Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation.

Mitochondria are multifaceted organelles crucial for cellular homeostasis that contain their own genome. Mitochondrial DNA (mtDNA) replication is a spatially regulated process essential for the maintenance of mitochondrial function, its defect causing mitochondrial diseases. mtDNA replication occurs at endoplasmic reticulum (ER)-mitochondria contact sites and is affected by mitochondrial dynamics: The absence of mitochondrial fusion is associated with mtDNA depletion whereas loss of mitochondrial fission causes the aggregation of mtDNA within abnormal structures termed mitobulbs. Here, we show that contact sites between mitochondria and ER sheets, the ER structure associated with protein synthesis, regulate mtDNA replication and distribution within mitochondrial networks. DRP1 loss or mutation leads to modified ER sheets and alters the interaction between ER sheets and mitochondria, disrupting RRBP1-SYNJ2BP interaction. Importantly, mtDNA distribution and replication were rescued by promoting ER sheets-mitochondria contact sites. Our work identifies the role of ER sheet-mitochondria contact sites in regulating mtDNA replication and distribution.

A new automated tool to quantify nucleoid distribution within mitochondrial networks.

Mitochondrial DNA (mtDNA) maintenance is essential to sustain a functionally healthy population of mitochondria within cells. Proper mtDNA replication and distribution within mitochondrial networks are essential to maintain mitochondrial homeostasis. However, the fundamental basis of mtDNA segregation and distribution within mitochondrial networks is still unclear. To address these questions, we developed an algorithm, Mitomate tracker to unravel the global distribution of nucleoids within mitochondria. Using this tool, we decipher the semi-regular spacing of nucleoids across mitochondrial networks. Furthermore, we show that mitochondrial fission actively regulates mtDNA distribution by controlling the distribution of nucleoids within mitochondrial networks. Specifically, we found that primary cells bearing disease-associated mutations in the fission proteins DRP1 and MYH14 show altered nucleoid distribution, and acute enrichment of enlarged nucleoids near the nucleus. Further analysis suggests that the altered nucleoid distribution observed in the fission mutants is the result of both changes in network structure and nucleoid density. Thus, our study provides novel insights into the role of mitochondria fission in nucleoid distribution and the understanding of diseases caused by fission defects.

The R941L mutation in MYH14 disrupts mitochondrial fission and associates with peripheral neuropathy.

BACKGROUND: Peripheral neuropathies are often caused by disruption of genes responsible for myelination or axonal transport. In particular, impairment in mitochondrial fission and fusion are known causes of peripheral neuropathies. However, the causal mechanisms for peripheral neuropathy gene mutations are not always known. While loss of function mutations in MYH14 typically cause non-syndromic hearing loss, the recently described R941L mutation in MYH14, encoding the non-muscle myosin protein isoform NMIIC, leads to a complex clinical presentation with an unexplained peripheral neuropathy phenotype. METHODS: Confocal microscopy was used to examine mitochondrial dynamics in MYH14 patient fibroblast cells, as well as U2OS and M17 cells overexpressing NMIIC. The consequence of the R941L mutation on myosin activity was modeled in C. elegans. FINDINGS: We describe the third family carrying the R941L mutation in MYH14, and demonstrate that the R941L mutation impairs non-muscle myosin protein function. To better understand the molecular basis of the peripheral neuropathy phenotype associated with the R941L mutation, which has been hindered by the fact that NMIIC is largely uncharacterized, we have established a previously unrecognized biological role for NMIIC in mediating mitochondrial fission in human cells. Notably, the R941L mutation acts in a dominant-negative fashion to inhibit mitochondrial fission, especially in the cell periphery. In addition, we observed alterations to the organization of the mitochondrial genome. INTERPRETATION: As impairments in mitochondrial fission cause peripheral neuropathy, this insight into the function of NMIIC likely explains the peripheral neuropathy phenotype associated with the R941L mutation. FUND: This study was supported by the Alberta Children's Hospital Research Institute, the Canadian Institutes of Health Research and the Care4Rare Canada Consortium.