No abstract

Summary Mitochondrial Ca 2+ uptake is an important mediator of metabolism and cell death. Identification of components of the highly conserved mitochondrial Ca 2+ uniporter has opened it up to genetic analysis in model organisms. Here, we report a comprehensive genetic characterization of all known uniporter components conserved in Drosophila . While loss of pore-forming MCU or EMRE abolishes fast mitochondrial Ca 2+ uptake, this results in only mild phenotypes when young, despite shortened lifespans. In contrast, loss of the MICU1 gatekeeper is developmentally lethal, consistent with unregulated Ca 2+ uptake. Mutants for the neuronally restricted regulator MICU3 are viable with mild neurological impairment. Genetic interaction analyses reveal that MICU1 and MICU3 are not functionally interchangeable. More surprisingly, loss of MCU or EMRE does not suppress MICU1 mutant lethality, suggesting that this results from uniporter-independent functions. Our data reveal the interplay among components of the mitochondrial Ca 2+ uniporter and shed light on their physiological requirements in vivo .

Water is a fundamental resource, yet its spatiotemporal availability in East Africa is poorly understood. This is the area where most hominin first occurrences are located, and consequently the potential role of water in hominin evolution and dispersal remains unresolved. Here, we show that hundreds of springs currently distributed across East Africa could function as persistent groundwater hydro-refugia through orbital-scale climate cycles. Groundwater buffers climate variability according to spatially variable groundwater response times determined by geology and topography. Using an agent-based model, grounded on the present day landscape, we show that groundwater availability would have been critical to supporting isolated networks of hydro-refugia during dry periods when potable surface water was scarce. This may have facilitated unexpected variations in isolation and dispersal of hominin populations in the past. Our results therefore provide a new environmental framework in which to understand how patterns of taxonomic diversity in hominins may have developed.

Mitochondrial Ca 2+ uptake is an important mediator of metabolism and cell death. Identification of components of the highly conserved mitochondrial Ca 2+ uniporter has opened it up to genetic analysis in model organisms. Here we report a comprehensive genetic characterisation of the known uniporter components conserved in Drosophila. While loss of MCU or EMRE abolishes fast mitochondrial Ca 2+ uptake, this results in surprisingly mild phenotypes. In contrast, loss of the regulatory gatekeeper component MICU1 has a much more severe phenotype, being developmental lethal, consistent with unregulated Ca 2+ uptake. Mutants for MICU3 are viable with mild neurological phenotypes. Genetic interaction studies reveal that MICU1 and MICU3 are not functionally interchangeable. More surprisingly, loss of MCU or EMRE does not suppress MICU1 mutant lethality, suggesting that the lethality results from MCU-independent functions. This study helps shed light on the physiological requirements of the mitochondrial Ca 2+ uniporter, and provides a suite of tools to interrogate their interplay in homeostasis and disease conditions. B. DNA analysis of MCU 1 . Primer sequences are detailed in the 'Materials and Methods' section.The control genotype yielded a ~2.5 kb band, compared to ~900 bp for MCU 1 homozygotes. C. Western blot analysis of MCU 1 . Immunoblots were probed with the indicated antibodies. Asterisk denotes a non-specific band. Mitochondrial ATP5A is used as a loading control. D.Representative traces of Ca 2+ uptake in mitochondria isolated from adult flies of the indicated genotypes after addition of 45 μM CaCl2. Extramitochondrial Ca 2+ was measured by Calcium Green-5N fluorescence. Ca 2+ was released from mitochondria by addition of 1 μM FCCP. a.u.: arbitrary units. The control genotype is w 1118 . Addition of the MCU inhibitor Ruthenium Red (RuR; 2 μM) blocks mitochondrial Ca 2+ uptake, which is mirrored by MCU 1 . Mitochondrial Ca 2+ uptake is restored by transgenic re-expression of MCU. E. Relative uptake kinetics were determined through linear fits of the initial phase of Ca 2+ uptake and normalized to the wild type control (mean ± SEM, n = 3). Figure 2. MCU 1 shortens lifespan without impacting organismal phenotypes despite respiratory defects. A. The percentage of adult flies eclosing as homozygous MCU 1 mutants versus balanced heterozygotes, together with the expected Mendelian ratio in the offspring (n > 700). B. Lifespan curves of MCU 1 male flies compared with control (w 1118 ) and transgenic rescue (MCU 1 + MCU). Statistical analysis: Mantel-Cox log-rank test (n ≧ 74). C. Climbing assay of control (da/+) and MCU 1 flies, 2 and 20 days post-eclosion. Significance measured by Kruskal-Wallis test with Dunn's post-hoc correction for multiple comparisons (mean ± 95% confidence interval; n > 50; ns, non-significant). D. Relative ATP levels from control and MCU 1 flies. Statistical analysis: unpaired t-test (mean ± SD; n 2-3; **** P < 0.0001, ns, non-significant). E, F. Oxygen consumption rate (OCR) of control and MC...