import into the chloroplast, clearly visible though chlorophyll autofluorescence, and specifically into the algal pyrenoid. The mTP from a mitochondrial carbonic anhydrase generates import into the mitochondria, made visible here by a mitotracker. © Oliver Caspari

In natural ecosystems, algae mutualistically interact with a range of other organisms. © Oliver Caspari

Generating (endo-) symbiosis

Mutualistic symbioses have been documented for Chlamydomonas with a range of partners, and such interactions can give rise to co-dependencies through laboratory evolution. In the laboratory, we assemble microbial consortia and analyse the existing microbes for mutualistic interactions with the algae. In addition to known interaction partners from the literature, we isolate new partners by screening environmental samples. A positive effect of the microbial partners is evaluated by testing for an extension of survival and productive growth of the algae in dense culture. We also optimise the introduction of bacterial cells into Chlamydomonas. For this purpose, we use Azotobacter vinelandii as a naturally mutualistic bacterial partner, which can fix nitrogen from the air.

Protein import in bacteria

This idea builds on work on the evolution of protein import by mitochondria and chloroplasts from a bacterial defence system against antimicrobial peptides (Garrido und Caspari et al. 2020, Caspari und Lafontaine 2021, Caspari et al. 2023), which suggests that it may be possible to engineer bacteria to import proteins. To this end, we are investigating defensive import systems for antimicrobial peptides in Escherichia coli. We also plan to express the protein import system of mitochondria in bacteria to enable protein import. Ultimately, the aim is to introduce a protein-importing bacterium into Chlamydomonas. This would then allow us to find out which genes need to be transferred from the endosymbiont to the host cell in order to stabilise the interaction and thus generate synthetic organelles.

Molecular biology in progress! © Oliver Caspari

Publications

Here you can find a list of our previously published papers.

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Team

Here you can find a list of the current members of the research group.

To the Team Members