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Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...

University of Zaragoza

Adherent cells exert contractile forces which play an important role in the spatial organization of the extracellular matrix (ECM). Due to these forces, the substrate experiments a volume reduction leading to a characteristic shape. ECM contraction is a key process in many biological processes such as embryogenesis, morphogenesis and wound healing. However, little is known about the specific parameters that control this process. With this aim, we present a 3D computational model able to predict the contraction process of a hydrogel matrix due to cell-substrate mechanical interaction. It considers cell-generated forces, substrate deformation, ECM density, cellular migration and proliferation. The model also predicts the cellular spatial distribution and concentration needed to reproduce the contraction process and confirms the minimum value of cellular concentration necessary to initiate the process observed experimentally. The obtained continuum formulation has been implemented in a finite element framework. In parallel, in vitro experiments have been performed to obtain the main model parameters and to validate it. The results demonstrate that cellular forces, migration and proliferation are acting simultaneously to display the ECM contraction.

Structural biology response of a collagen hydrogel synthetic extracellular matrix with embedded human fibroblast: computational and experimental analysis.

Multiprotein complexes of the mitochondrial electron transport chain form associations to generate supercomplexes. The relationship between tumor cell ability to assemble mitochondrial supercomplexes, tumorigenesis and metastasis has not been studied thoroughly. The mitochondrial and metabolic differences between L929dt cells, which lost matrix attachment and MHC-I expression, and their parental cell line L929, were analyzed. L929dt cells have lower capacity to generate energy through OXPHOS and lower respiratory capacity than parental L929 cells. Most importantly, L929dt cells show defects in mitochondrial supercomplex assembly, especially in those that contain complex I. These defects correlate with mtDNA mutations in L929dt cells at the  subunit of complex I and are accompanied by a glycolytic shift. In addition, L929dt cells show higher in vivo tumorigenic and metastatic potential than the parental cell line. Cybrids with L929dt mitochondria in L929 nuclear background reproduce all L929dt properties, demonstrating that mitochondrial mutations are responsible for the aggressive tumor phenotype. In spite of their higher tumorigenic potential, L929dt or mitochondrial L929dt cybrid cells are sensitive both in vitro and in vivo to the PDK1 inhibitor dichloroacetate, which favors OXPHOS, suggesting benefits for the use of metabolic inhibitors in the treatment of especially aggressive tumors.

Mutations in the  Subunit of Mitochondrial Complex I Are Sufficient to Confer Increased Tumorigenic and Metastatic Potential to Cancer Cells.

Measurement of thermogenesis in individual cells is a remarkable challenge due to the complexity of the biochemical environment (such as pH and ionic strength) and to the rapid and yet not well-understood heat transfer mechanisms throughout the cell. Here, we present a unique system for intracellular temperature mapping in a fluorescence microscope (uncertainty of 0.2 K) using rationally designed luminescent Ln-bearing polymeric micellar probes (Ln = Sm, Eu) incubated in breast cancer MDA-MB468 cells. Two-dimensional (2D) thermal images recorded increasing the temperature of the cells culture medium between 296 and 304 K shows inhomogeneous intracellular temperature progressions up to ∼20 degrees and subcellular gradients of ∼5 degrees between the nucleolus and the rest of the cell, illustrating the thermogenic activity of the different organelles and highlighting the potential of this tool to study intracellular processes.

Real-Time Intracellular Temperature Imaging Using Lanthanide-Bearing Polymeric Micelles.

Biocompatibility restrictions have limited the use of magnetic nanoparticles for magnetic hyperthermia therapy to iron oxides, namely magnetite (FeO) and maghemite (γ-FeO). However, there is yet another magnetic iron oxide phase that has not been considered so far, in spite of its unique magnetic properties: ε-FeO. Indeed, whereas FeO and γ-FeO have a relatively low magnetic coercivity, ε-FeO exhibits a giant coercivity. In this report, the heating power of ε-FeO nanoparticles in comparison with γ-FeO nanoparticles of similar size (∼20 nm) was measured in a wide range of field frequencies and amplitudes, in uncoated and polymer-coated samples. It was found that ε-FeO nanoparticles primarily heat in the low-frequency regime (20-100 kHz) in media whose viscosity is similar to that of cell cytoplasm. In contrast, γ-FeO nanoparticles heat more effectively in the high frequency range (400-900 kHz). Cell culture experiments exhibited no toxicity in a wide range of nanoparticle concentrations and a high internalization rate. In conclusion, the performance of ε-FeO nanoparticles is slightly inferior to that of γ-FeO nanoparticles in human magnetic hyperthermia applications. However, these ε-FeO nanoparticles open the way for switchable magnetic heating owing to their distinct response to frequency.

Magnetic hyperthermia with ε-FeO nanoparticles.

PT-112 is a novel pyrophosphate-platinum conjugate, with clinical activity reported in advanced pretreated solid tumors. While PT-112 has been shown to induce robust immunogenic cell death (ICD) in vivo but only minimally bind DNA, the molecular mechanism underlying PT-112 target disruption in cancer cells is still under elucidation. The murine L929 in vitro system was used to test whether differential metabolic status alters PT-112's effects, including cell cytotoxicity. The results showed that tumor cells presenting mutations in mitochondrial DNA (mtDNA) (L929dt and L929 cybrid cells) and reliant on glycolysis for survival were more sensitive to cell death induced by PT-112 compared to the parental and cybrid cells with an intact oxidative phosphorylation (OXPHOS) pathway (L929 and dt cybrid cells). The type of cell death induced by PT-112 did not follow the classical apoptotic pathway: the general caspase inhibitor Z-VAD-fmk did not inhibit PT-112-induced cell death, alone or in combination with the necroptosis inhibitor necrostatin-1. Interestingly, PT-112 initiated autophagy in all cell lines, though this process was not complete. Autophagy is known to be associated with an integrated stress response in cancer cells and with subsequent ICD. PT-112 also induced a massive accumulation of mitochondrial reactive oxygen species, as well as changes in mitochondrial polarization-only in the sensitive cells harboring mitochondrial dysfunction-along with calreticulin cell-surface exposure consistent with ICD. PT-112 substantially reduced the amount of mitochondrial CoQ10 in L929 cells, while the basal CoQ10 levels were below our detection limits in L929dt cells, suggesting a potential relationship between a low basal level of CoQ10 and PT-112 sensitivity. Finally, the expression of HIF-1α was much higher in cells sensitive to PT-112 compared to cells with an intact OXPHOS pathway, suggesting potential clinical applications.

PT-112 Induces Mitochondrial Stress and Immunogenic Cell Death, Targeting Tumor Cells with Mitochondrial Deficiencies.

As the last step of the OXPHOS system, mitochondrial ATP synthase (or complex V) is responsible for ATP production by using the generated proton gradient, but also has an impact on other important functions linked to this system. Mutations either in complex V structural subunits, especially in mtDNA-encoded ATP6 gene, or in its assembly factors, are the molecular cause of a wide variety of human diseases, most of them classified as neurodegenerative disorders. The role of ATP synthase alterations in cancer development or metastasis has also been postulated. In this work, we reported the generation and characterization of the first mt-Atp6 pathological mutation in mouse cells, an m.8414A>G transition that promotes an amino acid change from Asn to Ser at a highly conserved residue of the protein (p.N163S), located near the path followed by protons from the intermembrane space to the mitochondrial matrix. The phenotypic consequences of the p.N163S change reproduce the effects of MT-ATP6 mutations in human diseases, such as dependence on glycolysis, defective OXPHOS activity, ATP synthesis impairment, increased ROS generation or mitochondrial membrane potential alteration. These observations demonstrate that this mutant cell line could be of great interest for the generation of mouse models with the aim of studying human diseases caused by alterations in ATP synthase. On the other hand, mutant cells showed lower migration capacity, higher expression of MHC-I and slightly lower levels of HIF-1α, indicating a possible reduction of their tumorigenic potential. These results could suggest a protective role of ATP synthase inhibition against tumor transformation that could open the door to new therapeutic strategies in those cancer types relying on OXPHOS metabolism.

A Mutation in Mouse MT-ATP6 Gene Induces Respiration Defects and Opposed Effects on the Cell Tumorigenic Phenotype.

Mitochondrial function generates an important fraction of the heat that contributes to cellular and organismal temperature maintenance, but the actual values of this parameter reached in the organelles is a matter of debate. The studies addressing this issue have reported divergent results: from detecting in the organelles the same temperature as the cell average or the incubation temperature, to increasing differences of up to 10 degrees above the incubation value. Theoretical calculations based on physical laws exclude the possibility of relevant temperature gradients between mitochondria and their surroundings. These facts have given rise to a conundrum or paradox about hot mitochondria. We have examined by Blue-Native electrophoresis, both in intact cells and in isolated organelles, the stability of respiratory complexes and supercomplexes at different temperatures to obtain information about their tolerance to heat stress. We observe that, upon incubation at values above 43 °C and after relatively short periods, respiratory complexes, and especially complex I and its supercomplexes, are unstable even when the respiratory activity is inhibited. These results support the conclusion that high temperatures (>43 °C) cause damage to mitochondrial structure and function and question the proposal that these organelles can physiologically work at close to 50 °C.

How hot can mitochondria be? Incubation at temperatures above 43 °C induces the degradation of respiratory complexes and supercomplexes in intact cells and isolated mitochondria.

The generation of temperature gradients on nanoparticles heated externally by a magnetic field is crucially important in magnetic hyperthermia therapy. But the intrinsic low heating power of magnetic nanoparticles, at the conditions allowed for human use, is a limitation that restricts the general implementation of the technique. A promising alternative is local intracellular hyperthermia, whereby cell death (by apoptosis, necroptosis, or other mechanisms) is attained by small amounts of heat generated at thermosensitive intracellular sites. However, the few experiments conducted on the temperature determination of magnetic nanoparticles have found temperature increments that are much higher than the theoretical predictions, thus supporting the local hyperthermia hypothesis. Reliable intracellular temperature measurements are needed to get an accurate picture and resolve the discrepancy. In this paper, we report the real-time variation of the local temperature on γ-FeO magnetic nanoheaters using a Sm/Eu ratiometric luminescent thermometer located on its surface during exposure to an external alternating magnetic field. We measure maximum temperature increments of 8 °C on the surface of the nanoheaters without any appreciable temperature increase on the cell membrane. Even with magnetic fields whose frequency and intensity are still well within health safety limits, these local temperature increments are sufficient to produce a small but noticeable cell death, which is enhanced considerably as the magnetic field intensity is increased to the maximum level tolerated for human use, consequently demonstrating the feasibility of local hyperthermia.

Article	Year
Structural biology response of a collagen hydrogel synthetic extracellular matrix with embedded human fibroblast: computational and experimental analysis. Medical & biological engineering & computing\| PubMed ID: 25835213	2015
Mutations in the Subunit of Mitochondrial Complex I Are Sufficient to Confer Increased Tumorigenic and Metastatic Potential to Cancer Cells. Cancers\| PubMed ID: 31330915	2019
Real-Time Intracellular Temperature Imaging Using Lanthanide-Bearing Polymeric Micelles. Nano letters\| PubMed ID: 32787172	2020
Magnetic hyperthermia with ε-FeO nanoparticles. RSC advances\| PubMed ID: 35520081	2020
PT-112 Induces Mitochondrial Stress and Immunogenic Cell Death, Targeting Tumor Cells with Mitochondrial Deficiencies. Cancers\| PubMed ID: 36010843	2022
A Mutation in Mouse MT-ATP6 Gene Induces Respiration Defects and Opposed Effects on the Cell Tumorigenic Phenotype. International journal of molecular sciences\| PubMed ID: 36674816	2023
How hot can mitochondria be? Incubation at temperatures above 43 °C induces the degradation of respiratory complexes and supercomplexes in intact cells and isolated mitochondria. Mitochondrion\| PubMed ID: 36764502	2023
Local Temperature Increments and Induced Cell Death in Intracellular Magnetic Hyperthermia. ACS nano\| PubMed ID: 36940429	2023
Reply to the Comment on "Local Temperature Increments and Induced Cell Death in Intracellular Magnetic Hyperthermia". ACS nano\| PubMed ID: 37605858	2023

Raquel Moreno-Loshuertos

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