Mitochondrial membrane tension governs fission

Abstract

2Mitochondria rely on cellular machinery for their division, which is an essential component of metabolic response of the cell. Many division factors have been identified; however, a framework accounting for the energetic requirements of the mitochondrial fission process is lacking. We report that the presence of an active constriction does not ensure fission. Instead, by measuring constrictions down to ~100 nm with time-lapse super-resolution microscopy, we found that 34% of constrictions failed to divide and 'reversed' to an unconstricted state. Higher local curvaturesreflecting an increased bending energy -made constriction sites more likely to divide, but often with a significant residual energy barrier to fission. Our data suggest that membrane tension, largely arising from pulling forces, could account for this missing energy. These results lead us to propose that mitochondrial fission is probabilistic, and can be modeled as arising from bending energy complemented by a fluctuating membrane tension.Mitochondria are highly dynamic organelles, transported through the cytoplasm along cytoskeletal networks as they change in size and shape. Mitochondrial morphologies can range from a filamentous, connected network to a fragmented collection of individuals. Underlying such morphological changes are altered equilibria between fusion and division 1,2 . These transformations have been linked to an adaptive response to cellular energy requirements, for example in response to stress [3][4][5] or the cell cycle 6 . As a vestige of their bacterial origins, mitochondria cannot be generated de novo, but must multiply by division, or fission, of existing mitochondria 7 . Division has also been suggested to act as part of a quality control mechanism 8,9 and an intracellular signal for mitophagy 10,11 .All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/255356 doi: bioRxiv preprint first posted online 3 In bacterial division systems, internal assembly of the fission machinery is tightly regulated and a series of cell cycle checkpoints ensure daughter cell viability. In contrast, the mitochondrial division machinery is external to the organelle, allowing cells to flexibly regulate fission. Initially, the division site is marked by a pre-constriction defined by contact with ER tubules 12 and deformed by targeted actin polymerization [13][14][15] Live-cell Structured Illumination Microscopy (SIM) of Drp1-mediated mitochondrial constriction events enabled us to measure dynamic changes in membrane geometry leading up to and following division. We discovered that a fraction of mitochondria constrict dramatically before relaxing to an unconstricted state, termed 'reversals'. Using a custom-written image analysis package and classical elasticity theory, we calculated the energy differences between fission and reversal events, which al...

NMR identification of a conserved Drp1 cardiolipin-binding motif essential for stress-induced mitochondrial fission

“…In the current work, our ssNMR and EM studies provide independent and orthogonal support. Based on these results and previous observations from other groups, we conclude that additional force factors, including contributions from cytoskeletal elements that regulate Drp1 activity, for example, F-actin or microtubules (80)(81)(82)(83), are likely required for physiologically relevant membrane fission. In summary, our comprehensive studies firmly establish an essential role for Drp1 VD-CL interactions in stress-induced mitochondrial fission in vivo and provide insights into the molecular mechanisms underlying CL recognition and binding.…”

Mechanosensitive Fluorescent Probes to Image Membrane Tension in Mitochondria, Endoplasmic Reticulum, and Lysosomes

“…A complementary analysis on the role of membrane tension in mitochondrial fission using Mito Flipper 4 will be reported elsewhere. 27 In conclusion, three organelle-selective fluorescent membrane tension probes were designed, synthesized and characterized both in vesicles and living cells. As proven by the sensitivity of their lifetime to osmotic shock, they report on changes in membrane tension and reveal different lifetimes for each target.…”

Event-driven acquisition for content-enriched microscopy

“…This dataset was enhanced 10-fold by rotating the images, to make up the final dataset (37000 images). This data set was recorded on a fast dual-color SIM setup at Janelia Research Campus 51 and published previously 17 , but processed via Gaussian filtering to imitate the expected resolution of raw iSIM data before deconvolution. The output ground truth dataset was generated from the fluorescence channels, followed by manual curation.…”

“…The slow and EDA imaging modes were compared by calculating the minimal width of constriction measured during an event as described above. The measurement method was similar to that described in 17 . The deconvolution of the images was performed using the Richardson Lucy algorithm as implemented by the flowdec Python package with 30 iterations 53 .…”