Publications

Beyond the matrix: structural and physiological advancements in mitochondrial calcium signaling

Melissa JS MacEwen, Yasemin Sancak. Beyond the matrix: structural and physiological advancements in mitochondrial calcium signaling, Biochemical Society Transactions, 24 March 2023, https://doi.org/10.1042/BST20220317

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Mitochondrial calcium (Ca2+) signaling has long been known to regulate diverse cellular functions, ranging from ATP production via oxidative phosphorylation, to cytoplasmic Ca2+ signaling to apoptosis. Central to mitochondrial Ca2+ signaling is the mitochondrial Ca2+ uniporter complex (MCUC) which enables Ca2+ flux from the cytosol into the mitochondrial matrix. Several pivotal discoveries over the past 15 years have clarified the identity of the proteins comprising MCUC. Here, we provide an overview of the literature on mitochondrial Ca2+ biology and highlight recent findings on the high-resolution structure, dynamic regulation, and new functions of MCUC, with an emphasis on publications from the last five years. We discuss the importance of these findings for human health and the therapeutic potential of targeting mitochondrial Ca2+ signaling.

Mathematical modeling and biochemical analysis support partially ordered CaM-MLCK binding

Melissa JS MacEwen, Domnita Valeria Rusnac, Henok Ermias, Timothy Locke M Locke, Hayden E. Gizinski, Joseph P Dexter, Yasemin Sancak. Mathematical modeling and biochemical analysis support partially ordered CaM-MLCK binding, iScience, available online 4 February 2023, https://doi.org/10.1016/j.isci.2023.106146

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Activation of myosin light-chain kinase (MLCK) by calcium ions (Ca2+) and calmodulin (CaM) plays an important role in numerous cellular functions including vascular smooth muscle contraction and cellular motility. Despite extensive biochemical analysis of this system, aspects of the mechanism of activation remain controversial, and competing theoretical models have been proposed for the binding of Ca2+ and CaM to MLCK. The models are analytically solvable for an equilibrium steady state and give rise to distinct predictions that hold regardless of the numerical values assigned to parameters. These predictions form the basis of a recently proposed, multi-part experimental strategy for model discrimination. Here we implement this strategy by measuring CaM-MLCK binding using an in vitro FRET system. This system uses the CaM-binding region of smooth muscle MLCK protein to link two fluorophores to form an MLCK FRET Reporter (FR). Biochemical and biophysical experiments have established that FR can be reliably used to analyze MLCK-CaM binding. We assessed the binding of either wild-type CaM, or mutant CaM with one or more defective EF-hand domains, to FR. Interpretation of binding data in light of the mathematical models suggests a partially ordered mechanism for binding of CaM to MLCK. Complementary data collected using orthogonal approaches that directly quantify CaM-MLCK binding further supports our conclusions.

Evolutionary divergence reveals the molecular basis of EMRE dependence of the human MCU

MacEwen MJ, Markhard AL, Bozbeyoglu M, et al. Evolutionary divergence reveals the molecular basis of EMRE dependence of the human MCU. Life Sci Alliance. 2020;3(10):e202000718. Published 2020 Aug 7. doi:10.26508/lsa.202000718

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The mitochondrial calcium uniporter (shown to right) is a calcium-activated calcium channel complex critical for cellular signaling and bioenergetics. MCU, the pore-forming subunit of the uniporter, contains two transmembrane domains and is found in all major eukaryotic taxa. In amoeba and fungi, MCU homologs are sufficient to form a functional calcium channel, whereas human MCU exhibits a strict requirement for the metazoan-specific, single-pass transmembrane protein EMRE for conductance. Here, we exploit this evolutionary divergence to decipher the molecular basis of EMRE dependence of human MCU. By systematically generating chimeric proteins that consist of EMRE-independent D. discoideum MCU (DdMCU) and H. sapiens MCU (HsMCU), we converged on a stretch of 10 amino acids in DdMCU that can be transplanted to HsMCU to render it EMRE-dependent. We call this region in human MCU the EMRE-dependence domain (EDD). Crosslinking experiments show that HsEMRE directly interacts with MCU at both of its transmembrane domains as well as the EDD. Based on previously published structures of fungal MCU homologs, the EDD segment is located distal to the selectivity filter of the calcium pore and appears flexible. We propose that EMRE stabilizes EDD of MCU, permitting both channel opening and calcium conductance.

MECHANISTIC MODEL OF TEMPERATURE INFLUENCE ON FLOWERING THROUGH WHOLE-PLANT ACCUMULATION OF FT

Hannah A Kinmonth-Schultz, Melissa J S MacEwen, Daniel D Seaton, Andrew J Millar, Takato Imaizumi, Soo-Hyung Kim. An explanatory model of temperature influence on flowering through whole-plant accumulation of FLOWERING LOCUS T in Arabidopsis thaliana, in silico Plants, Volume 1, Issue 1, 2019, diz006, https://doi.org/10.1093/insilicoplants/diz006

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In this paper, we assessed temperature influence on flowering by incorporating temperature-responsive flowering mechanisms across developmental age into an existing model. Temperature influences both the leaf production rate and expression of FLOWERING LOCUS T (FT), a photoperiodic flowering regulator, in leaves. The Arabidopsis Framework Model incorporated temperature influence on leaf growth but ignored the consequences of leaf growth on and direct temperature influence of FT expression. We measured FT production in differently aged leaves and modified the model, adding the mechanistic temperature influence on FT transcription, and linking FT to leaf growth. Our simulations suggest that in long days, the developmental timing (leaf number) at which the reproductive transition occurs is influenced by day length and temperature through FT, while temperature influences the rate of leaf production and the time (in days) the transition occurs. Further, we demonstrated that FT is mainly produced in the first 10 leaves in the Columbia ecotype, and that FT accumulation alone cannot explain flowering in conditions in which flowering is delayed. Our simulations supported our hypotheses that: 1) temperature regulation of FT, accumulated with leaf growth, is a component of thermal time, and 2) incorporating mechanistic temperature regulation of FT can improve model predictions in fluctuating temperatures.