Over the course of the project, advances were made on two interconnected fronts: nanotechnology development and experimental endosymbiosis biology. On the technology side, FluidFM, which combines atomic force microscopy, optical microscopy, and nanofluidics, was developed into a versatile platform for single-cell manipulation. Mitochondria were transplanted between living mammalian cells: the aspirated organelles fused with the host mitochondrial network and maintained functionality across generations, opening new directions in organelle physiology and laying groundwork for therapeutic applications. A further technological advance was Live-seq, a method for extracting cytoplasmic RNA from a living cell without destroying it, enabling temporal transcriptomic recording at single-cell resolution. FluidFM was also specifically adapted for fungal cells, which possess a rigid cell wall, establishing a method for injection into and extraction from individual cells across yeasts and filamentous fungi. Building on these foundations, bacteria were implanted directly into the cytoplasm of an early-diverging filamentous fungus. Bacterial strains differed markedly in behaviour: some proliferated rapidly and became entrapped, while others achieved vertical transmission and enabled endosymbiont-mediated biosynthesis of a natural product. Endosymbiosis imposed fitness costs on the host that were offset through positive selection and host adaptation, allowing the stabilising dynamics of a nascent endosymbiosis to be characterised experimentally. In addition, a free-living soil bacterium was used to probe the earliest phase of endosymbiosis formation. Despite an initial antagonistic phase marked by compromised host fitness and immune activation, vertical transmission and repeated selective passaging led to transcriptional relaxation of host defence responses, documenting a shift from antagonism to commensalism. Host responses to bacterial implantation were also studied in enteroids, providing insight into how mammalian host cells respond to intruding bacteria.
