bims-mitdyn 2021-09-05 papers

Issue of 2021‒09‒05
six papers selected by
Edmond Chan
Queen’s University, School of Medicine

J Cell Biol. 2021 Nov 01. pii: e202104073. [Epub ahead of print]220(11):

Vps13D functions in a Pink1-dependent and Parkin-independent mitophagy pathway.

James L Shen, Tina M Fortier, Ruoxi Wang, Eric H Baehrecke.

Defects in autophagy cause problems in metabolism, development, and disease. The autophagic clearance of mitochondria, mitophagy, is impaired by the loss of Vps13D. Here, we discover that Vps13D regulates mitophagy in a pathway that depends on the core autophagy machinery by regulating Atg8a and ubiquitin localization. This process is Pink1 dependent, with loss of pink1 having similar autophagy and mitochondrial defects as loss of vps13d. The role of Pink1 has largely been studied in tandem with Park/Parkin, an E3 ubiquitin ligase that is widely considered to be crucial in Pink1-dependent mitophagy. Surprisingly, we find that loss of park does not exhibit the same autophagy and mitochondrial deficiencies as vps13d and pink1 mutant cells and contributes to mitochondrial clearance through a pathway that is parallel to vps13d. These findings provide a Park-independent pathway for Pink1-regulated mitophagy and help to explain how Vps13D regulates autophagy and mitochondrial morphology and contributes to neurodegenerative diseases.

DOI: https://doi.org/10.1083/jcb.202104073

Elife. 2021 Aug 31. pii: e69312. [Epub ahead of print]10

The mechanism of MICU-dependent gating of the mitochondrial Ca2+ uniporter.

Vivek Garg, Junji Suzuki, Ishan Paranjpe, Tiffany Unsulangi, Liron Boyman, Lorin S Milescu, W Jonathan Lederer, Yuriy Kirichok.

Ca2+ entry into mitochondria is through the mitochondrial calcium uniporter complex (MCUcx), a Ca2+-selective channel composed of five subunit types. Two MCUcx subunits (MCU and EMRE) span the inner mitochondrial membrane, while three Ca2+-regulatory subunits (MICU1, MICU2 and MICU3) reside in the intermembrane space. Here we provide rigorous analysis of Ca2+ and Na+ fluxes via MCUcx in intact isolated mitochondria to understand the function of MICU subunits. We also perform direct patch clamp recordings of macroscopic and single MCUcx currents to gain further mechanistic insight. This comprehensive analysis shows that the MCUcx pore, composed of the EMRE and MCU subunits, is not occluded nor plugged by MICUs during the absence or presence of extramitochondrial Ca2+ as has been widely reported. Instead, MICUs potentiate activity of MCUcx as extramitochondrial Ca2+ is elevated. MICUs achieve this by modifying the gating properties of MCUcx allowing it to spend more time in the open state.

Keywords: molecular biophysics; mouse; structural biology

DOI: https://doi.org/10.7554/eLife.69312

Elife. 2021 09 01. pii: e63453. [Epub ahead of print]10

The mitochondrial permeability transition pore activates the mitochondrial unfolded protein response and promotes aging.

Suzanne Angeli, Anna Foulger, Manish Chamoli, Tanuja Harshani Peiris, Akos Gerencser, Azar Asadi Shahmirzadi, Julie Andersen, Gordon Lithgow.

Mitochondrial activity determines aging rate and the onset of chronic diseases. The mitochondrial permeability transition pore (mPTP) is a pathological pore in the inner mitochondrial membrane thought to be composed of the F-ATP synthase (complex V). OSCP, a subunit of F-ATP synthase, helps protect against mPTP formation. How the destabilization of OSCP may contribute to aging, however, is unclear. We have found that loss OSCP in the nematode Caenorhabditis elegans initiates the mPTP and shortens lifespan specifically during adulthood, in part via initiation of the mitochondrial unfolded protein response (UPRmt). Pharmacological or genetic inhibition of the mPTP inhibits the UPRmt and restores normal lifespan. Loss of the putative pore-forming component of F-ATP synthase extends adult lifespan, suggesting that the mPTP normally promotes aging. Our findings reveal how an mPTP/UPRmt nexus may contribute to aging and age-related diseases and how inhibition of the UPRmt may be protective under certain conditions.

Keywords: C. elegans; F-ATP synthase; aging; c-subunit; cell biology; mitochondrial permeability transition pore; mitochondrial unfolded protein response; oscp/atp-3

DOI: https://doi.org/10.7554/eLife.63453

J Biol Chem. 2021 Aug 27. pii: S0021-9258(21)00935-2. [Epub ahead of print] 101134

Proteomic analysis demonstrates the role of the quality control protease LONP1 in mitochondrial protein aggregation.

Karen Pollecker, Marc Sylvester, Wolfgang Voos.

The mitochondrial matrix protease LONP1 is an essential part of the organellar protein quality control system. LONP1 has been shown to be involved in respiration control and apoptosis. Furthermore, a reduction in LONP1 level correlates with ageing. Up to now, the effects of a LONP1 defect were mostly studied by utilizing transient, siRNA-mediated knockdown approaches. We generated a new cellular model system for studying the impact of LONP1 on mitochondrial protein homeostasis by a CRISPR/Cas-mediated genetic knockdown (gKD). These cells show a stable reduction of LONP1 along with a mild phenotype characterized by absent morphological differences and only small negative effects on mitochondrial functions under normal culture conditions. To assess the consequences of a permanent LONP1 depletion on the mitochondrial proteome, we analyzed the alterations of protein levels by quantitative mass spectrometry, demonstrating small adaptive changes, in particular with respect to mitochondrial protein biogenesis. In an additional proteomic analysis, we determined the temperature-dependent aggregation behavior of mitochondrial proteins and its dependence on a reduction of LONP1 activity, demonstrating the important role of the protease for mitochondrial protein homeostasis in mammalian cells. We identified a significant number of mitochondrial proteins that are affected by LONP1 activity especially with respect to their stress-induced solubility. Taken together, our results suggest a very good applicability of the LONP1 gKD cell line as a model system for human ageing processes.

Keywords: Human; LONP1 protease; cell biology; mitochondria; protein aggregation; proteostasis

DOI: https://doi.org/10.1016/j.jbc.2021.101134

STAR Protoc. 2021 Sep 17. 2(3): 100767

Protocol for assessing real-time changes in mitochondrial morphology, fission and fusion events in live cells using confocal microscopy.

Irene Bertolini, Frederick Keeney, Dario C Altieri.

Changes in mitochondrial size, shape, and subcellular position, a process collectively known as mitochondrial dynamics, are exploited for various cancer traits. Modulation of subcellular mitochondrial trafficking and accumulation at the cortical cytoskeleton has been linked to the machinery of cell movements, fueling cell invasion and metastatic spreading. Here, we detail a technique to track changes in mitochondrial volume using a commercial CellLight™ Mitochondria-RFP/GFP reporter and live confocal microscopy. This allows a real-time study of mitochondrial dynamics in live cells. For complete details on the use and execution of this protocol, please refer to Bertolini et al. (2020).

Keywords: Cancer; Microscopy; Molecular/Chemical Probes

DOI: https://doi.org/10.1016/j.xpro.2021.100767

STAR Protoc. 2021 Sep 17. 2(3): 100752

Isolation and analysis of fractions enriched in WrappER-associated mitochondria from mouse liver.

Nicolò Ilacqua, Irene Anastasia, Luca Pellegrini.

The endoplasmic reticulum (ER) plays a central role in lipid homeostasis, but the role of individual ER subdomains in lipid biology has not been elucidated. WrappER is a curved wrapping type of rough-ER that establishes extensive contacts with almost every mitochondria of the hepatocyte in the mouse liver. Here, we describe a protocol for isolation of fractions enriched in wrappER-associated mitochondria from the mouse liver. We also provide techniques for assessing its quality by electron microscopy and biochemical/proteomic analysis. For complete information on the use and execution of this protocol, please refer to Anastasia et al. (2021).

Keywords: Cell Biology; Cell separation/fractionation; Metabolism; Protein Biochemistry

DOI: https://doi.org/10.1016/j.xpro.2021.100752