scholarly journals MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca2+ Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Mayara S. Bertolini ◽  
Miguel A. Chiurillo ◽  
Noelia Lander ◽  
Anibal E. Vercesi ◽  
Roberto Docampo
Keyword(s):  
Trypanosoma Cruzi ◽  
Cell Lines ◽  
Citrate Synthase ◽  
Host Cells ◽  
Mitochondrial Calcium ◽  
Content Type ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Eukaryotic Supergroup

ABSTRACT The mitochondrial Ca2+ uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used Trypanosoma cruzi to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca2+ uptake. T. cruzi MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca2+ uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca2+ concentrations ([Ca2+]cyt), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles. IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca2+ sensors and gatekeepers of the MCU, preventing Ca2+ uptake under resting conditions and favoring it at high cytosolic Ca2+ concentrations ([Ca2+]cyt). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca2+]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.

scholarly journals Different Roles of Mitochondrial Calcium Uniporter Complex Subunits in Growth and Infectivity of Trypanosoma cruzi

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Miguel A. Chiurillo ◽  
Noelia Lander ◽  
Mayara S. Bertolini ◽  
Melissa Storey ◽  
Anibal E. Vercesi ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Human Cells ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Host Cells ◽  
Dominant Negative Effect ◽  
Mitochondrial Calcium ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Molecular Nature

ABSTRACT Trypanosoma cruzi is the agent of Chagas disease, and the finding that this parasite possesses a mitochondrial calcium uniporter (TcMCU) with characteristics similar to that of mammalian mitochondria was fundamental for the discovery of the molecular nature of MCU in eukaryotes. We report here that ablation of TcMCU , or its paralog TcMCUb , by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 led to a marked decrease in mitochondrial Ca 2+ uptake without affecting the membrane potential of these cells, whereas overexpression of each gene caused a significant increase in the ability of mitochondria to accumulate Ca 2+ . While TcMCU- knockout (KO) epimastigotes were viable and able to differentiate into trypomastigotes, infect host cells, and replicate normally, ablation of TcMCUb resulted in epimastigotes having an important growth defect, lower rates of respiration and metacyclogenesis, more pronounced autophagy changes under starvation, and significantly reduced infectivity. Overexpression of TcMCUb , in contrast to what was proposed for its mammalian ortholog, did not result in a dominant negative effect on TcMCU. IMPORTANCE The finding of a mitochondrial calcium uniporter (MCU) in Trypanosoma cruzi was essential for the discovery of the molecular nature of this transporter in mammals. In this work, we used the CRISPR/Cas9 technique that we recently developed for T. cruzi to knock out two components of the uniporter: MCU, the pore subunit, and MCUb, which was proposed as a negative regulator of MCU in human cells. In contrast to what occurs in human cells, MCU is not essential, while MCUb is essential for growth, differentiation, and infectivity; has a bioenergetic role; and does not act as a dominant negative subunit of MCU.

scholarly journals Different Sensitivity of Control and MICU1- and MICU2-Ablated Trypanosoma cruzi Mitochondrial Calcium Uniporter Complex to Ruthenium-Based Inhibitors

2020 ◽  
Vol 21 (23) ◽  
pp. 9316
Author(s):  
Mayara S. Bertolini ◽  
Roberto Docampo
Keyword(s):  
Trypanosoma Cruzi ◽  
Ruthenium Red ◽  
Host Cells ◽  
Mitochondrial Calcium ◽  
Low Permeability ◽  
Intact Cells ◽  
Mitochondrial Calcium Uniporter ◽  
Biochemical Characteristics ◽  
Permeabilized Cells ◽  
Calcium Uniporter

The mitochondrial Ca2+ uptake in trypanosomatids shares biochemical characteristics with that of animals. However, the composition of the mitochondrial Ca2+ uniporter complex (MCUC) in these parasites is quite peculiar, suggesting lineage-specific adaptations. In this work, we compared the inhibitory activity of ruthenium red (RuRed) and Ru360, the most commonly used MCUC inhibitors, with that of the recently described inhibitor Ru265, on Trypanosoma cruzi, the agent of Chagas disease. Ru265 was more potent than Ru360 and RuRed in inhibiting mitochondrial Ca2+ transport in permeabilized cells. When dose-response effects were investigated, an increase in sensitivity for Ru360 and Ru265 was observed in TcMICU1-KO and TcMICU2-KO cells as compared with control cells. In the presence of RuRed, a significant increase in sensitivity was observed only in TcMICU2-KO cells. However, application of Ru265 to intact cells did not affect growth and respiration of epimastigotes, mitochondrial Ca2+ uptake in Rhod-2-labeled intact cells, or attachment to host cells and infection by trypomastigotes, suggesting a low permeability for this compound in trypanosomes.

scholarly journals Functional analysis and importance for host cell infection of the Ca2+-conducting subunits of the mitochondrial calcium uniporter ofTrypanosoma cruzi

2019 ◽  
Vol 30 (14) ◽  
pp. 1676-1690 ◽  
Author(s):  
Miguel A. Chiurillo ◽  
Noelia Lander ◽  
Mayara S. Bertolini ◽  
Anibal E. Vercesi ◽  
Roberto Docampo
Keyword(s):  
Citrate Synthase ◽  
Etiologic Agent ◽  
Host Cells ◽  
Mitochondrial Calcium ◽  
Amino Acid Residues ◽  
Cell Infection ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Low Glucose ◽  
Membrane Spanning

We report here that Trypanosoma cruzi, the etiologic agent of Chagas disease, possesses two unique paralogues of the mitochondrial calcium uniporter complex TcMCU subunit that we named TcMCUc and TcMCUd. The predicted structure of the proteins indicates that, as predicted for the TcMCU and TcMCUb paralogues, they are composed of two helical membrane-spanning domains and contain a WDXXEPXXY motif. Overexpression of each gene led to a significant increase in mitochondrial Ca2+uptake, while knockout (KO) of either TcMCUc or TcMCUd led to a loss of mitochondrial Ca2+uptake, without affecting the mitochondrial membrane potential. TcMCUc-KO and TcMCUd-KO epimastigotes exhibited reduced growth rate in low-glucose medium and alterations in their respiratory rate, citrate synthase activity, and AMP/ATP ratio, while trypomastigotes had reduced ability to efficiently infect host cells and replicate intracellularly as amastigotes. By gene complementation of KO cell lines or by a newly developed CRISPR/Cas9-mediated knock-in approach, we also studied the importance of critical amino acid residues of the four paralogues on mitochondrial Ca2+uptake. In conclusion, the results predict a hetero-oligomeric structure for the T. cruzi MCU complex, with structural and functional differences, as compared with those in the mammalian complex.

scholarly journals The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter

2013 ◽  
Vol 15 (12) ◽  
pp. 1464-1472 ◽  
Author(s):  
Xin Pan ◽  
Jie Liu ◽  
Tiffany Nguyen ◽  
Chengyu Liu ◽  
Junhui Sun ◽  
...  
Keyword(s):  
Physiological Role ◽  
Mitochondrial Calcium ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter

scholarly journals Actin enhances ER-mitochondrial calcium transfer and IMM constriction during mitochondrial division

2017 ◽  
Author(s):  
Rajarshi Chakrabarti ◽  
Wei-Ke Ji ◽  
Radu V. Stan ◽  
Jaime de Juan Sanz ◽  
Timothy A. Ryan ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Mitochondrial Calcium ◽  
Mitochondrial Membranes ◽  
Independent Manner ◽  
Mitochondrial Division ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Transport Chain

SummaryMitochondrial division requires division of both the inner and outer mitochondrial membranes (IMM and OMM, respectively). Interaction with endoplasmic reticulum (ER) promotes OMM division by recruitment of the dynamin Drp1, but effects on IMM division are not well characterized. We previously showed that actin polymerization through the ER-bound formin INF2 stimulates Drp1 recruitment in mammalian cells. Here, we show that INF2-mediated actin polymerization stimulates a second mitochondrial response independent of Drp1: a rise in mitochondrial matrix calcium through the mitochondrial calcium uniporter. ER stores supply the increased mitochondrial calcium, and the role of actin is to increase ER-mitochondria contact. Myosin IIA is also required for this mitochondrial calcium increase. Elevated mitochondrial calcium in turn activates IMM constriction in a Drp1-independent manner. IMM constriction requires electron transport chain activity. IMM division precedes OMM division. These results demonstrate that actin polymerization independently stimulates the dynamics of both membranes during mitochondrial division: IMM through increased matrix calcium, and OMM through Drp1 recruitment.

Abstract 409: Investigating the Role of the Mitochondrial Calcium Uniporter (mcu) in Calcium Regulation of Cardiac Mitochondria Using Transmural Spectroscopy and an Integrating Sphere

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Anna Kosmach ◽  
Junhui Sun ◽  
Armel Femnou ◽  
Robert S Balaban ◽  
Elizabeth Murphy
Keyword(s):  
White Light ◽  
Mitochondrial Outer Membrane ◽  
Integrating Sphere ◽  
Mitochondrial Calcium ◽  
Mouse Heart ◽  
Light Sources ◽  
Cardiac Mitochondria ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter

Cardiac mitochondria uptake calcium through the mitochondrial calcium uniporter (MCU). To better understand the role of MCU and mitochondrial calcium in regulating heart physiology and pathophysiology, we developed a method to measure mitochondrial matrix calcium in beating, perfused hearts. Langendorff perfusing hearts are loaded with 4.5 uM rhod-2-AM at 30°C and then perfused at 37°C to washout uncleaved dye. We determined that rhod-2 localized primarily to the mitochondria under our loading conditions, as shown by loading in a heart expressing a GFP-tagged mitochondrial outer membrane protein and analyzing heart slices under super resolution microscopy. Further, addition of Mn 2+ , which quenches cytosolic rhod-2, has little effect on rhod-2 signal. We insert an optical catheter with both white light and 532nm laser into the left ventricle and interleaved the collection of spectra from both light sources. The perfused heart is center mounted in an integrating sphere for spectra collection. The light passes through the ventricle and reflects off the integrating sphere resulting in a near uniform sampling of the transmitted light. Spectral properties from both light sources are determined using a rapid scanning spectrophotometer. Myoglobin oxygenation, cytochrome redox state, and rhod-2 loading are determined by white light absorbance. Ca 2+ bound rhod-2 emission is determined by removing background tissue effects from laser emission spectra and normalizing to tissue absorbance. Using this method we are able to measure changes in Ca 2+ and cytochromes during treatments such as isoproterenol and ischemia-reperfusion. The use of an integrating sphere transmural spectroscopy provides us an unique method to study mitochondrial Ca 2+ signaling in perfused mouse heart loaded with Rhod-2.

scholarly journals Mitochondrial Calcium Uniporter Deficiency in Zebrafish Causes Cardiomyopathy With Arrhythmia

2020 ◽  
Vol 11 ◽  
Author(s):  
Adam D. Langenbacher ◽  
Hirohito Shimizu ◽  
Welkin Hsu ◽  
Yali Zhao ◽  
Alexandria Borges ◽  
...  
Keyword(s):  
Energy Production ◽  
Mitochondrial Calcium ◽  
Sinus Arrest ◽  
Compact Layer ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Trabecular Density ◽  
And Function ◽  
Production Cell

Mitochondrial Ca2 + uptake influences energy production, cell survival, and Ca2 + signaling. The mitochondrial calcium uniporter, MCU, is the primary route for uptake of Ca2 + into the mitochondrial matrix. We have generated a zebrafish MCU mutant that survives to adulthood and exhibits dramatic cardiac phenotypes resembling cardiomyopathy and sinus arrest. MCU hearts contract weakly and have a smaller ventricle with a thin compact layer and reduced trabecular density. Damaged myofibrils and swollen mitochondria were present in the ventricles of MCU mutants, along with gene expression changes indicative of cell stress and altered cardiac structure and function. Using electrocardiography, we found that MCU hearts display conduction system defects and abnormal rhythm, with extended pauses resembling episodes of sinus arrest. Together, our findings suggest that proper mitochondrial Ca2 + homeostasis is crucial for maintaining a healthy adult heart, and establish the MCU mutant as a useful model for understanding the role of mitochondrial Ca2 + handling in adult cardiac biology.

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