Prioritizing Genes for Mitochondrial Calcium Uptake

Candidate human genes required for mitochondrial Ca²⁺ uptake were prioritized based on the expression of their homologues across mouse tissues27 (link), localization to the inner mitochondrial membrane28 (link),29 (link), and evolutionary conservation in kinetoplastids22 (link),23 (link),24 (link) but not in yeast22 (link),25 (link),26 (link). A focused RNAi screen was performed against 13 human genes in a commercially available, HeLa cell line expressing mitochondrial aequorin (mt-AEQ, Aequotech Catalog No. AT-002-H) using lentiviral constructs available from the Broad Institute’s RNAi Consortium44 (link). To rescue the MICU1 RNAi Ca²⁺ phenotype, we custom synthesized a cDNA containing synonymous mutations at all 8 codons complementary to the strongest hairpin (Blue Heron Biotechnology). Agonist-induced rises in mitochondrial Ca²⁺ were measured in mt-AEQ HeLa cells by luminescence30 (link),31 (link). Measurements of Ca²⁺ uptake in permeabilized HeLa cells were made using Calcium Green 5N33 (link). FRET based measurements of mitochondrial, cytosolic, and ER Ca²⁺ in single HeLa cells were performed using the 4mtD3cpv, D3cpv, and D1ER FRET sensors, respectively 32 (link). Mitochondrial respiration, morphology, and mtDNA copy number were measured as previously described45 (link). Mitochondrial membrane potential was measured by flow cytometry on cells stained with JC-1 according to manufacturer’s protocol. Single cell measurements of NADH were performed via established protocols46 (link). Crude mitochondria, Percoll purified mitochondria and mitoplasts were prepared from cultured HEK293 cells expressing MICU1-V5 as previously described47 (link),48 (link). Unless otherwise indicated, data are summarized as mean+/−standard deviation, and P-values correspond to t-tests.