scholarly journals Constriction and Dnm1p Recruitment Are Distinct Processes in Mitochondrial Fission

2003 ◽  
Vol 14 (5) ◽  
pp. 1953-1963 ◽  
Author(s):  
Aster Legesse-Miller ◽  
Ramiro H. Massol ◽  
Tom Kirchhausen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescence Imaging ◽  
Hot Spots ◽  
Mitochondrial Membrane ◽  
Mitochondrial Fission ◽  
Three Dimensional ◽  
Time Lapse ◽  
Outer Mitochondrial Membrane ◽  
Mitochondrial Matrix ◽  
Different Shapes

Mitochondria undergo cycles of fusion and fission crucial for organelle homeostasis. Fission is regulated partially by recruitment of the large GTPase Dnm1p to the outer mitochondrial membrane. Using three-dimensional time-lapse fluorescence imaging of Saccharomyces cerevisiae cells, we found that Dnm1p-EGFP appears and disappears at “hot spots” along mitochondrial tubes. It forms patches that convert rapidly into different shapes regardless of whether mitochondrial fission ensues or not. Moreover, the thickness of the mitochondrial matrix displays frequent temporal fluctuations apparently unrelated to fission or to recruitment of Dnm1p-EGFP. These results suggest that mitochondrial fission requires coordination of at least two distinct processes.

scholarly journals PINK1 disables the anti-fission machinery to segregate damaged mitochondria for mitophagy

2016 ◽  
Vol 213 (2) ◽  
pp. 163-171 ◽  
Author(s):  
Kenneth R. Pryde ◽  
Heather L. Smith ◽  
Kai-Yin Chau ◽  
Anthony H.V. Schapira
Keyword(s):  
Protein Kinase ◽  
Parkinson Disease ◽  
Protein Kinase A ◽  
Mitochondrial Membrane ◽  
Mitochondrial Fission ◽  
Scaffold Protein ◽  
Outer Mitochondrial Membrane ◽  
Related Protein ◽  
Anchoring Protein ◽  
Kinase A

Mitochondrial fission is essential for the degradation of damaged mitochondria. It is currently unknown how the dynamin-related protein 1 (DRP1)–associated fission machinery is selectively targeted to segregate damaged mitochondria. We show that PTEN-induced putative kinase (PINK1) serves as a pro-fission signal, independently of Parkin. Normally, the scaffold protein AKAP1 recruits protein kinase A (PKA) to the outer mitochondrial membrane to phospho-inhibit DRP1. We reveal that after damage, PINK1 triggers PKA displacement from A-kinase anchoring protein 1. By ejecting PKA, PINK1 ensures the requisite fission of damaged mitochondria for organelle degradation. We propose that PINK1 functions as a master mitophagy regulator by activating Parkin and DRP1 in response to damage. We confirm that PINK1 mutations causing Parkinson disease interfere with the orchestration of selective fission and mitophagy by PINK1.

scholarly journals Mitochondrial membrane tension governs fission

2018 ◽  
Author(s):  
Dora Mahecic ◽  
Lina Carlini ◽  
Tatjana Kleele ◽  
Adai Colom ◽  
Antoine Goujon ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mitochondrial Membrane ◽  
Mitochondrial Fission ◽  
Outer Mitochondrial Membrane ◽  
Membrane Tension ◽  
Structured Illumination ◽  
Bending Energy ◽  
Structured Illumination Microscopy ◽  
Cellular Factors ◽  
Tension Sensor

AbstractDuring mitochondrial fission, key molecular and cellular factors assemble on the outer mitochondrial membrane, where they coordinate to generate constriction. Constriction sites can eventually divide, or reverse upon disassembly of the machinery. However, a role for membrane tension in mitochondrial fission, although speculated, has remained undefined. We captured the dynamics of constricting mitochondria in mammalian cells using live-cell structured illumination microscopy (SIM). By analyzing the diameters of tubules that emerge from mitochondria and implementing a fluorescence lifetime-based mitochondrial membrane tension sensor, we discovered that mitochondria are indeed under tension. Under perturbations that reduce mitochondrial tension, constrictions initiate at the same rate, but are less likely to divide. We propose a model based on our estimates of mitochondrial membrane tension and bending energy in living cells which accounts for the observed probability distribution for mitochondrial constrictions to divide.

scholarly journals Measurement of perimitochondrial Ca2+ concentration in bovine adrenal glomerulosa cells with aequorin targeted to the outer mitochondrial membrane

1999 ◽  
Vol 341 (3) ◽  
pp. 745-753 ◽  
Author(s):  
Yves BRANDENBURGER ◽  
Jean-François ARRIGHI ◽  
Michel F. ROSSIER ◽  
Andrès MATURANA ◽  
Michel B. VALLOTTON ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Mitochondrial Membrane ◽  
Proteinase K ◽  
Outer Mitochondrial Membrane ◽  
Mitochondrial Matrix ◽  
Ang Ii ◽  
Peak Response ◽  
Strong Gradient ◽  
Glomerulosa Cells ◽  
Stimulation Of

Microdomains of high cytosolic free Ca2+ concentration in the proximity of mitochondria might have an important role in the stimulation of steroidogenesis in bovine adrenal glomerulosa cells. In the present study we have investigated local changes of free Ca2+ concentration near the outer mitochondrial membrane ([Ca2+]om) under stimulation with angiotensin II (Ang II) and K+. Glomerulosa cells in primary culture were transfected with a recombinant cDNA encoding the N-terminal region of the human translocase protein 20 of the outer mitochondrial membrane, in frame with the Ca2+-sensitive photoprotein aequorin. This chimaeric aequorin (TomAeq) was associated with mitochondria-enriched subcellular fractions of transfected COS-7 cells and was susceptible to proteinase K, showing that it was targeted to the outer mitochondrial membrane, facing the cytosolic space. In bovine adrenal glomerulosa cells transfected with TomAeq cDNA, Ang II induced a transient [Ca2+]om peak reaching 1.42±0.28 μM, which decreased immediately to the basal resting value. The peak response to Ang II was strikingly lower than the peak response of mitochondrial free Ca2+ concentration, which increased to 5.4±1.2 μM. The smaller response of [Ca2+]om to Ang II compared with the elevated matrix response did not result from buffering effects of the organelle, from altered mechanisms of intramitochondrial Ca2+ transport or from differences in the affinity of the chimaeric aequorins for Ca2+. This approach has allowed us to follow perimitochondrial Ca2+ homeostasis in bovine glomerulosa cells under stimulation with Ca2+-mobilizing agonists and to reveal a strong gradient of Ca2+ concentration between the mitochondrial matrix and the immediate environment of the organelle.

scholarly journals A novel motif in the yeast mitochondrial dynamin Dnm1 is essential for adaptor binding and membrane recruitment

2012 ◽  
Vol 199 (4) ◽  
pp. 613-622 ◽  
Author(s):  
Huyen T. Bui ◽  
Mary A. Karren ◽  
Debjani Bhar ◽  
Janet M. Shaw
Keyword(s):  
Mitochondrial Membrane ◽  
Mitochondrial Fission ◽  
Adaptor Proteins ◽  
Structural Features ◽  
Outer Mitochondrial Membrane ◽  
Membrane Recruitment ◽  
Suppressor Mutations ◽  
Guanosine Triphosphatase ◽  
Related Proteins ◽  
Binding Interfaces

To initiate mitochondrial fission, dynamin-related proteins (DRPs) must bind specific adaptors on the outer mitochondrial membrane. The structural features underlying this interaction are poorly understood. Using yeast as a model, we show that the Insert B domain of the Dnm1 guanosine triphosphatase (a DRP) contains a novel motif required for association with the mitochondrial adaptor Mdv1. Mutation of this conserved motif specifically disrupted Dnm1–Mdv1 interactions, blocking Dnm1 recruitment and mitochondrial fission. Suppressor mutations in Mdv1 that restored Dnm1–Mdv1 interactions and fission identified potential protein-binding interfaces on the Mdv1 β-propeller domain. These results define the first known function for Insert B in DRP–adaptor interactions. Based on the variability of Insert B sequences and adaptor proteins, we propose that Insert B domains and mitochondrial adaptors have coevolved to meet the unique requirements for mitochondrial fission of different organisms.

scholarly journals The Mitochondrial TOM Complex Is Required for tBid/Bax-induced Cytochrome c Release

2007 ◽  
Vol 282 (38) ◽  
pp. 27633-27639 ◽  
Author(s):  
Martin Ott ◽  
Erik Norberg ◽  
Katharina M. Walter ◽  
Patrick Schreiner ◽  
Christian Kemper ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Outer Membrane ◽  
Recombinant Proteins ◽  
Mitochondrial Membrane ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Outer Mitochondrial Membrane ◽  
Cytochrome C Release ◽  
Unknown Mechanism

Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.

scholarly journals Mitochondrial event localiser (MEL) to quantitatively describe fission, fusion and depolarisation in the three-dimensional space

2020 ◽  
Author(s):  
Rensu P Theart ◽  
Jurgen Kriel ◽  
Ben Loos ◽  
Thomas R Niesler
Keyword(s):  
Dimensional Space ◽  
Mitochondrial Fission ◽  
Three Dimensional ◽  
Time Lapse ◽  
Cell Periphery ◽  
Mitochondrial Network ◽  
Average Volume ◽  
Mitochondrial Homeostasis ◽  
Cellular Context ◽  
Three Dimensional Space

AbstractMitochondrial fission and fusion play an important role not only in maintaining mitochondrial homeostasis but also in preserving overall cellular viability. However, quantitative analysis based on the three-dimensional localisation of these highly dynamic mitochondrial events in the cellular context has not yet been accomplished. Moreover, it remains largely uncertain where in the mitochondrial network depolarisation is most likely to occur. We present the mitochondrial event localiser (MEL), a method that allows high-throughput, automated and deterministic localisation and quantification of mitochondrial fission, fusion and depolarisation events in large three-dimensional microscopy time-lapse sequences. In addition, MEL calculates the number of mitochondrial structures as well as their combined and average volume for each image frame in the time-lapse sequence. The mitochondrial event locations can subsequently be visualised by superposition onto the fluorescence micrograph z-stack. We apply MEL to both control samples as well as cells that have been treated with hydroxychloroquine sulphate (HCQ) and observe that fission and fusion events mostly occur around the terminal branches of the mitochondrial network, with few events detected in the strongly networked areas such as the perinuclear region. Depolarisation was most abundant in the HCQ treated samples, with the majority of the depolarisation events occurring in the cell periphery.

scholarly journals Mitochondrial event localiser (MEL) to quantitativelydescribe fission, fusion and depolarisation in the three-dimensional space

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0229634
Author(s):  
Rensu P. Theart ◽  
Jurgen Kriel ◽  
André du Toit ◽  
Ben Loos ◽  
Thomas R. Niesler
Keyword(s):  
Dimensional Space ◽  
Mitochondrial Fission ◽  
Three Dimensional ◽  
Time Lapse ◽  
Event Analysis ◽  
Representative Cell ◽  
Before And After ◽  
Cellular Context ◽  
Pre Treatment ◽  
And Control

Mitochondrial fission and fusion play an important role not only in maintaining mitochondrial homeostasis but also in preserving overall cellular viability. However, quantitative analysis based on the three-dimensional localisation of these highly dynamic mitochondrial events in the cellular context has not yet been accomplished. Moreover, it remains largely uncertain where in the mitochondrial network depolarisation is most likely to occur. We present the mitochondrial event localiser (MEL), a method that allows high-throughput, automated and deterministic localisation and quantification of mitochondrial fission, fusion and depolarisation events in large three-dimensional microscopy time-lapse sequences. In addition, MEL calculates the number of mitochondrial structures as well as their combined and average volume for each image frame in the time-lapse sequence. The mitochondrial event locations can subsequently be visualised by superposition over the fluorescence micrograph z-stack. We apply MEL to both control samples as well as to cells before and after treatment with hydrogen peroxide (H2O2). An average of 9.3/7.2/2.3 fusion/fission/depolarisation events per cell were observed respectively for every 10 sec in the control cells. With peroxide treatment, the rate initially shifted toward fusion with and average of 15/6/3 events per cell, before returning to a new equilibrium not far from that of the control cells, with an average of 6.2/6.4/3.4 events per cell. These MEL results indicate that both pre-treatment and control cells maintain a fission/fusion equilibrium, and that depolarisation is higher in the post-treatment cells. When individually validating mitochondrial events detected with MEL, for a representative cell for the control and treated samples, the true-positive events were 47%/49%/14% respectively for fusion/fission/depolarisation events. We conclude that MEL is a viable method of quantitative mitochondrial event analysis.

scholarly journals The role of Fis1p–Mdv1p interactions in mitochondrial fission complex assembly

2005 ◽  
Vol 171 (2) ◽  
pp. 291-301 ◽  
Author(s):  
Mary Anne Karren ◽  
Emily M. Coonrod ◽  
Teresa K. Anderson ◽  
Janet M. Shaw
Keyword(s):  
Mitochondrial Membrane ◽  
Mitochondrial Fission ◽  
Coiled Coil ◽  
Cytoplasmic Domain ◽  
Tetratricopeptide Repeat ◽  
Outer Mitochondrial Membrane ◽  
Complex Assembly ◽  
Binding Surface ◽  
New Evidence

Mitochondrial division requires coordinated interactions among Fis1p, Mdv1p, and the Dnm1p GTPase, which assemble into fission complexes on the outer mitochondrial membrane. The integral outer membrane protein Fis1p contains a cytoplasmic domain consisting of a tetratricopeptide repeat (TPR)–like fold and a short NH2-terminal helix. Although it is known that the cytoplasmic domain is necessary for assembly of Mdv1p and Dnm1p into fission complexes, the molecular details of this assembly are not clear. In this study, we provide new evidence that the Fis1p–Mdv1p interaction is direct. Furthermore, we show that conditional mutations in the Fis1p TPR-like domain cause fission complex assembly defects that are suppressed by mutations in the Mdv1p-predicted coiled coil. We also define separable functions for the Fis1p NH2-terminal arm and TPR-like fold. These studies suggest that the concave binding surface of the Fis1p TPR-like fold interacts with Mdv1p during mitochondrial fission and that Mdv1p facilitates Dnm1p recruitment into functional fission complexes.

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