Spatial proteomics reveals lipid droplet reorganization in symbiotic Paramecium bursaria cells

Endosymbiosis is an important adaptive mechanism allowing organisms to exploit novel niches. The flexible relationship between the ciliate Paramecium bursaria and green algae represents a model system for studying early endosymbiosis evolution. However, the mechanisms underlying how P. bursaria cells maintain this endosymbiotic relationship remain unclear. Here, we use mass spectrometry-based proteomics to generate a spatial proteomic atlas of the host P. bursaria cells with and without endosymbionts. This atlas defines the protein composition of the endosymbiont-containing compartment (perialgal vacuole) and reveals pronounced remodeling of host lipid droplets upon symbiosis. Imaging analyses confirm that lipid droplets change in size, morphology, and positioning, accumulating near intracellular algae. Perturbing symbiotic cells with chemical inhibitors of lipid metabolism reduces endosymbiotic algal numbers, revealing a role for lipid droplets in the host-endosymbiont interaction. Our data provide a comprehensive resource for protein localization in P. bursaria cells and elucidate how those cells remodel an existing cellular compartment to maintain endosymbiosis. Competing Interest Statement The authors have declared no competing interest. Footnotes

and Discussion have been separated into distinct sections. The Results section has been substantially expanded with two major additions. First, unsupervised clustering (HDBSCAN) of the proteomics data is now presented, revealing that proteins from distinct subcellular compartments form coherent clusters without prior annotation. This analysis enabled identification of a candidate perialgal vacuole (PV) cluster (cluster 18), which was validated by immunofluorescence microscopy using VPS4A as a PV marker. Second, transmembrane protein prediction using DeepTMHMM was added, showing that membrane-bound compartments are enriched in transmembrane proteins while the PV is not, consistent with its protein composition. Gene ontology analysis of symbiotic cell-specific proteins was expanded to cover the PV, lipid droplet, Golgi apparatus, peroxisome, and mitochondria, providing a broader functional characterization of organelle remodeling during endosymbiosis. The marker protein set was revised from 279 proteins defining 13 compartments to 252 proteins defining 14 compartments, with the PV added as a new compartment. The protein translocation analysis and nitric oxide/cGMP-PKG signaling sections present in version 1 have been removed. Two main figures and four supplementary figures have been added.