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Most unexpectedly, there is now increasing evidence that mitochondria have novel and crucial functions in the regulatory machinery of the growth/differentiation transition, cell-type determination, cellular movement and pattern formation. Here we created ρΔ cells with a reduced amount (about 1/4) of mitochondrial DNA (mtDNA) from Dictyostelium discoideum Ax-2 cells, by exposing Ax-2 cells to ca. 30 μg/ml of ethidium bromide (EtBr) in axenic growth medium. Importantly, the ρΔ cells exhibited a series of fascinating behaviors: when they were starved, they showed a marked delay of differentiation and stopped their development at the slug stage, thus failing to construct fruiting bodies. Moreover, cell patterning and cell-type proportioning were found to be greatly modified in slugs (referred to as ρΔ slugs) derived from ρΔ cells. That is, prestalk differentiation was significantly enhanced in ρΔ slugs, while prespore differentiation was markedly inhibited. In addition, the clear anterior prestalk/posterior prespore pattern was considerably disturbed in ρΔ slugs, presumably because of incomplete sorting between the two types of differentiated cells. After the assay of phototaxis, ρΔ slugs also exhibited highly disordered movement towards the light source. Taken together, these results suggest that mtDNA might have important multiple functions in a variety of cellular processes during Dictyostelium development.

Dd-TRAP1 is a Dictyostelium homologue of tumor necrosis factor receptor-associated protein 1 (TRAP-1). Dd-TRAP1 is located in the cortex of cells growing at a low density, but was found to be translocated to mitochondria with the help of a novel prestarvation factor that was accumulated in growth medium along with increased cell densities. The knockdown mutant of Dd-TRAP1 (TRAP1-RNAi cells) exhibited a significant defect in prestarvation response. Although TRAP1-RNAi cells showed normal expressions of classical prestarvation genes [dscA (discoidin I) and car1 (carA; cAMP receptor)], the expression of differentiation-associated genes (dia1 and dia3) induced by the prestarvation response were markedly repressed. By contrast, transformants overexpressing Dd-TRAP1 showed an early prestarvation response and also increased expression of dia1 and dia3 in a cell-density-dependent manner. Importantly, introduction of Dd-TRAP1 antibody into D. discoideum Ax-2 cells by electroporation inhibited the translocation of Dd-TRAP1 from the cortex to mitochondria and greatly inhibited the initiation of differentiation. Taken together, these results indicate that Dd-TRAP1 is translocated to mitochondria by sensing the cell density in growth medium and enhances the early developmental program through a novel prestarvation response.

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