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Role of calcium fluxes in mitochondrial dynamics

Periodic Reporting for period 1 - MITODYN (Role of calcium fluxes in mitochondrial dynamics)

Reporting period: 2017-07-01 to 2019-06-30

The MBU mitochondria Biology Unit (MBU) is an Institute dedicated to the study of mitochondria, a crucial organelle in the cell responsible for energy production. The MBU is composed of 8 different research groups exploring different aspects of mitochondria physiology. Numerous mutations in human mitochondrial DNA (mtDNA) have been identified and assigned to various deficiencies of the respiratory chain leading to severe diseases. Moreover, mitochondria house hundreds of biochemical reactions involved in processes critical for the survival and homeostatic adaptation of the cell. They are highly dynamic organelles, which adapt their shape and network depending on the cellular needs. As mitochondria play a central and unique role in metabolism, our understanding of the complex network of interrelations between mitochondria and other organelles has crucially changed our view on the cell biology of mitochondria and a new biology and pathophysiology of this organelle are emerging. Our research team is specialized in that aspect of mitochondria that is called mitochondrial dynamics.

Mitochondria are in dynamic contact with the endoplasmic reticulum (ER) and this specialized contact is essential for a number of relevant processes, including mitochondrial dynamics, lipid exchange, calcium homeostasis and the programmed cell death (apoptosis). The Mitochondrial Calcium Uniporter (MCU) and its regulators subunits (MICU1-2-3, EMRE) have recently been identified as forming the key complex regulating mitochondrial calcium uptake after calcium release form the ER. The amount of calcium in the mitochondrial matrix is tightly regulated as it controls the activities of three dehydrogenases of the Krebs cycle allowing the respiratory chain to function properly. In contrast, an excess of calcium can lead to mitochondria swelling and induce cell death. Thus, mitochondrial calcium uptake and energy production are directly connected and our goal is to understand how the MCU complex interacts with other proteins or protein complexes in the inner membrane to drive its functions.

Mitochondrial diseases are one of the most common groups of genetic diseases with a minimum prevalence of greater than 1 in 5000 in adults. They are characterized most of the time by severe neurological manifestation. Recently mutations in MICU-1, the major regulator of MCU, have been identified in patients suffering either from
proximal myopathy associated to learning difficulties or general fatigue and lethargy associated and difficulties to adapt to effort, confirming the importance of mitochondrial calcium homeostasis in human pathology.
By shedding lights on MCU complex function, we hope to contribute to the understanding of mitochondrial calcium related diseases and more broadly to all the mitochondrial diseases characterized by energy production deficiencies.

The overall objective of this project is to define the complete interactome of the Mitochondrial Calcium Uptake Machinery (MCUM) in different physiological conditions and to characterize the functions associated to this complex.
To address these goals, an unbiased proteomic analysis has been performed, both in cellulo (human cells) and in vivo (zebrafish), to identify the interactors of the MCUM at steady state and under different cellular stresses using the BioID technic. The mechanisms involved in the regulation of mitochondrial dynamics, apoptosis and cell migration have been studied using molecular and cell biology experiments coupled with the most recent advances in microscopic analysis. Finally, we have explored the relevance of these findings in the physiopathology associated to mitochondrial diseases using cell lines derived from patients harbouring a MCUM dysfunction.
From July 2017 to July 2019.

WP1: Study of MCUM function at steady state and during apoptosis:
We have characterized effect of the loss of the different components of the MCUM (MCU, MICU1/2 and EMRE) on mitochondrial morphology. We have analysed MCU localization in the MAM using confocal microscopy coupled to molecular biology approaches (MAM fractionation). Finally, we have studied the role of the MCUM in apoptosis and mitochondrial respiratory chain activity.

WP2: Cellular BioID MCUM interactome studies:
We have generated all the BirA constructs of each MCUM subunits and stably expressed them in HEK cells lines. The proper expression and localisation of each protein has been checked by molecular biology technics and microscopy. The conditions for mass spectrometry analyses have been set up and experimental results have been validated for all MCUM subunits. We have now finished conducted the steady state interactome analyses.

WP3: Zebrafish BioID MCUM interaction study
We have generated a MCU-BirA and MICU1-BirA constructs and check their proper mitochondrial localization in Zebrafish.

Conclusions regarding the milestones to reach.
1. Transgene expression in cell lines: This step has been been fully achieved.
2. Consistent mass spectrometry results in cell lines. This step has been completed for each of the subunits of the MCU complex.
3. transgene innocuity in Zebrafish: We have assessed the innocuity of BirA transgenes during zebrafish development for MCU and MICU1 subunits and conluded that the expression of these transgenes affected zebrafish early development.
4. Consistent mass spec. results in zebrafish : generating transgene for all the MCUM subunits and studying the interactome in vivo by the Bira technic have shown to be technically challenging. This part of the project has been now dropped.

Mitochondrial dynamics: overview of molecular mechanisms.
Tilokani L, Nagashima S, Paupe V, Prudent J.
Essays Biochem. 2018 Jul 20;62(3):341-360.

2 manuscripts in preparation: 1. “A complete interactome mapping of the MCU machinery”. This study will allow the scientific community to access to the complete interactome database of the MCUM machinery.
2. “Specific interaction between the MCUM and respiratory chain complex regulates mitochondrial bioenergetics”
During these 2 years of work, we have reached 3 out of 4 milestones depicted in the original project. We have conducted successfully the interactome analysis of the whole MCU complex in a human cell line. We have obtained consistent data for each of the subunits of the MCU complex that allowed us to build a complete interaction map of the MCUM interactome in human cell lines. As this part of the project has been successfully achieved, we are now exploiting the results obtained by this technic investigating the specific MCUM interactome at steady state, during specific cellular events and in the context of mitochondrial diseases.

An unexpected result has emerged from this study aiming to unravel MCUM interactome. Indeed, we have established that the MCUM complex can form super-complexes with some complex of the respiratory chain. We are now exploring the function of such a structure. In most cases, mitochondrial DNA mutations in humans affect the structure and the activity of the complexes of the respiratory chain, which lead to very severe conditions. Considering our results, exploring the role of MCUM in respiratory chain activity is likely to increase our knowledge on mitochondrial bioenergetics, which is crucial for a better understanding of mitochondrial diseases and developing potential cures.