This was a collaborative project by the entire Cleves Lab. We have worked hard to develop methods for RNAi, CRISPR/Cas9, and lab-induced spawning of the coral, Galaxea fascicularis. Thank you to @pcleves.bsky.social for supporting me during this project!

12/12 🧵

We conclude that symbiosis relies on the function of lysosomal proteins. How the algae resist digestion will require further investigation, but the co-option of lysosomal proteins for symbiosis may provide insight into the repeated evolution of photosymbiosis across animal taxa. 11/12 🧵

Excitingly, we found that mutating this gene in Galaxea also causes a loss of symbiosis, despite the independent evolution of photosymbiosis in these taxa! The defect is likely caused by breaking the supply of inorganic carbon and sulfate to the algae, required for the symbiont to thrive. 10/12 🧵

Finally, we asked if this gene is also required for symbiosis in the coral, Galaxea fascicularis, which we can now spawn in the lab. Look forward to a new preprint on this work! 9/12 🧵

We performed CRISPR/Cas9 mutagenesis on this gene in Aiptasia and found it caused a mosaic phenotype, with mutant animals exhibiting both low and high symbiont density regions that correlated with mutation frequency. 8/12 🧵

In the final section of this work, we wanted to test the function of SLC26A11, a lysosomal bicarbonate/sulfate transporter that we discovered in our proteome. We hypothesized that this protein plays a key role in providing inorganic carbon to fuel algal photosynthesis. IF confirmation below. 7/12 🧵

Next, using RNAi, we found that two key lysosomal proteins (LAMP1B, and a subunit of V-ATPase) are required for proper symbiosis formation. 6/12 🧵

The enrichment of lysosomal proteins was intriguing, so we developed new antibodies to confirm the lysosomal identity of the symbiosome with new super-resolution microscopy methods. 5/12 🧵

We found that the symbiosome has dynamic membrane trafficking, lysosomal, digestive enzymes, and transporter proteins, providing insights into how the symbiosome is formed, its cellular origins, and the types of molecules that are exchanged. 4/12 🧵

We then analyzed the symbiosome proteome. Remarkably, we found most of the proteins that were previously known to be symbiosomal based on careful antibody work (annotated on fig)! In addition, we found ~180 new symbiosome proteins. So now let’s look their predicted functions… 3/12 🧵

We first developed a method to isolate the symbiosome from the sea anemone, Aiptasia, using simple biochemistry techniques. The key was the use of an antibody for RHBG, a symbiosomal ammonium transporter, to track the isolation of the symbiosome. 2/12 🧵