bims-mitdyn 2022-12-25 papers

bims-mitdyn

Biomed News

on Mitochondrial dynamics: mechanisms

Issue of 2022–12–25
seven papers selected by
Edmond Chan, Queen’s University, School of Medicine

Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain.
Live-cell imaging and analysis of actin-mediated mitochondrial fission.
The N-terminal domain of human mitochondrial helicase Twinkle has DNA-binding activity crucial for supporting processive DNA synthesis by Polymerase γ.
Moms' mitochondria may refresh cells in sick kids.
Mitochondria Clumping vs. Mitochondria Fusion in CMT2A Diseases.
From passage to inhibition: Uncovering the structural and physiological inhibitory mechanisms of MCUb in mitochondrial calcium regulation.
Calcium Overload and Mitochondrial Metabolism.

Proc Natl Acad Sci U S A. 2022 Dec 27. 119(52): e2215799119

Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain.

Tianyan Liu, Till Stephan, Peng Chen, Jan Keller-Findeisen, Jingting Chen, Dietmar Riedel, Zhongtian Yang, Stefan Jakobs, Zhixing Chen.

  Capturing mitochondria's intricate and dynamic structure poses a daunting challenge for optical nanoscopy. Different labeling strategies have been demonstrated for live-cell stimulated emission depletion (STED) microscopy of mitochondria, but orthogonal strategies are yet to be established, and image acquisition has suffered either from photodamage to the organelles or from rapid photobleaching. Therefore, live-cell nanoscopy of mitochondria has been largely restricted to two-dimensional (2D) single-color recordings of cancer cells. Here, by conjugation of cyclooctatetraene (COT) to a benzo-fused cyanine dye, we report a mitochondrial inner membrane (IM) fluorescent marker, PK Mito Orange (PKMO), featuring efficient STED at 775 nm, strong photostability, and markedly reduced phototoxicity. PKMO enables super-resolution (SR) recordings of IM dynamics for extended periods in immortalized mammalian cell lines, primary cells, and organoids. Photostability and reduced phototoxicity of PKMO open the door to live-cell three-dimensional (3D) STED nanoscopy of mitochondria for 3D analysis of the convoluted IM. PKMO is optically orthogonal with green and far-red markers, allowing multiplexed recordings of mitochondria using commercial STED microscopes. Using multi-color STED microscopy, we demonstrate that imaging with PKMO can capture interactions of mitochondria with different cellular components such as the endoplasmic reticulum (ER) or the cytoskeleton, Bcl-2-associated X protein (BAX)-induced apoptotic process, or crista phenotypes in genetically modified cells, all at sub-100 nm resolution. Thereby, this work offers a versatile tool for studying mitochondrial IM architecture and dynamics in a multiplexed manner.

Keywords:  STED nanoscopy; cristae; mitochondria

DOI:  https://doi.org/10.1073/pnas.2215799119
STAR Protoc. 2022 Dec 20. pii: S2666-1667(22)00838-3. [Epub ahead of print]4(1): 101958

Live-cell imaging and analysis of actin-mediated mitochondrial fission.

Daisuke Shimura, Robin M Shaw.

  Current approaches, such as fixed-cell imaging or single-snapshot imaging, are insufficient to capture cytoskeleton-mediated mitochondrial fission. Here, we present a protocol to capture actin-mediated mitochondrial fission using high-resolution time-lapse imaging. We describe steps starting from cell preparation and mitochondria labeling through to live-cell imaging and final analysis. This approach is also applicable for analysis of multiple cytoskeleton-mediated organelle events such as vesicle trafficking, membrane fusion, and endocytic events in live cells. For complete details on the use and execution of this protocol, please refer to Shimura et al. (2021).1.

Keywords:  Cell Biology; Microscopy; Molecular Biology

DOI:  https://doi.org/10.1016/j.xpro.2022.101958
J Biol Chem. 2022 Dec 14. pii: S0021-9258(22)01240-6. [Epub ahead of print] 102797

The N-terminal domain of human mitochondrial helicase Twinkle has DNA-binding activity crucial for supporting processive DNA synthesis by Polymerase γ.

Laura C Johnson, Anupam Singh, Smita S Patel.

  Twinkle is the ring-shaped replicative helicase within the human mitochondria with high homology to bacteriophage T7 gp4 helicase-primase. Unlike many orthologs of Twinkle, the N-terminal domain (NTD) of human Twinkle has lost its primase activity through evolutionarily acquired mutations. The NTD has no demonstrated activity thus far; its role has remained unclear. Here, we biochemically characterize the isolated NTD and C-terminal domain with linker (CTD) to decipher their contributions to full-length (FL) Twinkle activities. This novel CTD construct hydrolyzes ATP, has weak DNA unwinding activity, and assists DNA Polymerase γ (Polγ)-catalyzed strand-displacement synthesis on short replication forks. However, CTD fails to promote multi-kilobase length product formation by Polγ in rolling-circle DNA synthesis. Thus, CTD retains all the motor functions but struggles to implement them for processive translocation. We show that NTD has DNA binding activity, and its presence stabilizes Twinkle oligomerization. CTD oligomerizes on its own, but the loss of NTD results in heterogeneously-sized oligomeric species. The CTD also exhibits weaker and salt-sensitive DNA binding compared to FL Twinkle. Based on these results, we propose that NTD directly contributes to DNA binding and holds the DNA in place behind the central channel of the CTD like a 'doorstop', preventing helicase slippages and sustaining processive unwinding. Consistent with this model, mitochondrial single-stranded DNA binding protein (mtSSB) compensate for the NTD loss and partially restore kilobase length DNA synthesis by CTD and Polγ. The implications of our studies are foundational for understanding the mechanisms of disease-causing Twinkle mutants that lie in the NTD.

Keywords:  DNA polymerase; Twinkle; helicase; mitochondria; mitochondrial diseases; replication; replisome

DOI:  https://doi.org/10.1016/j.jbc.2022.102797
Science. 2022 Dec 23. 378(6626): 1267

Moms' mitochondria may refresh cells in sick kids.

Mitch Leslie.

Technique is designed to treat mitochondrial disease.

DOI: https://doi.org/10.1126/science.adg3936
Life (Basel). 2022 Dec 15. pii: 2110. [Epub ahead of print]12(12):

Mitochondria Clumping vs. Mitochondria Fusion in CMT2A Diseases.

Antonietta Franco, Caroline E Walton, Xiawei Dang.

  Phenotypic variations in Charcot-Marie-Tooth disease type 2A (CMT2A) result from the many mutations in the mitochondrial fusion protein, mitofusin 2 (MFN2). While the GTPase domain mutations of MFN2 lack the ability to hydrolyze GTP and complete mitochondrial fusion, the mechanism of dysfunction in HR1 domain mutations has yet to be explored. Using Mfn1/Mfn2 double null cells and Mfn2 knock out (KO) fibroblasts, we measured the ability of this variant protein to change conformations and hydrolyze GTP. We found that a mutation in the HR1 domain (M376A) of MFN2 results in conformational change dysfunction while maintaining GTPase ability. Prolonged exposure to mitofusin agonist MiM 111 reverses mitochondrial fusion dysfunction in the HR1 mutant through encouraging an open conformation, resulting in a potential therapeutic model in this variant. Herein, we describe a novel mechanism of dysfunction in MFN2 variants through exploring domain-specific mitochondrial characteristics leading to CMT2A.

Keywords:  Charcot-Marie-Tooth disease type 2A; mitochondria fitness; mitochondria fusion defects; mitofusin 2

DOI:  https://doi.org/10.3390/life12122110
FASEB J. 2023 Jan;37(1): e22678

From passage to inhibition: Uncovering the structural and physiological inhibitory mechanisms of MCUb in mitochondrial calcium regulation.

Danielle M Colussi, Peter B Stathopulos.

  Mitochondrial calcium (Ca2+ ) regulation is critically implicated in the regulation of bioenergetics and cell fate. Ca2+ , a universal signaling ion, passively diffuses into the mitochondrial intermembrane space (IMS) through voltage-dependent anion channels (VDAC), where uptake into the matrix is tightly regulated across the inner mitochondrial membrane (IMM) by the mitochondrial Ca2+ uniporter complex (mtCU). In recent years, immense progress has been made in identifying and characterizing distinct structural and physiological mechanisms of mtCU component function. One of the main regulatory components of the Ca2+ selective mtCU channel is the mitochondrial Ca2+ uniporter dominant-negative beta subunit (MCUb). The structural mechanisms underlying the inhibitory effect(s) exerted by MCUb are poorly understood, despite high homology to the main mitochondrial Ca2+ uniporter (MCU) channel-forming subunits. In this review, we provide an overview of the structural differences between MCUb and MCU, believed to contribute to the inhibition of mitochondrial Ca2+ uptake. We highlight the possible structural rationale for the absent interaction between MCUb and the mitochondrial Ca2+ uptake 1 (MICU1) gatekeeping subunit and a potential widening of the pore upon integration of MCUb into the channel. We discuss physiological and pathophysiological information known about MCUb, underscoring implications in cardiac function and arrhythmia as a basis for future therapeutic discovery. Finally, we discuss potential post-translational modifications on MCUb as another layer of important regulation.

Keywords:  MCU; MCU dominant-negative beta subunit; MCUb; mitochondrial calcium uniporter; mitochondrial calcium uptake; post-translational modifications; structure-function

DOI:  https://doi.org/10.1096/fj.202201080R
Biomolecules. 2022 Dec 17. pii: 1891. [Epub ahead of print]12(12):

Calcium Overload and Mitochondrial Metabolism.

Lauren L Walkon, Jasiel O Strubbe-Rivera, Jason N Bazil.

  Mitochondria calcium is a double-edged sword. While low levels of calcium are essential to maintain optimal rates of ATP production, extreme levels of calcium overcoming the mitochondrial calcium retention capacity leads to loss of mitochondrial function. In moderate amounts, however, ATP synthesis rates are inhibited in a calcium-titratable manner. While the consequences of extreme calcium overload are well-known, the effects on mitochondrial function in the moderately loaded range remain enigmatic. These observations are associated with changes in the mitochondria ultrastructure and cristae network. The present mini review/perspective follows up on previous studies using well-established cryo-electron microscopy and poses an explanation for the observable depressed ATP synthesis rates in mitochondria during calcium-overloaded states. The results presented herein suggest that the inhibition of oxidative phosphorylation is not caused by a direct decoupling of energy metabolism via the opening of a calcium-sensitive, proteinaceous pore but rather a separate but related calcium-dependent phenomenon. Such inhibition during calcium-overloaded states points towards mitochondrial ultrastructural modifications, enzyme activity changes, or an interplay between both events.

Keywords:  bioenergetics; calcium overload; calcium phosphate; calcium precipitates; mitochondria; mitochondrial ATP production; mitochondrial function; mitochondrial ultrastructure; oxidative phosphorylation

DOI:  https://doi.org/10.3390/biom12121891