Real-time flow cytometry analysis of permeability transition in isolated mitochondria

Lecœur, Hervé; Langonné, Alain; Baux, Ludwig; Rebouillat, Dominique; Rustin, Pierre; Prévost, Marie‐Christine; Brenner, Catherine; Edelman, L; Jacotot, Étienne

doi:10.1016/j.yexcr.2003.10.030

Cited by 69 publications

(66 citation statements)

References 47 publications

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“…Classically, light-scattering assays of rodent liver mitochondria in sucrose media have been employed [95]. In this case, large-amplitude swelling is monitored by measuring the absorbance of a mitochondrial suspension at 545 nm, for instance in a conventional spectrophotometer [96]. This Fig.…”

Section: Detection Of Im Permeabilizationmentioning

confidence: 99%

“…Recently, a multiparametric flow cytometry approach has been proposed to measure the modifications of mitochondrial size, structure and m associated with the onset of MMP [96]. Here, the authors correlated the structural rearrangements that characterize large-amplitude swelling with an increase in the forward scatter parameter (FSC) paralleled by a decrease in the side scatter parameter (SSC) of isolated mitochondria.…”

Section: Detection Of Im Permeabilizationmentioning

confidence: 99%

“…Reportedly, the ratio FSC/SSC represents a sensitive marker for PT, being able to detect morphological modifications on a per-organelle basis. Additional advantages of this method include the need for small amounts of mitochondrial suspensions (which may be important for the evaluation of rare samples from patients with mitochondrial disorders) and the possibility to analyze the heterogeneity of mitochondrial populations (which is not the case of classical spectrophotometric and fluorometric techniques) [96].…”

Section: Detection Of Im Permeabilizationmentioning

confidence: 99%

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Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

et al. 2007

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Mitochondrial membrane permeabilization (MMP) is considered as the "point-of-no-return" in numerous models of programmed cell death. Indeed, mitochondria determine the intrinsic pathway of apoptosis, and play a major role in the extrinsic route as well. MMP affects the inner and outer mitochondrial membranes (IM and OM, respectively) to a variable degree. OM permeabilization culminates in the release of proteins that normally are confined in the mitochondrial intermembrane space (IMS), including caspase activators (e.g. cytochrome c) and caspase-independent death effectors (e.g. apoptosis-inducing factor). Partial IM permeabilization disrupts mitochondrial ion and volume homeostasis and dissipates the mitochondrial transmembrane potential ( m ). The assessment of early mitochondrial alterations allows for the identification of cells that are committed to die but have not displayed yet the apoptotic phenotype. Several

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Section: Detection Of Im Permeabilizationmentioning

confidence: 99%

Section: Detection Of Im Permeabilizationmentioning

confidence: 99%

Section: Detection Of Im Permeabilizationmentioning

confidence: 99%

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Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

et al. 2007

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“…Liver mitochondria were isolated from 3-4 week old Balb-c mice (IFFA CREDO) as described previously. 38 Mitochondria were also isolated from HeLa cells (3 Â 10 6 cells) using a protocol adapted from reference. 39 Briefly, cells were harvested with PBS containing 1 mM EDTA, centrifuged at 750 g for 10 min, washed and resuspended in isotonic MB buffer (210 mM mannitol, 70 mM sucrose, 1 mM EDTA and 10 mM HEPES, pH 7.5) supplemented with protease inhibitors.…”

mentioning

confidence: 99%

“…Swelling was determined by measuring absorbance at 545 nm with an Ultrospec 3300 pro spectrofluorometer (Amersham Pharmacia Biotech). 38 The percentage of specific swelling was calculated as follows: (A init ÀA reagent ) 100/(A init ÀA Ca2 þ ) where A Ca2 þ , A reagent and A init correspond to the absorbance value obtained for CaCl 2 -treated, reagenttreated and untreated mitochondria, respectively. Mitochondrial transmembrane potential (DC m ) was assessed by incorporation of 5,5 0 ,6,6 0 ,-tetrachloro 1,1,3,3 0 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1; excitation 488 nm; emission 525-535 nm (low DC m ) and 560-605 nm (high DC m ); Molecular Probes; 1 mM) and flow cytometry acquisition with a FACScalibur (Becton Dickinson) as described previously.…”

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Targeted Vpr-derived peptides reach mitochondria to induce apoptosis of αVβ3-expressing endothelial cells

Borgne‐Sanchez¹,

Dupont²,

Langonné³

et al. 2006

Cell Death Differ

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The HIV-1 encoded apoptogenic protein Vpr induces mitochondrial membrane permeabilization (MMP) via interactions with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). We have designed a peptide, TEAM-VP, composed of two functional domains, one a tumor blood vessel RGD-like 'homing' motif and the other an MMP-inducing sequence derived from Vpr. When added to isolated mitochondria, TEAM-VP interacts with ANT and VDAC, reduces oxygen consumption and overcomes Bcl-2 protection to cause inner and outer MMP. TEAM-VP specifically recognizes cell-surface expressed a V b 3 integrins, internalizes, temporarily localizes to lysosomes and progressively co-distributes with the mitochondrial compartment with no sign of lysosomal membrane permeabilization. Finally TEAM-VP reaches mitochondria of angiogenic endothelial cells to induce mitochondrial fission, dissipation of the mitochondrial transmembrane potential (DW m ), cytochrome c release and apoptosis hallmarks. Hence, this chimeric peptide constitutes the first example of a virus-derived mitochondriotoxic compound as a candidate to kill selectively tumor neo-endothelia. Within the integrin family, a V b 3 receptors are considered to be an important target for anticancer therapy being upregulated in certain invasive tumors.1 a V b 3 recognizes a specific consensus sequence, an RGD motif, which is present in a number of extracellular matrix proteins like vitronectin, fibronectin, fibrinogen and thrombospondin.2 RGD-containing peptides have been shown to reduce angiogenesis induced by cytokines and to mediate apoptosis in endothelial cells.3 It appears that RGD peptides that are constrained in a cyclic conformation, show an increased affinity for binding to integrins. 4 One example is the cyclic RGD-4C peptide, a potent binder of a 5 b 1 , a V b 3 and a V b 5 used to selectively deliver doxorubicin to tumor blood vessels 5 and to internalize the amphipatic peptide (KLAKLAK) 2 in endothelial cells resulting in cellular apoptosis.

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J

2010

Handbook of Biological Dyes and Stains

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Real-time flow cytometry analysis of permeability transition in isolated mitochondria

Cited by 69 publications

References 47 publications

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

Targeted Vpr-derived peptides reach mitochondria to induce apoptosis of αVβ3-expressing endothelial cells

J

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