Carbonic anhydrase IX proteoglycan-like and intracellular domains mediate pulmonary microvascular endothelial cell repair and angiogenesis.

The lungs of patients with acute respiratory distress syndrome (ARDS) have hyperpermeable capillaries that must undergo repair in an acidic microenvironment. Pulmonary microvascular endothelial cells (PMVECs) have an acid-resistant phenotype, in part due to carbonic anhydrase IX (CA IX). CA IX also facilitates PMVEC repair by promoting aerobic glycolysis, migration, and network formation. Molecular mechanisms of how CA IX performs such a wide range of functions are unknown. CA IX is composed of four domains known as the proteoglycan-like (PG), catalytic (CA), transmembrane (TM), and intracellular (IC) domains. We hypothesized that the PG and CA domains mediate PMVEC pH homeostasis and repair, and the IC domain regulates aerobic glycolysis and PI3k/Akt signaling. The functions of each CA IX domain were investigated using PMVEC cell lines that express either a full-length CA IX protein or a CA IX protein harboring a domain deletion. We found that the PG domain promotes intracellular pH homeostasis, migration, and network formation. The CA and IC domains mediate Akt activation but negatively regulate aerobic glycolysis. The IC domain also supports migration while inhibiting network formation. Finally, we show that exposure to acidosis suppresses aerobic glycolysis and migration, even though intracellular pH is maintained in PMVECs. Thus, we report that 1) the PG and IC domains mediate PMVEC migration and network formation, 2) the CA and IC domains support PI3K/Akt signaling, and 3) acidosis impairs PMVEC metabolism and migration independent of intracellular pH homeostasis.

Correction: Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

[This corrects the article DOI: 10.1371/journal.pone.0152705.].

Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions.

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA.