Inside-out model for the evolution of eukaryotic cell organization. Model showing the stepwise evolution of eukaryotic cell organization from (A) an eocyte ancestor with a single bounding membrane and a glycoprotein rich cell wall (S-layer) interacting with epibiotic α-proteobacteria (proto-mitochondria). (B) We envision the eocyte cell forming protrusions, aided by protein-membrane interactions at the protrusion neck. These protrusions facilitated material exchange with proto-mitochondria. (C) Selection for a greater area of contact between the symbionts would have led to bleb enlargement and the eventual loss of the S-layer from the protrusions. (D) Blebs would have then been further stabilized by the development of a symmetric nuclear pore outer ring complex (Figure 2) and through the establishment of LINC complexes that, following the gradual loss of the S-layer, physically connected the original cell body (the nascent nuclear compartment) to the inner bleb membranes. (E) With the expansion of blebs to enclose the proto-mitochondria, a process that would have facilitated the acquisition of bacterial lipid biosynthesis machinery by the host, the site of cell growth would have progressively shifted to the cytoplasm, facilitated by the development of regulated traffic through the nuclear pore. At the same time, the spaces between blebs would have enabled the gradual maturation of proteins secreted into the environment via the perinuclear space through glycosylation and proteolytic cleavage. (F) Finally, bleb fusion would have connected cytoplasmic compartments and driven the formation of an intact plasma membrane, perhaps through a process akin to phagocytosis whereby one bleb enveloped the whole. This simple topological transition would have isolated the endoplasmic reticulum from the outside world, driven the full development of a system of vesicular trafficking, and established strict vertical transmission of mitochondria, leading to a cell with modern eukaryotic cell organization.