US20130164774A9 - Methods, compositions and kits for assaying mitochondrial function - Google Patents

Methods, compositions and kits for assaying mitochondrial function Download PDF

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US20130164774A9
US20130164774A9 US13/441,418 US201213441418A US2013164774A9 US 20130164774 A9 US20130164774 A9 US 20130164774A9 US 201213441418 A US201213441418 A US 201213441418A US 2013164774 A9 US2013164774 A9 US 2013164774A9
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cells
pfo
cell
mitochondrial
respiration
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US20120301912A1 (en
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Nagendra Yadava
Alejandro Pablo Heuck
Chul Kim
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University of Massachusetts UMass
Baystate Medical Center Inc
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University of Massachusetts UMass
Baystate Medical Center Inc
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Publication of US20130164774A9 publication Critical patent/US20130164774A9/en
Priority to US14/519,119 priority patent/US9513281B2/en
Priority to US15/087,521 priority patent/US9915647B2/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: BAYSTATE MEDICAL CENTER
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)

Definitions

  • the invention relates to methods for evaluating intracellular functions as well as cell health and viability.
  • Mitochondrial dysfunction is at the core of many encephalomyopathies, diabetes and cancer. Inherited mutations in over 100 genes constituting the oxidative phosphorylation (OxPhos) machinery are linked with mitochondrial encephalomyopathies in humans. The diseases resulting from defective OxPhos are also referred to as mitochondrial diseases or mitochondrial disorders. They are usually multisystemic fatal disorders with progressive onset with age. Impaired mitochondrial metabolism is also thought to play a key role in the pathophysiology of diabetes and cancer. Mutational analysis of mitochondrial (mt)DNA suggests that mitochondrial dysfunction is a widespread phenomenon, which occurs in almost all types of cancers. Somatic mutations in the mtDNA, which encodes proteins essential for OxPhos, are found associated with almost all types of cancer. However, their functional relevance is yet to be determined in most cases.
  • the COSMIC (Catalogue Of Somatic Mutations In Cancer) database reveals that mutations in nuclear genes associated with oxidative phosphorylation functions is common. Somatic mutations in over 25 nuclear genes associated with respiratory Complex I (NADH-ubiquinone oxidoreductase) structure/function have been identified. In addition, switching of the cellular metabolism from oxidative metabolism to glycolysis, which is known as metabolic reprogramming, is one of the hallmarks of cancer phenotypes. This could be due to somatic mutations in mtDNA, nDNA or negative regulation of the oxidative phosphorylation due to host and environmental factors. Mitochondrial dysfunction is also implicated in other age-associated diseases such as Parkinson's disease, and Alzheimer's disease.
  • aspects of the invention relate to methods, compositions, and devices that are useful for measuring mitochondrial functions accurately and reproducibly under intracellular conditions.
  • the invention relates to methods, compositions, and devices that are useful for measuring mitochondrial functions accurately and reproducibly under different conditions without isolating the mitochondria from cells.
  • cholesterol-dependent cytolysins e.g., perfringolysin O (PFO)
  • PFO perfringolysin O
  • This selective permeabilization allows accurate evaluations of intracellular mitochondrial activities, by assaying, for example, the uptake and/or release of mitochondrial substrates and/or products that can be measured outside a selectively permeabilized cell.
  • analyses with permeabilized cells provide a more accurate assessment of the biological or physiological status of mitochondria, while allowing direct correlation with whole cells containing the same amount of mitochondria as in permeabilized cells.
  • methods for analyzing intracellular mitochondrial activity involve using detergents or other permeabilization agents that are damaging to one or both of the cellular and mitochondrial membranes.
  • Detergents typically solubilize mitochondrial membranes, and even with careful titrations below ⁇ 0.01% it is difficult to establish concentrations of detergents under which mitochondrial function is sustained.
  • Cholesterol-dependent cytolysin-based (e.g., PFO-based) methods provide effective permeabilization of cellular membranes without disrupting the mitochondrial membranes.
  • permeabilization with cytolysins avoids unwanted release of mitochondrial molecules into the cell and surrounding cellular environment. This also avoids cytosolic molecules entering the mitochondria and interfering with mitochondrial function.
  • cholesterol-dependent cytolysins are surprisingly effective at creating conditions suitable for measuring mitochondrial activity in cells.
  • cholesterol-dependent cytolysin-based (e.g., PFO-based) methods provide effective permeabilization of cellular membranes without disrupting the mitochondrial membranes at concentrations over a wide dynamic range up to 50 nM or more in some cases (e.g., 0.1 to 20 nM). Also, since the cytolysins are proteins, they can typically be handled very accurately at different dilutions.
  • cholesterol-dependent cytolysin-based permeabilization techniques can be used to evaluate one or more mitochondrial activities without disrupting the cellular environment, because mitochondrial membranes remain largely intact when exposed to a cholesterol-dependent cytolysin (e.g., PFO) as described herein.
  • Substrate uptake and/or product release can be assayed to evaluate one or more mitochondrial-specific functions (e.g., oxidative phosphorylation).
  • mitochondrial-specific functions e.g., oxidative phosphorylation
  • cholesterol-dependent cytolysin-based (e.g., PFO-based) assay results can provide an accurate assessment of mitochondrial activity in a natural cellular environment.
  • cholesterol-dependent cytolysin-based (e.g., PFO-based) assay results can provide an accurate assessment of mitochondrial activity in cells obtained from subjects suspected of having a mitochondrial disorder.
  • cholesterol-dependent cytolysin-based (e.g., PFO-based) cell permeabilization allows for analysis of the effects of exogenous agents (e.g., molecules that are impermeable across the plasma membrane) on one or more intracellular functions.
  • exogenous agents e.g., molecules that are impermeable across the plasma membrane
  • cholesterol-dependent cytolysins e.g., PFOs
  • cholesterol-dependent cytolysin-based may facilitate the use of exogenous agents such as ion sensitive dyes (e.g., calcium sensing dyes, pH sensing dyes, etc.) or ion sensitive fluorescent proteins to probe one or more intracellular functions.
  • exogenous agents such as ion sensitive dyes (e.g., calcium sensing dyes, pH sensing dyes, etc.) or ion sensitive fluorescent proteins to probe one or more intracellular functions.
  • cholesterol-dependent cytolysin-based e.g., PFOs
  • Methods are provided, in some embodiments, that are based on selective permeabilization of the plasma membrane with a cholesterol-dependent pore forming protein, perfringolysin O (PFO) from the Clostridium perfringens .
  • PFO perfringolysin O
  • intracellular membranes remain largely unaffected by CDCs such as to PFO.
  • PFO-permeabilized cells preserve mitochondrial integrity, and produce reproducible results across a wide range of cell types and buffer conditions.
  • methods are provided that utilize PFO-based cell permeabilization (e.g., in a microplate format) that are useful for determining spare oxidative phosphorylation (OxPhos) capacity and other features of mitochondrial bioenergetics.
  • the methods permit assessment of mitochondrial function without isolating mitochondria from cells.
  • the methods are useful for determining (i) spare and total OxPhos capacities, (ii) spare and total respiratory capacities, (iii) specific defects in electron transport/respiratory chain (ETC/RC) capacities, (iv) TCA cycle function, and/or (v) cell-specific features of mitochondrial metabolism.
  • PFO-based assays may be used for measuring spare OxPhos (SOC) capacity and spare respiratory (ETC/RC) capacity (SRC) in a single assay.
  • evaluating spare or total OxPhos capacity involves using adequate amounts of ADP (e.g., 1 mM for cells, ⁇ 2 mM for myoblasts), and Pi (e.g., ⁇ 10 mM) in the respiration medium along with a desired substrate.
  • the methods are useful for determining spare OxPhos capacity and other bioenergetic features without the need to isolate mitochondria from cells.
  • methods are useful for determining: (i) cell-specific features of mitochondrial metabolism, (ii) direct and/or indirect effects of drugs on ETC/RC function and other mitochondrial functions, (iii) defects OxPhos complexes, (iv) uncoupling activity of compounds and/or (v) conditions that can impair functional integrity of mitochondria.
  • the methods disclosed herein are useful for assessing mitochondrial function and metabolism in cell lines, primary cells and tissues. In some embodiments, the methods are useful for determining oxidative phosphorylation and respiratory chain capacities. In some embodiments, the methods are useful for determining the capacity of individual respiratory OxPhos Complexes (I-V). In some embodiments, the methods are useful for evaluating mitochondrial permeability transitions. In some embodiments, the methods are useful for diagnosing impaired mitochondrial metabolism, e.g., due to genetic mutations or drug toxicity. In some embodiments, the methods are useful for evaluating organelles' (e.g., mitochondria, Lysosomes, nucleus, endoplasmic reticulum etc.) function under conditions maintain mitochondrial function.
  • organelles' e.g., mitochondria, Lysosomes, nucleus, endoplasmic reticulum etc.
  • the methods are useful for evaluating protein topology in internal membranes by assessing protease sensitivity of internal membrane to proteins following PFO-based permeabilization. In some embodiments, the methods are useful for facilitating delivery of macromolecules inside cells using conditional pore-formation.
  • PFO-permeabilized cells are used to determine conditions for maximal respiratory response with different NADH substrates (e.g., pyruvate, isocitrate, ⁇ -ketoglutarate and glutamate) when added individually.
  • NADH substrates e.g., pyruvate, isocitrate, ⁇ -ketoglutarate and glutamate
  • the methods disclosed herein are useful for assessing mitochondrial function with small samples applicable to clinical research.
  • the methods are useful for assessing mitochondrial permeability transition and other cellular processes, and organelles under the conditions that do not destroy mitochondrial functions.
  • PFO derivatives are provided that can be used at as low as 0.1 nM concentrations for mitochondrial function assays. In some embodiments, PFO derivatives are provided that can be used in the absence of DTT or other reducing agents. In such embodiments, O 2 consumption resulting from DTT or other reducing agents is avoided. In some embodiments, a minimal amount of DTT is used (e.g., ⁇ 100 nM of DTT).
  • PFO-based assays are provided that are applicable across different cells, e.g., ⁇ cells, fibroblasts, neuronal cells, primary myoblasts/tubes, mammary epithelial cells, embryonic fibroblasts, macrophages, splenocytes and thymocytes, and different species of cells, e.g., mouse, rat, hamster and human.
  • different variants of PFO are provided (See, e.g., Tables 3 and 5).
  • methods comprise contacting a preparation comprising a cell with a cholesterol-dependent cytolysin and measuring an intracellular function of the cell.
  • the intracellular function is a metabolic rate of the cell, a respiratory rate of the cell, a proportion of aerobic to anaerobic respiration, a rate of consumption of a molecule, or a production rate of a molecule.
  • the intracellular function is indicative of cell health or cell viability.
  • the intracellular function is mitochondrial function.
  • the methods are used as an apoptosis (a cell death mechanism) assay.
  • a preparation typically comprises a plurality of cells.
  • Such preparations may comprise one or more cells of any type.
  • the cells may be animal cells or plant cells.
  • the cells are primary cells.
  • the cells are obtained from an individual.
  • the cells may be any mammalian cells.
  • the cells may be any human cells.
  • the cells may be selected from the group consisting of to Lymphocytes, B cells, T cells, cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells, myeloid cells, granulocytes, basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes, hypersegmented neutrophils, monocytes, macrophages, reticulocytes, platelets, mast cells, thrombocytes, megakaryocytes, dendritic cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, oxyphil cells, adrenal cells, chromaffin cells, pineal cells, pinealocytes, glial cells, glioblasts, astrocytes, oligodendrocytes, microglial cells, magnocellular neurosecretory cells, stellate cells, boettcher cells; pituitary cells, gonadotropes, corticotropes, thy
  • the cells may be of mesenchymal, ectodermal, and endodermal origin.
  • the cells may be selected from the group consisting of cord-blood cells, stem cells, embryonic stem cells, adult stem cells, progenitor cells, induced progenitor cells, autologous cells, isograft cells, allograft cells, xenograft cells, and genetically engineered cells.
  • methods comprise contacting a preparation comprising a cell with a cholesterol-dependent cytolysin (CDC) and determining the level of a molecule in the preparation, in which the level of the molecule is indicative of an intracellular function of the cell.
  • the level of the molecule is indicative of mitochondrial function.
  • the cholesterol-dependent cytolysin is a protein or variant or derivative thereof selected from Table 1.
  • the cholesterol-dependent cytolysin is a protein having an amino acid sequence corresponding to a GenBank Accession Number listed in Table 1, or a variant or derivative thereof.
  • the cholesterol-dependent cytolysin is a protein that is a member of the family of cholesterol-dependent cytolysins listed in Table 1, or a variant or derivative thereof.
  • the cholesterol-dependent cytolysin is a Perfringolysin O (PFO).
  • PFO Perfringolysin O
  • the term “Perfringolysin O (PFO)” refers to a cytolysin of a Clostridium perfringens bacterium, or a variant, derivative or recombinant form of such a cytolysin that is capable of forming pores in a plasma membrane of a cell.
  • PFOs form pores in the plasma membrane of cells in a cholesterol-dependent manner.
  • the PFO comprises an amino acid sequence as set forth in any one of SEQ ID NOS: 1-12, or a variant or derivative thereof.
  • the PFO comprises an amino acid sequence that is a fragment of an amino acid sequence as set forth in any one of SEQ ID NOS: 1-12.
  • the fragment comprises an amino acid sequence as set forth in any one of SEQ ID NOS: 1-7 that does not include an N-terminal signal sequence.
  • the N-terminal signal sequences is the first 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids of the amino acid sequence.
  • the PFO comprises an amino acid sequence that has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more sequence identity or sequence homology with a sequence as set forth in any one of SEQ ID NOS: 1-12.
  • Sequence homology can be calculated using various, publicly available software tools developed by NCBI (Bethesda, Md.), for example, that can be obtained through the internet. Exemplary tools include “protein blast” available online at blast.ncbi.nlm nih.gov/Blast.cgi, which may be utilized with its default settings.
  • the PFO comprises an amino acid sequence that has an amino acid substitution at up to 25, up to 20, up to 15, up to 10, up to 5, or up to 2 positions compared with the sequence as set forth in any one of SEQ ID NOS: 1-12.
  • the PFO comprises an amino acid sequence that has one or more conservative amino acid substitutions, e.g., one or more conservative amino acid substitutions compared with any one of SEQ ID NO: 1-12.
  • a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art.
  • Conservative substitutions of amino acids include substitutions made among amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Accordingly, conservative amino acid substitutions may provide to functionally equivalent variants, or homologs of a protein.
  • the PFO comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2 with a cysteine to alanine substitution at amino acid position 459.
  • the PFO comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2 with a threonine to cysteine substitution at amino acid position 319 and/or a valine to cysteine substitution at amino acid position 334.
  • methods that utilize the PFO may further comprise adding a reducing reagent to the preparation.
  • the reducing reagent is Dithiothreitol (DTT) or 2-Mercaptoethanol.
  • the PFO comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2 with a cysteine to alanine substitution at amino acid position 459, and one or more other amino acid substitutions. In some embodiments of the methods, the PFO comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2 with a threonine to cysteine substitution at amino acid position 319 and/or a valine to cysteine substitution at amino acid position 334, and one or more other amino acid substitutions.
  • an isolated perfringolysin O (PFO) is provided that comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2 with an aspartate to serine substitution at amino acid position 434. In some embodiments, an isolated perfringolysin O (PFO) is provided that comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2 with an aspartate to serine substitution at amino acid position 434 and one or more other amino acid substitutions.
  • the molecule is O 2 . In some embodiments, the level of O 2 in the preparation is indicative of the oxygen consumption rate of the cell. In some embodiments, the oxygen consumption rate is indicative of mitochondrial function. In some embodiments of the methods involving determining the level of a molecule in a preparation, the molecule is H. In some embodiments, the level of H + in the preparation is indicative of the H + production rate of the cell.
  • the methods further comprise contacting the cell with a cellular-respiration effector.
  • the cellular-respiration effector is a nucleotide or a protonophore.
  • the nucleotide is adenosine diphosphate (ADP).
  • the methods may further comprise measuring the oxygen consumption rate of the cell, wherein the ADP-stimulated oxygen consumption rate is indicative of oxidative phosphorylation in the cell.
  • the protonophore is cyanide-p-trifluoromethoxyphenylhydrazone (FCCP).
  • the methods may further comprise measuring the oxygen consumption rate of the cell, wherein the FCCP-stimulated oxygen consumption rate is indicative of electron transport and respiratory chain function in the cell.
  • the methods further comprise contacting the cell with glutamate, pyruvate, malate, isocitrate, alpha-ketoglutarate or a combination thereof.
  • results of the methods may be indicative of the enzymatic activity of Complex I (NADH-ubiquinone oxidoreductase) in the cell.
  • the methods further comprise contacting the cell with succinate.
  • the results of the methods may be indicative of the enzymatic activity of Complex II (succinate-ubiquinone oxidoreductase) in the cell.
  • the methods further comprise contacting the cell with Glycerol 3-phosphate.
  • results of the methods may be indicative of the enzymatic activity of Complex III (ubiquinol-cytochrome c oxidoreductase) in the cell.
  • the methods further comprise contacting the cell with N,N,N′,N′-Tetramethyl-p-Phenylenediamine (TMPD) and/or ascorbate.
  • TMPD N,N,N′,N′-Tetramethyl-p-Phenylenediamine
  • results of the methods may be indicative of the enzymatic activity of Complex IV (cytochrome c oxidase) in the cell.
  • the methods further comprise contacting the cell with succinate and ADP and/or contacting the cell with glycerol-3-phosphate. In some embodiments, the methods further comprise contacting the cell with succinate, glycerol-3-phosphate and ADP. In such embodiments, results of the methods may be indicative of the enzymatic activity of Complex V (ATP synthase) in the cell.
  • Complex V ATP synthase
  • the methods further comprise contacting the cell with a respiratory chain inhibitor, an oxidative phosphorylation inhibitor, an uncoupling agent, a transport inhibitor, an ionophore or a krebs cycle inhibitor. In some embodiments, the methods further comprise contacting the cell with rotenone, malonate, antimycin A, KCN, and oligomycin.
  • the methods further comprise contacting the cell with a test agent and determining an effect of the test agent on an intracellular function (e.g., mitochondrial function).
  • the test agent is a drug candidate.
  • the methods provide assays for drug screens. The methods may be used in some to embodiments to screen libraries of unknown or known compounds (e.g., known bioactive compounds) to identify compounds that affect intracellular function, such as uncoupling ATP synthesis with respiratory chain function. In other embodiments, the methods may be used to evaluate the toxicity of compounds. Accordingly, in some embodiments, the methods may be used for evaluating lead compounds.
  • the methods further comprise identifying one or more genetic mutations in the cell.
  • the results of the methods are indicative of whether the one or more genetic mutations affect the intracellular function (e.g., mitochondrial function) of the cell.
  • the one or more genetic mutations are associated with a mitochondrial disorder.
  • methods involve contacting a preparation comprising a cell obtained from a subject with a cholesterol-dependent cytolysin; and determining the level of a molecule in the preparation, wherein the level of the molecule is indicative of the presence or absence of a respiratory chain deficiency in the cell.
  • the cell is obtained from an individual having or suspected of having a mitochondrial disorder.
  • the results of the methods aid in diagnosing an individual as having one or more mitochondrial disorders.
  • the mitochondrial disorder is selected from the group consisting of Myoclonic Epilepsy with Ragged Red Fibers (MERRF); Mitochondrial Myopathy, Encephalopathy, Lactacidosis, and Stroke (MELAS); Diabetes mellitus and deafness (DAD) a combination, which at an early age can be due to mitochondrial disease; Maternally Inherited Diabetes and Deafness (MIDD), Leber's Hereditary Optic Neuropathy (LHON); chronic progressive external ophthalmoplegia (CPEO); Leigh Disease; Kearns-Sayre Syndrome (KSS); Friedreich's Ataxia (FRDA); Co-Enzyme QIO (Co-QIO) Deficiency; Neuropathy, ataxia, retinitis pigmentosa, and p
  • an individual having one or more symptoms of a mitochondrial disorder may be suspected of having the mitochondrial disorder.
  • Symptoms of mitochondrial disorders include muscle weakness or exercise intolerance, heart failure or rhythm disturbances, to dementia, movement disorders, stroke-like episodes, deafness, blindness, droopy eyelids, limited mobility of the eyes, vomiting, and seizures.
  • muscles may become easily fatigued or weak. Muscle cramping may occur. Nausea, headache, and breathlessness are also associated with these disorders.
  • mitochondrial disorders occur or first manifest at a young age (e.g., during childhood, before the age of 20). Accordingly, in some embodiments, the methods may be useful for diagnosing or aiding in diagnosing a mitochondrial disorder in an individual who is a child.
  • the diagnostic methods disclosed herein may be used with individuals of any age.
  • a “subject,” “individual,” or “patient,” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
  • the subject, individual or patient is a child.
  • the subject, individual or patient is a young child.
  • the subject, individual or patient is an infant.
  • the term “child” or “children” as used herein means persons over the age of 3 years and prior to adolescence.
  • the term “young child” or “young children” means persons from the age of more than 12 months up to the age of three years.
  • the term “infant” means a person not more than 12 months of age.
  • the subject, individual or patient is at or above the age of adolescence.
  • methods of transfecting a cell with an exogenous nucleic acid involve contacting the cell with any one or more of the cholesterol-dependent cytolysins disclosed herein; and contacting the cell with the exogenous nucleic acid (e.g., an expression vector, a cloning vector, etc.).
  • the methods are useful for transfecting cells that are difficult to transfect with conventional transfection reagents (e.g., liposome based reagents).
  • the methods are useful for transfecting cells grown in suspension, e.g., hematopoietic cells.
  • the methods are useful for transfecting stem cells or primary cells.
  • the preparation is contained in a container, e.g., a well.
  • the container is a well in a multi-well plate.
  • the methods may be performed in a multiplex format (e.g., high-throughput format).
  • the methods are conducted using a multi-well extracellular flux analyzer.
  • kits that comprise a container to housing a cholesterol-dependent cytolysin and a container housing a reagent for evaluating an intracellular function of a cell.
  • the intracellular function is a mitochondrial function.
  • kits are provided that comprise a container housing a cholesterol-dependent cytolysin and a container housing a cellular-respiration effector.
  • the cellular-respiration effector is a nucleotide or a protonophore.
  • the nucleotide is adenosine diphosphate (ADP).
  • the protonophore is cyanide-p-trifluoromethoxyphenylhydrazone (FCCP).
  • kits are provided that comprise a container housing glutamate, pyruvate, malate, isocitrate, alpha-ketoglutarate or a combination thereof. In some embodiments, kits are provided that comprise a container housing succinate. In some embodiments, kits are provided that comprise a container housing Glycerol 3-phosphate. In some embodiments, kits are provided that comprise a container housing N,N,N′,N′-Tetramethyl-p-Phenylenediamine (TMPD). In some embodiments, kits are provided that comprise a container housing ascorbate.
  • TMPD N,N,N′,N′-Tetramethyl-p-Phenylenediamine
  • kits are provided that comprise a container housing a respiratory chain inhibitor, a oxidative phosphorylation inhibitor, an uncoupling agent, a transport inhibitor, an ionophore or a krebs cycle inhibitor.
  • kits are provided that comprise a container housing rotenone, malonate, antimycin A, KCN, or oligomycin.
  • kits are provided that comprise a container housing assay buffer.
  • the cholesterol-dependent cytolysin is selected from Table 1 or a variant or derivative thereof.
  • the cholesterol-dependent cytolysin is Perfringolysin O (PFO).
  • the PFO comprises an amino acid sequence as set forth in any one of SEQ ID NOS: 1-12, or a variant or derivative thereof.
  • the PFO comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2 with a cysteine to alanine substitution at amino acid position 459.
  • kits comprise a container housing a reducing reagent.
  • the reducing reagent is Dithiothreitol (DTT) or 2-Mercaptoethanol.
  • At least one container is a reusable container. In some embodiments of the kits, at least one container is a single-use container. In some embodiments of the kits, at least one container is a tube or bottle. In some embodiments of the kits, the tube is a snap-top tube or a screw-top tube. In some embodiments of the kits, the bottle is a snap-top bottle or a screw-top bottle. In some embodiments of the kits, at least one container is a glass vial. In some embodiments of the kits, the containers are housed together in a box or a package. In some embodiments, the kits further comprise instructions for permeabilizing a cell.
  • kits further comprise instructions for storing at least one container at a particular temperature (e.g., less than 0° C., room temperature). In some embodiments, the kits further comprise instructions for carrying out any of the methods disclosed herein using reagents provided in the kits.
  • kits disclosed herein are useful for research purposes, in other embodiments, the kits disclosed herein are useful for diagnostic purposes. Accordingly, in some embodiments, the kits contain one or more reagents or components useful in methods for diagnosing or aiding in diagnosing an individual as having a mitochondrial disorder, including, for example, any of the mitochondrial disorders disclosed herein.
  • FIG. 1 provides a non-limiting overview of glucose metabolism in a typical mammalian cell. Relationships of the glycolysis, TCA cycle and OxPhos system are shown. The reactions generating and oxidizing NADH and FADH 2 are shown. CC: Cytochrome c; Q: ubiquinone; CI-V: OxPhos Complexes I-V; MAS: malate-aspartate shuttle; GPS: glycerol-3-phosphate shuttle. The measurements of lactate, O 2 and CO 2 levels in the immediate surroundings of cells are expected to give the rates of glycolysis, respiration and the TCA cycle.
  • FIG. 2 illustrates digitonin (DIG)-based cell permeabilization.
  • DIG digitonin
  • FIG. 3 illustrates PFO performance compared with digitonin performance.
  • FIG. 3A shows primary pancreatic ⁇ -cells.
  • FIG. 3B shows Chinese hamster lung fibroblasts. Injections were as follows: “a” was 1 mM ADP+10 ⁇ M Cytochrome c (Cyt C or CC) with nPFO (25 nM) or Digitonin (DIG, 0.010% in FIG. 3A , 0.005% in FIG. 3B ) in the presence of succinate as to respiratory substrate; and “b” was 1 ⁇ g/ml oligomycin. OCR rates either shown as absolute value ( FIG. 3A ) or normalized to 3rd rate ( FIG. 3B ).
  • FIG. 4 shows that PFO-based assays do not need exogenous Cytochrome c. Succinate supported, ADP stimulated INS1E cells respiration is shown. Injections were as follows: “a” was nPFO, “b” was ADP with or without Cytochrome c.
  • FIG. 5 illustrates OxPhos capacity and respiratory (ETC/RC) capacity.
  • ADP— and FCCP— stimulated respirations give OxPhos and respiratory capacities, respectively.
  • FIG. 5A shows relative levels of ADP— and FCCP-stimulated respiration in Chinese hamster lung fibroblasts, the V79-G3. Injections were as follows: “a” was 1 mM ADP; “b” was 2 ⁇ M FCCP.
  • FIG. 5B shows oligomycin sensitivity (coupling test) of the ADP-stimulated respiration in permeabilized V79-G3 cells compared to the sensitivity of basal respiration in intact cells.
  • Injections were as follows “a” was 1 mM ADP or ADP+nPFO; and “b” was 2 ⁇ g/ml oligomycin.
  • FIG. 5C shows respiratory decline following nPFO permeabilization is due to ADP limitation resulting from the dilution of the cytosol. Injections were as follows: “a” was 5 nM nPFO; and “b” was buffer (nPFO) or 1 mM ADP (nPFO+ADP) or 1 mM ADP+104M Cytochrome c (nPFO+ADP+CC).
  • FIG. 6 shows comparable levels of ADP-stimulated and FCCP-stimulated respiration in rat pancreatic ⁇ cells.
  • FIG. 6A shows rat insulinoma INS1E cells. Injections were as follows: “a” was nPFO+ADP; and “b” was FCCP.
  • FIG. 6B shows primary rat ⁇ /islet-cells. Injections were as follows “a” was nPFO, “b” was succinate+ADP, and “c” was FCCP. The lower rates of basal respiration in primary cells are due to pre-starvation and the absence of any exogenous substrates prior to injection arrow “b”.
  • FIG. 7 shows functional assays for Complex I deficiency using the PFO-based method.
  • FIG. 7A shows results of a complex I mutant showing the lack of respiration on Complex I substrates.
  • FIG. 7B shows complemented control cells showing the restoration respiration on Complex I substrates.
  • FIG. 7C shows the data from panel B with the basal rate before arrow “a” is set to “0”. Injections were as follows: “a” was nPFO, and “b” was succinate (Suc), or glutamate+malate (Glu+Mal).
  • FIG. 8 illustrates limitations in substrate supply to ⁇ -cell ETC/RC following oligomycin treatment, and sensitivity to Ca2+-mediated mitochondrial permeability transition. All groups were treated with oligomycin (2 ⁇ g/ml) and then after >60 min cells were succinate and/or FCCP with or without nPFO. Groups are as followings.
  • “Oligo_Buffer” indicates oligomycin treated only; “Oligo_SF” indicates oligomycin+succinate+FCCP only; “Oligo_PF” indicates oligomycin+nPFO+FCCP followed by succinate addition later; “Oligo_PSF” indicates oligomycin+nPFO+succinate+FCCP; “Oligo_PSFCa” indicates oligomycin and 1.3 mM CaCl 2 added after nPFO+succinate+FCCP additions.
  • FIG. 9 provides an overview of a PFO purification scheme.
  • FIG. 10 provides an outline of a working model showing that ⁇ -cell respiration is primarily supported by the redox shuttles when cells are intact. Complex I-dependent respiration should drop significantly when ⁇ -cells are permeabilized if it is primarily supported by redox shuttle.
  • FIG. 11 shows that ⁇ -cells display low level of Complex I-dependent respiration.
  • FIG. 11A shows Complex I, II, & III-dependent respiration determined by measuring respiratory inhibition with the increasing concentrations of rotenone (Rot), thenoyltrifluoroacetone (TTFA), antimycin A (Ant A) respectively compared to control (Con).
  • FIG. 11B shows percent maximal respiratory inhibition by rotenone, TTFA and antimycin A in INS1E cells.
  • FIG. 12 shows that permeabilized ⁇ -cells do not display Complex I-dependent respiration:
  • FIG. 12A shows results from primary ⁇ -cells.
  • FIG. 12B shows results from primary astrocytes.
  • FIG. 12C shows results from CCL16 lung fibroblasts.
  • FIG. 12D shows NAD(P)H levels in isolated mitochondria in the presence of glutamate+malate and rotenone.
  • Arrows indicate the following: “a” indicates cell permeabilization; and “b” indicates substrate+ADP (for FIGS. 12A , 12 B) or ADP (for FIG. 12C ).
  • FIG. 12C all substrates were added in the respiration buffer before permeabilization. Pyruvate, glutamate, malate, isocitrate, ⁇ -ketoglutarate and rotenone are indicated with one letter symbols: P, G, M, I, K and R respectively.
  • FIG. 13 shows that ⁇ -cell'sr elative respiratory (ETC/RC) and oxidative phosphorylation (OxPhos) capacities are comparable across cell types.
  • FIG. 13A shows INS1E cells.
  • FIG. 13B shows V79 Chinese hamster lung fibroblasts.
  • FIG. 13C shows primary ⁇ -cells. Arrows indicate the following: “a” indicate permeabilization with ADP (for FIGS. 13A , 13 B) or without ADP (for FIG. 13C ); “b” indicates FCCP (for FIGS. 13A , 13 B) or ADP (for FIG. 13C ); “c” indicates FCCP. Maximal respiratory stimulations with ADP and FCCP were taken as the indices of the oxidative phosphorylation and respiratory capacities, respectively.
  • FIG. 14 shows that ⁇ -cell respiration gradually declines when oxidative phosphorylation is inhibited. Test of substrate limitation vs. respiratory chain dysfunction.
  • FIG. 14A shows that glucose (Glu) and pyruvate (Pyr) stimulate respiration in INS1E cells, which gradually declines in the presence of oligomycin. Lo & Hi indicates 2 mM and 16.7 mM glucose.
  • FIG. 14B shows the respiratory decline in the presence of oligomycin is due to the limitations in substrate supply to the respiratory chain. Letters P, S, F indicate permeabilization, succinate and FCCP, respectively.
  • FIG. 15 shows inter-relationships among the glycolysis, TCA cycle and OxPhos. Emphasis is given to reactions that generate electron donors such as NADH and FADH 2 to support OxPhos as indicated.
  • the NADH and FADH 2 generating reactions are shown by broken lines, while those consuming them are shown by solid lines. Reversible reactions are shown by lines with arrowheads on both ends.
  • F-1, 6-BP fructose-1, 6-bisphosphate
  • G-3-P glyceraldehyde-3-phosphate
  • GPS glycerol-3-phosphate shuttle
  • MAS malate aspartate shuttle
  • CI-V OxPhos complexes I-V
  • .p proton motive force
  • mitochondrial membrane potential mitochondrial membrane potential
  • .pH pH difference across mitochondrial inner membrane
  • Pi inorganic phosphate
  • Q ubiquinone.
  • FIG. 16 shows results of an assay of mitochondrial function with digitonin-permeabilized cells.
  • ⁇ cells were grown as described in Example 8. Ca2+-free LKB buffer containing 10 mM succinate was used in the presence of 2 mM ( FIGS. 16A-16C ) or 15 mM glucose ( FIGS. 16D-16F ).
  • FIG. 16A shows exogenous Cyt C used for maximal respiration.
  • ⁇ cells were first permeabilized with 0.01% digitonin (DIG) and then 1 mM ADP either alone (ADP) or with 10 ⁇ M Cyt C (ADP+CC) was added at right arrow. Control group did not receive either ADP or Cyt C.
  • DIG 0.01% digitonin
  • FIG. 16B shows respiratory coupling with ATP synthesis under the assay conditions used in FIG. 16A .
  • Cells were first permeabilized with 0.01% DIG in the presence (Oligo-FCCP) or absence (Control, Oligo, FCCP) of 2 ⁇ g/ml Oligo and then following additions were made:“Control”, which is ADP+CC; “Oligo”, which is control plus Oligo; “FCCP”, which is control plus 2 ⁇ M FCCP; “Oligo-FCCP”, which is Oligo plus 2 ⁇ M FCCP.
  • FIG. 16C shows data from FIG. 16B with basal rate set at 100% for the comparison of ADP- and FCCP-stimulated respiration rates.
  • FIGS. 16D-16F show results from primary rat astrocytes ( FIG.
  • FIG. 16D hamster B2-MWFE cells
  • FIG. 16E hamster B2-MWFE cells
  • FIG. 16F human H1080 cells
  • FIG. 17 shows results of an assay of mitochondrial function with nPFO-permeabilized to cells.
  • Cells were prepared as described in Example 8 and the assays were performed in Ca2+-free LKB buffer containing 2 mM glucose ( FIGS. 17A-17D ) or 15 mM glucose ( FIG. 17E-17F ).
  • the ADP- and FCCP-stimulated respiration over basal respiration rates (BRR) were indicative of Spare OxPhos capacity (SOC) and spare respiratory capacity (SRC), respectively.
  • FIG. 17A shows the respiratory response of primary pancreatic ⁇ cells permeabilized with 25 nM nPFO or 0.01% digitonin (DIG).
  • FIG. 17B shows results of nPFO titration using INS1E cells. Assay conditions were the same as in FIG. 17A except Cyt C was not added. ADP with varying concentrations of nPFO (@ADP) and 2 ⁇ M FCCP (@FCCP) were added.
  • FIG. 17C shows SOC compared with SRC in INS1E cells.
  • FIG. 17D shows nPFO concentration compared with SOC in INS1E cells.
  • FIG. 17E shows SOC compared with SRC in V79-G3 cells.
  • FIG. 17F shows nPFO concentration compared with SOC and SRC in V79-G3 cells.
  • FIGS. 18A-18H shows an assessment of mitochondrial integrity and respiratory coupling in nPFO-permeabilized cells.
  • respiration was measured in Ca2+-free LKB buffer containing 10 mM succinate with 2 mM glucose ( FIGS. 18A-18C ) or 15 mM glucose ( FIGS. 18D-18H ) ⁇ cells were first permeabilized with at least 1 nM nPFO, and then changes in respiration rates were measured following addition of the indicated compounds.
  • FIG. 18A shows mitochondrial integrity in nPFO-permeabilized INS1E cells (“ADP” indicates 1 mM ADP alone; “ADP+CC” indicates 1 mM ADP+10 ⁇ M Cyt C).
  • FIG. 18A shows mitochondrial integrity in nPFO-permeabilized INS1E cells (“ADP” indicates 1 mM ADP alone; “ADP+CC” indicates 1 mM ADP+10 ⁇ M Cyt C).
  • FIG. 18B shows respiratory coupling in nPFO-permeabilized INS1E cells (“ADP” indicates 1 mM ADP alone; “oligo” indicates 1 mM ADP with 1 ⁇ g/ml oligomycin; “ADP+FCCP” indicates 1 mM ADP+1 ⁇ g/ml oligomycin+2 ⁇ M FCCP).
  • FIG. 18C shows data from FIG. 18B normalized with basal rate set at 100% for comparison of ADP- and FCCP-stimulated respiration rates.
  • FIG. 18D shows integrity in nPFO-permeabilized V79-G3 cells (“control” indicates buffer only; “ADP” indicates 1 mM ADP; “ADP+CC” indicates 1 mM ADP with 10 ⁇ M Cyt C).
  • FIG. 18E shows respiratory decline in nPFO-permeabilized (nPFO) compared with oligomycin-treated intact (Oligo) V79-G3 cells.
  • FIG. 18F shows oligomycin-insensitive respiration in intact compared with permeabilized V79-G3 cells (“Oligo” indicates 1 mM ADP followed by oligomycin; “nPFO+ADP_Oligo” indicates nPFO with 1 mM ADP followed by oligomycin).
  • FIGS. 18G and 18H show mitochondrial integrity in nPFO-permeabilized HEK293 and SHSY-5Y cells, respectively (“ADP” indicates 1 mM ADP; “ADP+CC” indicates 1 mM ADP+10 ⁇ M Cyt C).
  • FIGS. 19A-19F show estimations of the spare (reserve) and total capacities of OxPhos and ETC/RC.
  • ⁇ cells were grown as described in Example 8 and assays were measured in Ca2+-free LKB ( FIGS. 19A , and 19 D- 19 F) or LPBT ( FIGS. 19B and 19C ) buffers containing 15 mM glucose.
  • 10 mM succinate was added with 1 mM ADP to measure spare OxPhos capacity (SOC).
  • FCCP was added to measure spare ETC/RC capacity (SRC).
  • FIGS. 19A-19C the succinate and ADP were added with nPFO, while in FIGS.
  • FIG. 19D-19F they were added after permeabilization with nPFO.
  • FIG. 19A shows SOC and SRC in intact (SRCi) and permeabilized (SRCp) HEK293 cells. In “Control,” 2 ⁇ M FCCP was added to non-permeabilized cells; In “nPFO,” 3 ⁇ M FCCP was added to permeabilized cells after measuring succinate+ADP-stimulated respiration.
  • FIG. 19B shows an estimation of total OxPhos (TOC) capacity in HEK293 cells by adding SOC to the oligomycin-sensitive portion of basal respiration rate.
  • TOC total OxPhos
  • FIG. 19C shows an coupling efficiency of HEK293 cells determined by monitoring oligomycin-sensitive portion of the ADP-stimulated respiration (“PSA_O” indicates nPFO+succinate+ADP followed by oligomycin addition; “O_PSAF” indicates oligomycin followed by nPFO+succinate+ADP+FCCP addition).
  • 19D-19F shows a strategy for determination of TOC by serial additions of nPFO, ADP, and FCCP in different cells such as HEK293 ( FIG. 19D ), SHSY-5Y ( FIG. 19E ) and ⁇ cells ( FIG. 19F ).
  • FIGS. 20A-20I show the effects of different factors on OxPhos capacity. Experimental conditions were as described in Example 8 and with reference to FIG. 19 . Respiratory substrates (10 mM each) and ADP (1 mM) were either added simultaneously with nPFO ( FIGS. 20A , 20 B, 20 C, 20 G, 20 H) or afterwards ( FIGS. 20E , 20 F).
  • FIG. 20A depicts substrate-dependent variation in OxPhos capacity of primary ⁇ cells in Ca2+-free LKB buffer containing 2 mM glucose (“succ” indicates succinate; “G+M” indicates glutamate+malate; “G3-P” indicates glycerol-3-phosphate).
  • succ indicates succinate
  • G+M indicates glutamate+malate
  • G3-P indicates glycerol-3-phosphate
  • FIG. 20B shows the effect of free Ca2+ on succinate-supported OxPhos in ⁇ cells under assay conditions used in the experiments associated with FIG. 20A (“control” indicates succinate+ADP; “CaCl 2 ” indicates 700 ⁇ M CaCl 2 added with succinate+ADP).
  • FIG. 20C shows the effect of different respiration buffers on the OxPhos capacity of HEK293 cells. Cells were incubated in different buffers (see Table 6) and then ADP-stimulated respiration was to measured. 2 ⁇ g/ml oligomycin was added to test the coupling efficiency.
  • FIG. 20D shows coupling efficiency (CE) in different buffers. Data from FIG. 20C are plotted.
  • FIG. 20E shows the effect of phosphate (KH 2 PO 4 ) on OxPhos and respiratory capacity. See Table 6 for LKB, HKB, and MAS buffers. KH 2 PO 4 is the LKB with 10 mM KH 2 PO 4 instead of 0.4 mM.
  • FIG. 20F shows data from FIG. 20E re-plotted after setting basal respiration rate to 100%.
  • FIG. 20G shows, for INS1E cells, spare OxPhos capacity in different buffers, comparing LKB with LPBT, and HKB with HPBT. Basal respiration rate before permeabilization was set at 100%.
  • FIG. 20H shows differences in the basal and ADP-stimulated respiration rates of INS1E cells in high Na+ and high K+ buffers. Actual respiration rates in LPBT and HPBT buffers for FIG. 20G are shown.
  • FIG. 20I shows the effect of different buffers on Complex I- and Complex II-dependent respirations in INS1E cells.
  • FIGS. 21A-21F show data relating to mitochondrial dysfunction and specific features of mitochondrial metabolism.
  • FIGS. 21A-21C Complex I dependence of the glutamate+malate (Glu+Mal)-supported respiration is shown. Assays were performed in free Ca2+ LKB buffer containing 15 mM glucose. ⁇ cells were permeabilized with 1 nM nPFO and ADP-stimulated respiration was measured using the indicated substrates.
  • FIG. 21A a rotenone sensitivity test of the Glu+Mal-supported respiration is shown. 2 ⁇ M rotenone (Rot) was used to inhibit Complex I.
  • Rot ⁇ M rotenone
  • FIG. 21B a lack of Glu+Mal-supported respiration in Complex I-deficient CCL16-B2 cells is shown.
  • FIG. 21C shows complex I activity rescued in B2-MWFE cells (which are CCL16-B2 cells complemented with wild type Chinese hamster Ndufal cDNA).
  • FIG. 21D provides data showing that malate effects efficient utilization of NADH generating substrates such as pyruvate (P), isocitrate (I), glutamate (G), and .-ketoglutarate (K (“PF”, “IF”, “GF” and “KF” indicate, respectively, P, I, G, and K being added separately with 2 ⁇ M FCCP; “PGMIKF” indicates P, I, G, and K added together with FCCP).
  • P pyruvate
  • I isocitrate
  • G glutamate
  • KF .-ketoglutarate
  • FIG. 21E shows a lack of Complex I-dependent respiration on individual substrates (P, G, M, I, K) in primary ⁇ cells (“PA”, “GA”, “MA”, “IA” and “KA” indicate, respectively, P, G, I, M, and K added separately with 1 mM ADP).
  • SA Succinate-supported respiration
  • FIG. 21F shows complex I-dependent respiration in rat astrocytes compared with INS1E cells.
  • Glu+Mal supported respiration was monitored in astrocytes and IS1E cells in the presence (Astro_GM+ROT, INS 1E_GM+ROT) and absence (Astro_GM, INS 1E_GM) of 2 ⁇ M rotenone.
  • FIGS. 22A-22B show the effects of oligomycin treatment on substrate supply to ⁇ cell ETC/RC.
  • INS1E cells were grown and starved as described in the Examples.
  • FIG. 22A shows progressive respiratory decline in INS1E cells in the presence of oligomycin.
  • LKB buffer containing 2 mM glucose in the presence of 1.3 mM CaCl 2 was used for the assays; EGTA was not added.
  • After measuring the basal respiration rates in 2 mM glucose 14.7 mM glucose or 10 mM pyruvate were added (left arrow) to measure respiratory stimulations. Subsequently, approximately 90 min later, 2 ⁇ g/ml oligomycin (Oligo) was added. Control group received buffer only.
  • FIG. 1 shows progressive respiratory decline in INS1E cells in the presence of oligomycin.
  • LKB buffer containing 2 mM glucose in the presence of 1.3 mM CaCl 2 was used for the assays; EGTA was
  • 22B shows effects of oligomycin on ETC/RC function of INS1E cells.
  • Ca2+-free LKB buffer with 16.7 mM glucose and without added EGTA was used for the assays.
  • ⁇ cells were incubated for approximately 60 min in 16.7 mM glucose before the assay was performed. All groups were treated with 2 ⁇ g/ml oligomycin (Oligo) at indicated time point, and then received 10 mM succinate and/or 2 ⁇ M FCCP with ⁇ nPFO.
  • Control indicates buffer only; “Succ+FCCP” indicates succinate+FCCP only (no PFO); “nPFO+FCCP_Succ” indicates nPFO+FCCP followed by succinate addition; “nPFO+Succ+FCCP” indicates nPFO+succinate+FCCP added together.
  • FIGS. 23A-23B show results of FCCP titrations using intact cells.
  • HEK293 cells were seeded at indicated cells densities, and then respiration assay was performed as described in Example 8 using LKB buffer with 1.3 mM CaCl 2 and 15 mM glucose without added EGTA.
  • FIG. 23A shows an actual graph obtained from the XF24 analyzer. Successive additions of 1 ⁇ M FCCP were made using ports A, B, C, and D resulting in cumulative concentrations of 1, 2, 3, and 4 ⁇ M FCCP respectively.
  • FIG. 23B shows a linear relationship of the basal and maximal respiration rates with cell density. Data from rates 3 (without FCCP) and 6 (with 2 ⁇ M FCCP) in FIG. 23A were used.
  • FIGS. 24A-24B shows the relative performance of certain cell permeabilizing agents compared to digitonin.
  • INS1E cells were grown and assays were performed as described in Example 8. ⁇ cells were permeabilized with indicated agents and respiration was monitored in the presence of succinate (10 mM), ADP (1 mM) and Cyt c (10 ⁇ M).
  • FIG. 23A shows relative mitochondrial function in digitonin (0.01% DIG) compared with saponin (5-25 ⁇ g/ml SAP) permeabilized cells.
  • FIG. 23B shows relative mitochondrial function in digitonin (0.01% DIG) compared with alamethicin (3-30 ⁇ g/ml ALA) permeabilized cells.
  • FIGS. 25A-25F shows comparative performances of different PFO variants and the effects of DTT on respiration. Respiratory rates in nPFO- and rPFO-permeabilized HEK293 cells were determined in Ca2+-free LKB ( FIG. 25A ) and HKB ( FIG. 25B ) buffers. The rPFO data from panels FIGS. 25A and 25B are shown after setting basal respiration rate to 100% in FIG. 25C .
  • FIG. 25D shows a comparison of nPFO and dbPFO using INS1E cells. Assays were performed in Ca2+-free LKB with 2 mM glucose and cells were permeabilized with 1 nM nPFO or 1 nM dbPFO+1 mM DTT.
  • FIG. 25E shows the effects of 1 mM DTT on respiration of permeabilized INS1E cells.
  • FIG. 25F shows the effect of 1 mM DTT on the respiration of intact INS1E cell.
  • FIG. 26A shows rPFO-permeabilized INS1E cells.
  • Cells permeabilized with 1 nM rPFO were observed under microscope at the end of experiments in V7 culture plates. Propidium iodide (PI) staining was used to confirm complete permeabilization (bottom), which was evident from cytoplasmic swelling (top).
  • FIG. 26B shows dbPFO-mediated permeabilization of B2-MWFE cells. 100 nM DTT was used to induce the pore formation in 1 nM dbPFO-treated cells.
  • FIG. 27 shows cholesterol-dependence of Cysteine-free PFO derivatives for membrane binding.
  • the fraction of bound PFO derivatives (0.1 ⁇ M final concentration) to liposomes of varying cholesterol content and POPC, POPE and SM in a constant 1:1:1 ratio (0.2 mM total lipid final concentration) was determined using intrinsic Trp fluorescence as described in experimental procedures.
  • the cholesterol-dependent binding isotherms for various derivatives: nPFO (PFO) (squares), rPFO (circles), rPFO D434S (upward triangles) and dbPFO D434S-C459A (downward triangles) are shown. Data points are the average of at least two measurements ⁇ standard deviation.
  • FIG. 28 shows the relative performance of Cysteine-free PFO derivatives in mitochondrial function assays.
  • HEK293 cells were permeabilized using nPFO, rPFO and rPFO D434S (see Table 5) at two different concentrations (0.1, 1.0 nM). Subsequently succinate (Succ: 10 mM) and FCCP (3 mM) were added together to measure maximal respiratory activity.
  • FIG. 28A shows the relative performance at 0.1 nM.
  • FIG. 28B shows the relative performance at 1.0 nM.
  • FIG. 28C shows a comparison of activity of nPFO at both 0.1 and 1.0 nM concentrations.
  • FIG. 28D shows a comparison of activity among PFO variants at both concentrations (0.1 & 1.0 nM). Assays were performed in LPBT buffer (see Table 6).
  • FIG. 29 shows that digitonin mediated cell permeabilization does not give stable respiration.
  • Cells were permeabilized in the presence of different concentrations of digitonin (% DIG) and ADP stimulated respiration using succinate as substrate was monitored. Assays were performed either in LKB or LPBT buffer (see Table 6).
  • FIGS. 29A and 29B show inverse relationships between digitonin concentration and respiratory activity in LKB buffer.
  • succinate was present in the medium before permeabilization and in FIG. 29B it was added with ADP. Compare arrows ADP and Succ+ADP in both panels. Assay was done with starved INS1E cells.
  • FIGS. 29 shows that digitonin mediated cell permeabilization does not give stable respiration.
  • Cells were permeabilized in the presence of different concentrations of digitonin (% DIG) and ADP stimulated respiration using succinate as substrate was monitored. Assays were performed either in LKB or LPBT buffer (see Table 6).
  • FIGS. 29C and 29D show effects of the respiratory buffer used in non-starved INS1E cells. Respiration declines faster in LKB buffer ( FIG. 29C ) compared to that in LPBT ( FIG. 29D ) at all digitonin concentrations tested.
  • FIGS. 29E and 29F show that Cytochrome c loss with digitonin is associated with respiratory decline. Addition of exogenous Cytochrome c (CC) with succinate+ADP inhibits the respiratory decline even at highest digitonin concentration used (0.01%) in both LKB & LPBT buffers.
  • FIG. 30 shows effects of low K+ (LPBT buffer) compared with high K+ (HPBT) buffer on INS1E cell bioenergetics.
  • FIG. 30A shows respiratory decline following the addition of oligomycin (Oligo; 1 ⁇ g/ml) and response to uncoupler FCCP (2 ⁇ M).
  • FIG. 30B shows data in FIG. 30A re-plotted with base line set to 100% at rate 4. A faster decline and no response to FCCP is depicted in high K+ buffer (HPBT).
  • FIG. 31 shows dose-dependent effects of rotenone on overall cellular respiration in intact cells and on Complex I activity in PFO permeabilized cells.
  • FIGS. 31A and 31D show base-line normalized respiration rates of rotenone-treated HEK293 human cells in LKB ( FIG. 31A ) and LPBT ( FIG. 31D ) buffers (“Rot” indicates 0, 10, 20, 50 & 1000 nM rotenone added at arrow; “P/GM/A” indicates 1 nM rPFO, glutamate+malate (10 mM each), 1 mM ADP & 3 ⁇ M FCCP; “succ” indicates 10 mM succinate).
  • FIGS. 31A and 31D show base-line normalized respiration rates of rotenone-treated HEK293 human cells in LKB ( FIG. 31A ) and LPBT ( FIG. 31D ) buffers (“Rot” indicates 0, 10, 20, 50 & 1000 nM rotenone added at arrow; “
  • FIGS. 31A and 31D show respiration rates and rotenone concentrations plotted from data in FIGS. 31A and 31D , respectively (“Basal” indicates decline in respiration of intact cells; “Glu+Mal” indicates Complex I dependent respiration in the presence of FCCP; and “succ” indicates Complex II-dependent respiration in the presence of FCCP).
  • FIG. 31C shows percent inhibition of Complex I and II-dependent respirations at different rotenone doses (“LKB-CI, CII” indicates Complex I and II functions in LKB buffer; and “LPBT-CI, II” indicates Complex I and II functions in LPBT buffer).
  • 31F shows a relationship between the basal Complex I inhibition (in intact cells) and maximal Complex I inhibition (in permeabilized cells) in LKB (broken lines) and LPBT buffer (solid lines). At maximal rotenone dose (1 ⁇ M) no significant difference was observed between basal and maximal as well as between the buffers (see FIGS. 31B and 31E ).
  • FIG. 32 shows reduced respiratory activity in Ndufa1S55A-derived MEFs.
  • FIGS. 32A-32B show differences in the basal and maximal respiration rates of WT and S55A MEFs. Maximal respiration was determined by using the increasing dose of FCCP (2-4 ⁇ M).
  • FIGS. 32C-32D show respiratory activity in cells that were permeabilized by recombinant perfringolysin O (rPFO). Complex I and II activities were measured by using specific substrates in the presence of ADP+FCCP (“Glu+Mal” indicates glutamate+malate and “succ” indicates succinate). 100,000 cells/well were spun down in V7 plates and measurements were made after 4 hours of culture.
  • aspects of the invention relate to simple and reproducible assays for mitochondrial function.
  • one or more assay components can be provided as kits.
  • aspects of the invention relate to the surprising finding that cytolysin-based (e.g., perfringolysin O (PFO)-based) cell permeabilization eliminates the need for isolating mitochondria, and maintains the cellular microenvironment around mitochondria. This allows functional assays to be performed at close to physiological conditions.
  • cytolysin-based e.g., perfringolysin O (PFO)-based
  • the invention relates to cholesterol-dependent cytolysins.
  • cholesterol-dependent cytolysins are members of a family of proteins that form pores in lipid-based membranes in a cholesterol sensitive manner.
  • cholesterol-dependent cytolysins are pore-forming toxins secreted by Gram-positive bacteria.
  • cholesterol-dependent cytolysins have a characteristic ⁇ -barrel structure.
  • cholesterol-dependent cytolysins are monomeric proteins that oligomerize on the membrane surface of target cells.
  • cholesterol-dependent cytolysins form a ring-like pre-pore complex at the membrane surface of target cells, and insert a large ⁇ -barrel into the membrane.
  • the presence of cholesterol in the target membrane is required for pore-formation.
  • cholesterol-dependent cytolysins selectively permeabilize cellular plasma membranes without damaging mitochondrial membranes.
  • Non-limiting examples of cholesterol-dependent cytolysins are provided in Table 1. Other examples will be apparent to the skilled artisan.
  • the cholesterol-dependent cytolysin is a perfringolysin O (PFO).
  • the selective permeabilization of cellular to membranes by cholesterol-dependent cytolysins without damaging mitochondrial membranes can be used in assays to measure mitochondrial metabolites.
  • Many mitochondrial metabolites readily cross the mitochondrial membrane to and from the cytosol. However, these metabolites do not readily cross the cellular membrane. This makes it difficult to assay these metabolites without disrupting the cells and in the process disrupting the natural physiological environment of the mitochondria.
  • cholesterol-dependent cytolysins e.g., PFOs
  • this allows mitochondrial activity to be evaluated by measuring the extracellular levels of one or more mitochondrial metabolites.
  • the selectivity of cholesterol-dependent cytolysins e.g., PFOs is useful, because it allows the cellular membrane to be permeabilized with respect to mitochondrial metabolites without disrupting the mitochondrial membrane. In some embodiments, this allows the activity of the mitochondria to be evaluated in their natural cellular environment.
  • PFO-based cell permeabilization methods are provided for mitochondrial function assays.
  • the methods are generally applicabile across different cell types (e.g., established cell lines and primary cells).
  • Kits that provide reagents for the methods are also provided herein. In some embodiments, these kits are useful for assays of Complexes I, II, III, IV and/or V, Oxidation Phosphorylation (OxPhos) capacity, and/or Respiratory (ETC/RC) capacity.
  • simple and reproducible assays for mitochondrial function are provided.
  • the assays and kits provide diagnostic tools for mitochondrial dysfunction.
  • aspects of the invention are useful to understand the role of mitochondrial metabolism in pathophysiology.
  • aspects of the invention provide diagnostic tools for mitochondrial disorders.
  • cholesterol-dependent cytolysin-based (e.g., PFO-based) cell permeabilization eliminates the need for isolating mitochondria, and maintains the cellular microenvironment around them. This permits mitochondrial function assays to be performed at close to physiological conditions using assays techniques that involve measuring the uptake, release, consumption, and/or production of cellular metabolites (e.g., mitochondrial metabolites), for example, using microplate-based respirometry. This also permits examination of the effects of membrane impermeable agents on mitochondrial function.
  • cellular metabolites e.g., mitochondrial metabolites
  • weihenstephanensis WLO 74 (69) 87 (83) 462 (512) ABY46062 Listeriaceae Listeria L. monocytogenes LLO 43 (40) 66 (62) 469 (529) DQ838568.1 L. seeligeri LSO 45 (41) 67 (63) 469 (530) P31830.1 L. ivanovii ILO 46 (43) 66 (62) 469 (528) AAR97343.1 Planococcaceae Lysinibacillus L. sphaericus SPH 76 (72) 90 (87) 463 (506) YP_001699692.1 Paenibacillaceae Paenibacillus P.
  • butyricum BRY 69 (65) 85 (82) 462 (513) ZP_02950902.1 C.
  • tetani TLY 60 55) 78 (72) 464 (527) NP_782466.1 C. botulinum B BLYb 60 (49) 78 (63) 464 (602) YP_001886995.1 C. botulinum E3 BLYe 60 (48) 77 (60) 464 (602) YP_001921918.1 C. botulinum C BLYc 60 (56) 79 (74) 463 (518) ZP_02620972.1 C.
  • novyi NVL 58 (54) 78 (73) 463 (514) YP_878174.1 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Gardenella G. vaginallis VLY 40 (39) 65 (60) 466 (516) EU522488.1 Actinomycetales Actinomycetaceae Arcanobacterium A. pyogenes PLO 41 (38) 60 (56) 469 (534) U84782.2
  • Perfringolysin O [ Clostridium perfringens str. 13] gi
  • OxPhos one of the key functions of mitochondria, is carried out by five multimeric enzyme complexes (I-V, see below) with the help of electron donors (NADH, & FADH 2 ) and electron carriers (ubiquinone, Cytochrome c).
  • NADH, & FADH 2 electron donors
  • ubiquinone Cytochrome c
  • the ⁇ p is the driving force for ATP synthesis using ADP and Pi.
  • Four enzyme complexes constitute the ETC/RC.
  • Complex I is NADH-ubiquinone oxidoreductase
  • Complex II is succinate-ubiquinone oxidoreductase
  • Complex III is ubiquinol-Cytochrome c oxidoreductase
  • Complex IV is Cytochrome c-ubiquinone oxidoreductase. Coupled electron transfer with proton translocation across the mitochondrial membrane by Complexes I, III, and IV establishes ⁇ p, which drives ATP synthesis using the ATP synthase (Complex V) (see FIGS. 1 and 15 ).
  • NADH is also generated within cytosol from the reaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
  • GAPDH glyceraldehyde-3-phosphate dehydrogenase
  • NADH lactate dehydrogenase
  • MAS malate-aspartate
  • GPS glycerol-3-phosphate
  • Impairments in OxPhos are often referred to as mitochondrial dysfunction (and are associated with mitochondrial disorders), and can result from hereditary and somatic mutations in nuclear genes or mtDNA, or functional impairments by drugs or toxins. Mutations in over 100 genes constituting the oxidative phosphorylation machinery are linked with mitochondrial encephalopathies in humans, which are the most common metabolic diseases with an incidence of over ⁇ 1/5000 in live births. Respiratory chain Complex I deficiency is a cause of mitochondrial diseases in many cases. Twenty five of at least fifty known genes implicated in Complex I biogenesis are found associated with mitochondrial diseases.
  • NDUFV1,2 pathogenic mutations in structural subunits (e.g., NDUFA1, 2, 11; NDUFS1-4, 6-8; NDUFV1,2) and assembly factors (e.g., NDUFAF1-6) have been identified.
  • Neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease are also associated with mitochondrial dysfunction.
  • mtDNA mutations are found associated with almost all types of cancers.
  • Type 2 diabetes is also linked with declining mitochondrial function in relevant tissues such as ⁇ -cells and muscles. Type 2 diabetes represents a major clinical challenge due to the sharp rise in obesity-induced disease.
  • methods are provided for accurate assessment of mitochondrial function in the context of pathophysiology.
  • Plasma membrane permeabilization removes the permeability barrier of cells and allows accurate estimation of mitochondrial function in intact cells.
  • the selective permeabilization of the plasma membrane has been achieved by exploiting the differential distribution of the cholesterol in membranes. Because most cholesterol is present in the plasma membrane, intracellular membranes are expected to remain largely untouched by cholesterol dependent pore forming agents. Thus, it is possible to selectively permeabilize the plasma membrane in the presence of cholesterol-dependent pore forming agents.
  • mitochondrial function assay methods based on a method of cell permeabilization, using a cholesterol dependent pore forming protein (e.g., perfringolysin O (PFO) from Clostridium perfringens ). The mechanism of pore formation by PFOs has been characterized.
  • PFO perfringolysin O
  • methods and kits disclosed herein are useful with systems for analyzing extracellular flux (XF) (e.g., commercially available extracellular flux (XF) analyzers, e.g., available from Seahorse Bioscience) to assess mitochondrial function in cells.
  • XF extracellular flux
  • users of such analyzers who investigate bioenergetic pathways isolate cells or tissue, adhere them to a culture plate, and perform bioenergetic assessments. These techniques may be used to provide valuable information and insight into mitochondrial biology, under conditions that allow for experimental control of the substrate supply and demand.
  • whole cells may be selectively permeabilized directly in the plates (e.g., XF plates), allowing for control over substrate supply and demand, access to both oxidative phosphorylation and respiratory chain components, as well as eliminating the need to go through complicated and potentially damaging mitochondrial isolation techniques.
  • the plates e.g., XF plates
  • cholesterol-dependent cytolysins and assay-specific reagents are packaged into a kit format.
  • the kit design may vary with respect to specific assays rather than cells types.
  • these kits are designed to address specific aspects of mitochondrial metabolism such as the OxPhos, TCA cycle, and cell-specific OxPhos/TCA cycle features. Kits are provided for Complex I-IV assays, OxPhos capacity and ETC/RC capacity assays.
  • ADP-stimulated respiration is a measure of the Complex V activity, e.g., when the substrates are not limiting.
  • succinate with glycerol-3-phosphate together ensures that the ETC/RC activity is not limiting factor, and thus the ADP-stimulated, oligomycin-sensitive respiration is the output of Complex V function.
  • the carboxyatractiloside-sensitive respiration gives the functional output of the ATP/ADP nucleotide translocator (ANT).
  • the activity of Complex III is assayed using glycerol-3-phosphate+succinate supported, Antimycin A-sensitive respiration.
  • the activity of Complex IV is assayed in some embodiments using ascorbate+TMPD supported, KCN-sensitive respiration as described.
  • a kit has components selected from: PFO, ADP, FCCP and assay buffer.
  • the actual concentration of reagents will typically vary with the experimental design (e.g., 24-well vs. 96-well assay format) and the number of assays.
  • PFO can typically be used in a range of 1-100 nM.
  • ADP can typically be used in a range of 1-2 mM.
  • FCCP can typically be used in a range of 2-4 ⁇ M.
  • the components are typically provided as 100-1000-fold concentrated stocks, which can be used at desired concentrations within the recommended range by the user to get the maximal mitochondrial performance within the assays.
  • a kit may include one or more components (e.g., one or more substrates or inhibitors) for a specific Complex assay (e.g., one of Complex I-V) along with a cholesterol-dependent cytolysin (e.g., PFO), and optionally one or more reducing agents (e.g., DTT or other suitable reducing agent).
  • a specific Complex assay e.g., one of Complex I-V
  • a cholesterol-dependent cytolysin e.g., PFO
  • reducing agents e.g., DTT or other suitable reducing agent
  • the different components may be provided in separate containers in a kit. However, in some embodiments, two or more different components may be combined in a single container (e.g., sample tube, well, etc.).
  • Table 2 provides non-limiting examples of components of kits for mitochondrial complexes.
  • functional assays will be based on a microplate-based system or device, for example using the extracellular flux (XF) analyzer from Seahorse Biosciences.
  • XF extracellular flux
  • wild type PFO and/or its variants will be used for selective plasma membrane permeabilization to eliminate the substrate transport barrier, and characteristics of mitochondrial performance (e.g., maximal mitochondrial performance) can be determined. Methods for assaying specific mitochondrial functions that work across different cell types can be implemented.
  • one or more microplates may be preloaded (and/or provided) with one or more assay components (e.g., substrates, inhibitors, etc.) and one or more cholesterol-dependent cytolysins (e.g., PFO), and optionally one or more reducing agents (e.g., DTT or other suitable reducing agent).
  • assay components e.g., substrates, inhibitors, etc.
  • cholesterol-dependent cytolysins e.g., PFO
  • reducing agents e.g., DTT or other suitable reducing agent
  • the terms “approximately” or “about” in reference to a number are generally taken to include numbers that fall within a range of 1%, 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
  • Wild type PFOs and variants have been tested for maximal mitochondrial performance (see Table 1 for examples of PFOs) using methods disclosed herein.
  • Specific assays for different OxPhos components applicable to a wide variety of were developed based on cell permeabilization methods described herein. Both established and primary cells were utilized to test the general applicability of PFO-based assays.
  • a reducing agent may be used to increase shelf life of wild-type PFO.
  • PFO contains only one Cys residue at position 459.
  • a Cys free derivative, PFO C459A has been used. This variant has activity comparable to wild type PFO.
  • a mutant rPFO T319C-V334C provides for conditional cell permeabilization, as it does not form pore in the membrane following insertion until a reducing agent, such as DTT is added. After treatment with rPFO T319C-V334C the cells may be washed to remove the protein before the experiment.
  • FIG. 9 A non-limiting purification scheme that has been used for obtaining recombinant PFO (e.g., wild-type or variant) is illustrated in FIG. 9 .
  • the scheme involves over-expressing a PFO protein and/or its variants with a His-Tag for affinity purification.
  • E. coli BL21 (DE3) (Invitrogen) cells expressing PFO and rPFOs conditionally are grown in 2 L cultures at 37° C. with constant agitation. Expression of the PFO/rPFO are induced by the addition of isopropyl ⁇ -D-thiogalactopyranoside (IPTG, Gold Biochemicals, St.
  • IPTG isopropyl ⁇ -D-thiogalactopyranoside
  • the supernatant is loaded onto a column (1.5 cm I.D. ⁇ 10 cm) containing chelating Sepharose Fast Flow (GE Healthcare, Piscataway, N.J.) that had been preloaded with Ca 2+ and equilibrated with buffer B at room temperature.
  • the column is washed with 115 mL of buffer B (2 mL/min), and a linear gradient 0 to 50 mM imidazole pH 6.5 to remove additional contaminating proteins.
  • the bound PFO/rPFO is eluted with 55 mL of buffer B containing 300 mM imidazole.
  • the pooled fractions containing the bulk of the protein are dialyzed overnight at 4° C. against 4 L of buffer C [10 mM MES (pH 6.5), 1 mM EDTA], and loaded directly onto a SP Sepharose HP (GE Healthcare, Piscataway, N.J.) cation exchange column (1.5 cm I.D. ⁇ 10 cm) equilibrated with buffer C.
  • the column is washed with 60 mL of buffer B (3 mL/min), and 30 mL of 0.1 M NaCl in buffer C before the elution of the PFO/rPFO with a 100 mL linear gradient (3 mL/min) from 0.1 to 0.9 M NaCl in buffer C.
  • PFO eluted at ⁇ 0.5 M NaCl and the pooled fractions containing the PFO are dialyzed against buffer A made 10% (v/v) in glycerol, aliquoted into cryovials, quick-frozen in liquid nitrogen, and stored at ⁇ 80° C.
  • Dithiothreitol (5 mM) is included as a reducing agent when purifying PFO derivatives containing Cys residues. Cys-less derivatives (rPFO) do not need DTT additions.
  • a mouse model (Ndufa1 S55A ) of the partial Complex I deficiency ( ⁇ 50%) may be used.
  • primary cells neurons, mouse embryonic fibroblasts, blood monocytes, thymocytes and splenocytes
  • synaptosomes may be derived from the Ndufa1 K1 mice to determine the physiological effects of partial Complex I assembly.
  • Partial Complex I deficiencies are the most common cause of mitochondrial diseases in humans. However, other deficiencies also may be studied using these methods.
  • Non-adherent cells Peripheral blood May involve coating with coatings monocytes of plates with PEI or (PBMCs)/Lymphocytes similar reagents e.g.
  • Cells may be grouped with respect to their OxPhos capacity vs. respiratory (ETC/RC) capacity to provide a reference for each enlisted cell line.
  • ETC/RC OxPhos capacity vs. respiratory
  • excitable cells such as ⁇ cells
  • neurons and muscle cells have comparable OxPhos vs. respiratory (ETC/RC) capacity under low inorganic phosphate (Pi) medium/buffer.
  • non excitable cell such as fibroblasts have lower OxPhos capacity compared to respiratory capacity.
  • Increasing the Pi concentration to 10 mM significantly increases the OxPhos capacity in these cells, but it is still relatively lower than respiratory capacity.
  • mammary epithelial cells and adult stem cells have been evaluated with mammary epithelial cells and adult stem cells. Similar experiments may be performed with other cells.
  • different primary cells such as mammary epithelial cells, mouse embryonic fibroblasts, can be used.
  • these primary cells can be derived from the Ndufa1 S55A mice.
  • assays with ⁇ -cells which are also excitable cells, indicate such cells may be evaluated effectively in a wide range of assay conditions. Testing these parameters for a given cell type is possible while using ADP-stimulated and succinate supported respiration as output response for the mitochondrial performance.
  • FIG. 2 shows representative data for conditions for INS1E cells that involve the addition of exogenous Cytochrome c.
  • a Ca 2+ -free low K + respiration buffer [20 mM TES pH7.4, 3.5 mM KCL, 120 mM NaCl, 0.4 mM KH 2 PO 4 , 1.2 mM Na 2 SO 4 , 2 mM MgSO 4 , 1 mM EGTA with 0.4% fatty acid free BSA] was used unless otherwise specified.
  • a PFO-based assay was developed and found to overcome problems associated with digitonin-based assays. Initially, a wild type PFO was used for selective permeabilization of the plasma membrane. The performance of PFO was compared to digitonin in assays using INS1E cells and Chinese hamster lung fibroblasts. (V79-G3). Surprisingly, PFO outperformed digitonin in both cell types (see FIG. 3 ). The need for exogenous Cytochrome c in PFO-based assays was evaluated. This was accomplished by comparing the ADP-stimulated respiration in the presence and absence of Cytochrome c. The tests showed that even lower concentrations of PFO could be used without the addition of Cytochrome c ( FIG. 4 ).
  • FIG. 5C The OxPhos and ETC/RC capacities are measured by the ADP-stimulated and the protonophore FCCP-stimulated respiration, respectively. Further increase in the respiration over ADP-stimulation by FCCP suggests that the OxPhos capacity is lower compared to ETC/RC capacity ( FIG. 5A ). This may be a common feature of certain cells such as fibroblasts, which may not face an acute ATP shortage that requires a larger spare OxPhos capacity ( FIGS. 5A and 6A ).
  • FIG. 7 illustrate an example of Complex I mutants.
  • Complex I mutants lack respiration on substrates that generate NADH (e.g., glutamate+malate), while they show respiration comparable to control cells on succinate that generates FADH2.
  • NADH e.g., glutamate+malate
  • the NADH & FADH2 feed electrons to Complexes I & II respectively.
  • the activities of other complexes also can be assayed using specific substrates and inhibitors (see description of Complex III & IV assays provided herein).
  • the PFO-based assays can be tailored to determine the limitations of substrate supply to the ETC/RC, which can cause respiratory decline under certain conditions.
  • the respiratory decline caused by oligomycin in ⁇ cells was evaluated.
  • Data in FIG. 8 show that this oligomycin-induced respiratory decline in ⁇ cells is due to limitations in substrate supply. Respiration could be restored only in the PFO-permeabilized cells in the presence of both succinate and FCCP. Further, the addition of Ca 2+ dropped the respiration significantly, which is believed to be due at least in part to the opening of the permeability transition pore.
  • the data presented demonstrate the feasibility of the PFO-based functional assays of mitochondrial function. These assays can be performed using any suitable technique.
  • microplate-based respirometry using the XF analyzer from Seahorse Biosciences can be used.
  • the PFO can be used to permeabilize cells in a range of settings to study the mitochondrial metabolism apart from the XF analyzer based studies.
  • FIGS. 10-14 show results from assays described below involving PFO-based permeabilization.
  • the role of mitochondrial metabolism is widely recognized in glucose sensing. It is thought that the low affinity glucokinase, which is insensitive to feedback inhibition by ATP, increases the flux through glycolysis and enhances pyruvate production. Pyruvate is suggested to be metabolized by TCA cycle to generate electron donors (NADH & FADH2 for respiratory Complexes I & II, respectively) for ATP production, and provide additional signals for sustained release of insulin.
  • imaging based studies have suggested that pyruvate metabolism within mitochondria may not be efficient in generating NADH, its implication on mitochondrial function does not appear to have been explored in detail elsewhere.
  • NADH levels were lower compared to that found in fibroblast and astrocyte mitochondria. It is proposed that ⁇ -cells regulate NADH output per glucose within mitochondria by negatively regulating key steps to favor the reliance on redox shuttles, and help export of malate and citrate to cytosol for tight coupling of glucose metabolism with insulin secretion.
  • ⁇ -cell bioenergetics is dependent on oxidative phosphorylation. It plays a role in insulin secretion by ⁇ -cells.
  • the reliance of insulin secretion on cytosolic NADH oxidation via redox shuttles suggests that the Respiratory Chain function in ⁇ -cells is primarily dependent on the cytosolic electron donors. Therefore, whether NADH production within ⁇ -cell mitochondria is lower compared with other cells (such as astrocytes and fibroblasts) that are not glucose sensitive has been tested.
  • the differences in Complex I-dependent respiration using various NADH generating substrates were monitored.
  • the respiratory decline in the presence of oligomycin that blocks ATP synthase (Complex V) activity was monitored.
  • CDCs cholesterol-dependent cytolysin (CDC) perfringolysin O
  • PFO cholesterol-dependent cytolysin O
  • CDCs are secreted as water soluble monomers of 50-70 kDa that form large ring- and arc shaped homooligomeric pores (35-50 monomers/oligomer) in cholesterol containing membranes.
  • the formed pores are approximately 250 ⁇ in diameter and allow the passage of large molecules (e.g. antibodies, ⁇ -amylase, and thyroglobulin). Results described below indicate that mitochondrial integrity is better preserved when cells are permeabilized with PFO compared to detergents.
  • Rotenone was from Calbiochem. Other reagents were from Sigma unless otherwise stated.
  • nPFO Native (nPFO), the Cysteine-free rPFO (nPFO containing the C459A mutation), and the engineered disulphide-bond containing mutant dbPFO (rPFO containing the double T319C-V334C mutation) were purified using art known methods. These derivatives contain the polyhistidine tag from the pRSET-B vector (Invitrogen). No significant functional or structural differences were detected among PFO derivatives bearing or lacking the polyhistidine tag.
  • nPFO and rPFO are cytolytically active, but rPFO utilizes higher cholesterol concentrations when tested using model membranes.
  • dbPFO binds to membranes but does not form pores because one of the transmembrane ⁇ -hairpins is covalently linked to domain 2 via a disulphide bond. Reduction of the disulphide bond by the addition of (2S,3S)-1,4-bis(sulfanyl)butane-2,3-diol (DTT) releases the locked transmembrane ⁇ -hairpin triggering the insertion of a large transmembrane barrel.
  • DTT (2S,3S)-1,4-bis(sulfanyl)butane-2,3-diol
  • Cysteine-free rPFO and the dbPFO were stored in buffer A [50 mM HEPES pH 7.5, 100 mM NaCl, and 10% (v/v) glycerol] while the nPFO was stored in buffer A supplemented with 5 mM DTT to retain its cytolytic activity. Proteins were kept at ⁇ 80° C. until used. The protein concentration was calculated using a molar absorptivity (cm) of 74260 cm ⁇ 1 M ⁇ 1 .
  • Rat insulinoma INS1E cells were grown in RPMI1640 medium (Mediatech Inc, Manassas, Va.) which was supplemented with 11.1 mM of glucose, 10% fetal bovine serum (FBS), 1 mM HEPES (Invitrogen), and 50 ⁇ M of ⁇ -mercaptoethanol (2-Sulfanylethan-1-ol).
  • Starvation medium contained 4 mM glucose instead of 11.1 mM glucose in the presence of 1 mM Na-pyruvate.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS Invitrogen
  • nonessential amino acids Mediatech, Inc, Manassas, Va.
  • antibiotic mix PenStrep, Invitrogen
  • the human neuroblastoma SHSY5Y cells were cultured in DMEM/F12 media with 10% FBS and 1% antibiotics mix. Cells were harvested after washing once with Ca2+ and Mg2+-free phosphate buffered saline (PBS: pH 7.4) using 0.05% trypsin-EDTA (Invitrogen).
  • Pancreatic islets were isolated from Wistar rats using methods known in the art. Collagenase P enzyme solution (1.2-1.4 mg/ml; Roche Diagnostics Corporation, Indianapolis, Ind.) was injected into the distal end of the donor pancreas. After digestion, islets were gradient purified and then handpicked and cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 1% PenStrep (Mediatech, Manassas, Va.) in a 5% CO 2 incubator. Single cells from islets were prepared using art known methods. After 24-48 hr post culture, islets were collected at 1100 rpm for 5 min at 4° C. followed by washing in PBS twice.
  • Collagenase P enzyme solution 1.2-1.4 mg/ml; Roche Diagnostics Corporation, Indianapolis, Ind.
  • islets were gradient purified and then handpicked and cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 1% PenStrep (Mediatech, Manass
  • ⁇ -cells are the predominant cell types ( ⁇ 80%). Unless otherwise indicated dispersed rat islet cells are referred to herein as primary ⁇ -cells.
  • Cells grown ⁇ 80% confluence in V7 tissue culture plates were used for in situ microplate-based respirometry using the XF24 Flux Analyzer (Seahorse Biosciences). All assays were performed with V7 PS plates unless otherwise specified. Cells were seeded at the following densities/well: 10-20,000 (lung fibroblasts—V79-G3, B2-MWFE; HT1080); 25,000 (primary astrocytes); 30,000 (HEK293); 50,000/well (INS1E); and 100,000 (primary (3 cells).
  • Cells were grown for 24-72 hr after seeding, unless otherwise indicated, washed twice with 500 ⁇ l of the indicated respiration buffer (see Table 6) and then incubated in a non-CO 2 incubator at 37° C. for ⁇ 30-60 min.
  • XF24 cartridges pre-hydrated for 24 hr were calibrated according to the to manufacturer's instructions after loading injection ports with the indicated compounds. After calibration of the sensor cartridge per manufacturer's instructions, the V7 culture plate with cells was loaded into the XF24 analyzer. Respiratory activity of cells was measured using cycles of mixing, waiting and measuring at 0.5-2, 1.5-2 and 3-5 min, respectively, depending upon the cell type used.
  • FIGS. 23A-23B show a typical FCCP titration assay using HEK293 cells.
  • Mitochondria from INS1E and B2-MWFE cells were isolated using art known methods. Mitochondrial content was measured using a microplate-based BCA protein assay kit (Thermo Fisher). 20 g of mitochondria in 250 ⁇ l Ca2+ free LKB (without glucose) were incubated with glutamate+malate (10 mM each) in the presence of 2 ⁇ M rotenone for 115 min at 37° C. FLUOstar Omega fluorimeter (BMG Labtech) was used to measure the relative levels of NAD(P)H at 355 nm excitation and 460 nm emission wavelengths. An NADH standard curve was used to determine concentrations of NAD(P)H.
  • Mitochondrial Integrity is Compromised in Detergent-Permeabilized Cells:
  • INS1E rat insulinoma cell line
  • INS1E cells are an experimental model for rat pancreatic ⁇ cells.
  • Digitonin has been used for selective plasma membrane permeabilization to assess mitochondria function.
  • a digitonin-based assay was evaluated to determine whether it would be suitable for a microplate based respirometry that employs a limited number of cells (e.g., 5000 to 100,000 cells).
  • Oligomycin is an inhibitor of the ATP synthase (Complex V) that uses ⁇ p to synthesize ATP from ADP and Pi (see FIG. 15 ).
  • the majority of the ADP stimulated respiration ( ⁇ 75%) was oligomycin sensitive when oligomycin was added along with ADP. The remaining respiration is expected to be supported by the H+ leak across inner mitochondrial membrane.
  • FCCP FCCP
  • FCCP which is a protonophore, induces maximal respiration by dissipating the H+ gradient across the mitochondrial inner membrane.
  • a suitable FCCP concentration as determined by titration was used to determine the respiratory capacity (maximal respiration) of INS1E cells.
  • ADP and FCCP gave comparable respiratory stimulations indicating that spare OxPhos capacity and respiratory capacity, respectively, were comparable in INS1E cells ( FIG. 16B , 16 C).
  • Significant differences were not observed between the spare OxPhos and respiratory capacities using succinate as the respiratory substrate.
  • FCCP-stimulated respiration in the presence of oligomycin was not significantly different from that observed without oligomycin, which indicates that respiratory capacity was primarily determined by the functional capacity of ETC/RC.
  • ADP-stimulated, succinate-supported respiration was evaluated in various cell types, including primary rat astrocytes, Chinese hamster fibroblasts and human cells. Similar conditions were applicable to rat astrocytes ( FIG. 16D ), and digitonin titrations were utilized for hamster (B2-MWFE) and humans (HT1080) cells ( FIG. 16E , F).
  • CDCs Cholesterol-dependent cytolysins
  • PFO perfringolysin O
  • Pores formed by the insertion of transmembrane ⁇ -barrels of a CDC were found to be better controlled and more homogenous than those obtained with detergents. Intracellular organelles therefore remain structurally and functionally intact in the presence CDCs.
  • perfringolysin O (PFO) derivatives were developed for permeabilization assays. It was found that the respiratory response was more robust in ⁇ cells permeabilized with nPFO compared with digitonin ( FIG. 17A ). Such an improvement in mitochondrial function with nPFO was also observed in Chinese hamster lung fibroblasts as well (V79-G3). nPFO dose-response analyses were performed to determine a minimal concentration required for maximal mitochondrial function. Spare OxPhos and respiratory capacities of INS1E and V79-G3 cells were measured at different doses. It was found that, in some embodiments, 1 nM PFO was adequate for maximal mitochondrial performance ( FIGS. 17B-D ). A further increase in respiration was not observed by FCCP addition beyond ADP-stimulation at all nPFO doses tested ( FIG. 17B ).
  • OxPhos capacity of INS1E cells was comparable to respiratory capacity ( FIGS. 16B , C and FIGS. 17B , C).
  • the spare OxPhos capacity in V79-G3 cells was lower compared to respiratory capacity ( FIGS. 17E , F).
  • ADP-stimulated respiration was sensitive to oligomycin, indicating respiratory coupling with ATP synthesis ( FIG. 18F ).
  • ADP alone result in respiratory stimulation indicating that plasma membrane was impermeable to ADP.
  • FIG. 19A show a typical experiment for determining spare OxPhos and respiratory capacities.
  • Human HEK293 cells were used for these assays. ⁇ cells were incubated in Ca2+-free respiration buffer containing 15 mM glucose, and then succinate and ADP were added with nPFO simultaneously. The ADP-stimulated respiration over the basal respiration gives the estimate of spare OxPhos capacity.
  • SOC spare OxPhos capacity
  • SRC spare respiratory capacity
  • SOC spare OxPhos capacity
  • OSR oligomycin-sensitive respiration
  • TOC total OxPhos capacity
  • FIG. 18D In the presence of substrate respiratory decline in PFO-permeabilized cells is associated with ADP leaking from the cytoplasm, ( FIG. 18D , G, H).
  • An alternative assay design was used to determine total OxPhos capacity ( FIG. 19D ). This permits an analysis of the relationship between OxPhos and respiratory capacities on a given substrate.
  • Experimental conditions such as the time taken to achieve steady-state respiration rate following PFO-permeabilization may be optimized for a given cell type.
  • OxPhos capacity can be influenced by certain factors, including, for example, substrate(s) and experimental conditions. Having determined that the PFO-based assay was robust and reproducible, factors that could influence OxPhos capacity were assessed. Spare OxPhos capacity was found to vary with different substrates such as glutamate+malate, succinate, and glycerol-3-phosphate, which support ETC/RC function at Complex I, II and III respectively ( FIG. 20A ). OxPhos was significantly increased in the presence of free Ca2+ ( ⁇ 220 nM) even with succinate, which oxidation is considered insensitive to Ca2+ ( FIG. 20B ). Experiments were performed using LKB buffer to facilitate a comparison of mitochondrial bioenergetics in intact and permeabilized cells under the same conditions.
  • Table 6 shows a list of respiration buffers that were compared based on effects on OxPhos capacity of HEK293 cells. HEK293 cells were used to because they show lower OxPhos capacity than respiratory capacity ( FIG. 19A , B). ADP-stimulated respiration was minimal in the LKB buffer. The maximal respiration was observed in MAS buffer ( FIG. 20C ). Compared to LKB and HKB buffers, the coupling efficiency was significantly higher in the MAS buffer (p ⁇ 0.05, FIG. 20D ).
  • the choice of Na+ vs. K+ buffer may have relatively minor effects on the determination of spare Phos/respiratory capacities in non-excitable cells such as HEK293 ( FIG. 20C , E, F) compared to excitable cells such as INS1E ( FIG. 20G , H).
  • INS1E cells while the spare OxPhos capacity was significantly higher with succinate, a Complex II substrate, significant difference were not observed on Complex I substrates glutamate+malate ( FIG. 20I ).
  • B2-MWFE cells which are CCL16-B2 cells complemented with wild type MWFE protein, showed both glutamate+malate- and succinate-supported respirations under the same conditions ( FIG. 21C ).
  • TCA cycle is the main source of NADH within mitochondria
  • Complex I-dependent respiration can also provide information about the functional status of individual NADH generating steps, and the factors affecting transport of metabolites into mitochondria.
  • Malate is a TCA cycle metabolite that regulates transport of citrate/isocitrate, ⁇ -ketoglutarate and Pi across the mitochondrial inner membrane.
  • FIG. 21F Under conditions that showed Complex I-dependent respiration in primary rat astrocytes, no significant respiration in INS1E was observed ( FIG. 21F ). This may be due at least in part to ⁇ -cell specific negative regulation of NADH metabolism within mitochondria. Such a regulation would permit ⁇ cells to rely mostly on cytosolic NADH for their bioenergetic needs in insulin secretion.
  • rPFO binding utilized ⁇ 4 mol % more cholesterol than the native PFO (nPFO).
  • D434S mutation introduced into the rPFO derivative shifted the membrane binding properties of the mutant protein such that it could be used in the context of similar cholesterol levels as for nPFO.
  • rPFO T319C-V334C the binding properties of resulting protein dbPFO D434S-C459A were similar to the native PFO.
  • rPFO T319C-V334C or dbPFO D434S-C459A mutants may be used in the absence of reducing agents such as DTT in binding assays to avoid Cys oxidation or disulfide-bond formation between proteins.
  • the reducing agent may be added after the protein is bound to the target membranes to induce pore formation.
  • the unbound protein may be removed before triggering pore formation using the rPFO T319C-V334C or dbPFO D434S-C459A mutants.
  • the fraction of bound PFO derivatives (0.1 ⁇ M final concentration) to liposomes of varying cholesterol content and POPC, POPE and SM in a constant 1:1:1 ratio (0.2 mM total lipid final concentration) was determined using intrinsic Trp fluorescence as described in experimental procedures.
  • the FIG. 27 shows the cholesterol dependent binding isotherms for various derivatives: native PFO (squares), PFO C459A (circles), PFO D434S-C459A (upward triangles) and PFO T319C-V334C-D434S-C459A (or dbPFO D434S-C459A downward triangles).
  • native PFO squares
  • PFO C459A circles
  • PFO D434S-C459A upward triangles
  • PFO T319C-V334C-D434S-C459A or dbPFO D434S-C459A downward triangles.
  • data points are the average of at least two measurements and
  • PFO Pore PFO forma- SEQ derivative Comments tion ID NO PFO (nPFO) Native or wild type protein, Yes 8 utilizes DTT for maintain- ing activity and storage rPFO Recombinant, Cys less Yes 9 (Cysteine Free) derivative of PFO (PFO C459A ); it does not require DTT; cholesterol sensitivity is reduced slightly compared to PFO rPFO D434S rPFO with D434S mutation Yes 10 which restores cholesterol sensitivity comparable to that in wild type PFO; it does not require DTT rPFO T319C-V334C Disulfide bond introduced Yes, 11 in rPFO by double mutation trig- T319C-V334C; monomeric gered binding and gets inserted by DTT in the membrane; also referred as dbPFO; cholesterol sensitivity lower than PFO dbPFO D434S-C459A rPFO T319C-V334C with Yes, 12 (may
  • a PFO may have a truncated N-terminus (e.g., a PFO without an N-terminal signal sequence, e.g., without the first 28 amino acids of SEQ ID NO: 1 or 2) or an extended N-terminus (e.g., a PFO having an N-terminal peptide tag).
  • the same amino acids may be substituted although the relative position of those amino acids from the N-terminus may be different than in the context of the sequence set forth in SEQ ID NO: 1 or 2.
  • PFO derivatives were express and purified using appropriate methods known in the art for protein expression and purification.
  • the PFO to derivative containing a native PFO sequence (amino acids 29-500 of SEQ ID NO: 2) plus the polyhistidine tag that came from the pRSETB vector (Invitrogen) is named nPFO.
  • the PFO Cys-less derivative (nPFO C459A , where Cysteine 459 is replaced by Alanine) is named rPFO. Mutagenesis of PFO was done using the QuickChange (Stratagene) procedure as described previously. Table 5 provides additional information regarding PFO derivatives.
  • Binding to liposomes was performed using the change in the Trp emission intensity produced by the binding of PFO to cholesterol containing membranes as described previously. Briefly, emission for Trp fluorescence was recorded at 348 nm (4 nm bandpass) with the excitation wavelength fixed at 295 nm (2 nm bandpass). The signal of monomeric PFO derivatives were obtained with samples containing 200 nM protein in buffer A (HEPES 50 mM, NaCl 100 mM, DTT 1 mM, EDTA 0.5 mM, pH 7.5) using 4 mm ⁇ 4 mm quartz cuvettes. The net emission intensity (F 0 ) for monomers was obtained after subtracting the signal of the sample before the protein was added.
  • buffer A HEPPS 50 mM, NaCl 100 mM, DTT 1 mM, EDTA 0.5 mM, pH 7.5
  • Liposomes were added ( ⁇ 200 ⁇ M total lipids) and the samples were incubated 20 min at 37° C. Trp emission after membrane incubation was measured after re-equilibration of the sample at 25° C., and the signal from an equivalent sample lacking the protein was subtracted (F). Fraction of protein bound was determined as (F-F 0 )/(F f -F 0 ), where F f is the emission intensity when all the protein is bound. Binding of PFO derivatives to cholesterol dispersions in aqueous solutions was done as describe previously.
  • Nonsterol lipids were obtained from Avanti Polar to Lipids (Alabaster, Ala.), and cholesterol was from Steraloids (Newport, R.I.). Large unilamellar vesicles were generated as described previously. Briefly, equimolar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and sphingomyelin (SM, porcine brain), were combined with the indicated amount of cholesterol (5-cholesten-3 ⁇ -ol) in chloroform. The thin film of lipids formed after chloroform evaporation was resuspended in buffer A and passed through an extruder equipped with 0.1 ⁇ m filter 21 times. Liposomes were stored in ice and discarded after three weeks.
  • POPC 1-palmitoyl-2-oleoyl-sn-glycer
  • cysteine-free derivatives of PFO were produced and evaluated. Like native PFO, these derivatives can permeabilize mammalian cells. In some embodiments, it was found that the derivatives can permeabilize cells as efficiently as the native PFO. The cholesterol sensitivity of these derivatives was assessed using liposomes as artificial membranes. Side-by side comparisons of cysteine-free PFO derivatives show comparable or relatively better performance than native PFO.
  • FIG. 28 shows the relative performance of nPFO, rPFO and rPFO D434S in mitochondrial function assays using human HEK293 cells.
  • rPFO and rPFO D434S were comparable, and maintained their activity in the absence of added reducing agents.
  • a visual inspection under microscope indicated that the overall cellular morphology following permeabilization with rPFO D434S was better preserved compared to other derivatives under the conditions tested. Swelling of cytoplasm/cell ghosts was relatively low after permeabilization with rPFO D434S . In some embodiments, therefore, rPFO D434S is advantageous for general use.
  • rPFO is advantageous for permeabilization of cells because it has a relatively lower cholesterol sensitivity than other PFO derivatives.
  • cholesterol content in certain mammalian cells may rise under certain pathophysiological conditions (e.g., metabolic syndrome), in such cases cholesterol can accumulate in mitochondria, which may compromise mitochondrial function if the PFO is left in the assay medium.
  • pathophysiological conditions e.g., metabolic syndrome
  • rPFO has a relatively low cholesterol sensitivity it may be advantageous for cell permeablization of such cells. Derivatives of PFO that are conditionally active have been developed.
  • the derivatives of PFO are triggered to form pores in plasma membranes by ⁇ 50 nM DTT.
  • DTT at concentrations of approximately 50 nM or less does not significantly interfere with cell function when used with these derivatives.
  • These derivatives are useful in cells having high levels of cholesterol. In such cases, removing excess PFO before permeabilization can preserve mitochondrial function after permeabilization.
  • rPFO T319C-V334C and dbPFO D434S-C459 are useful for cell permeabilization because they can be removed before inducing pore formation with ⁇ 50 nM DTT. These are concentrations of DTT that are not expected to affect mitochondrial function significantly.
  • dbPFO D434S-C459A is functionally comparable rPFO T319C-V334C .
  • Assays for assessing mitochondrial function that utilize digitonin for cell permeabilization are, in some embodiments, limited because (i) observed effects of digitonin on cell permeabilization are concentration dependent, (ii) observed effects of digitonin are influenced by buffer composition, (iii) stable respiration is often not obtained even after careful titration of digitonin, and (iv) observed effects of digitonin have low reproducibility due to limited dynamic range of the digitonin concentration (see FIG. 29 ).
  • relatively high concentrations of digitonin can damage mitochondrial membranes and release Cytochrome c which is important for respiratory chain function.
  • PFO derivatives overcome these limitations of digitonin PFO is useful as a permeabilizing agent, in part, because it can be handled with high precision facilitating reliable and reproducible assays.
  • PFO derivatives produce reproducible results within a concentration range of 0.1-20 nM without significant difference in the experimental output (compare FIG. 28 and FIG. 29 ).
  • respiratory activity of cells permeabilized with PFO in contrast with digitonin is stable in all the buffers tested (See Example 8).
  • FIG. 20 shows that buffer choice can affect the oxidative phosphorylation (OxPhos) capacity of cells, which was maximal in MAS buffer.
  • OxPhos oxidative phosphorylation
  • FIG. 31A shows the dose-dependent inhibition of the cellular respiration. While in low phosphate LKB buffer (0.4 mM Pi), the ADP-stimulated respiration was not apparent ( FIG. 31A ), ADP-stimulated respiration was observed in high phosphate LBPT buffer (10 mM Pi). ADP-stimulated respiration was inhibited by rotenone dose-dependently ( FIG. 31D ). Complex I inhibition was measured in the presence of 3 ⁇ M FCCP using the respiration supported by glutamate+malate ( FIG. 31A , D). It was recognized that with increasing Complex I inhibition by rotenone, Complex II-dependent respiration was also significantly inhibited in LBPT buffer ( FIG. 31D , E) and was minimal in LKB buffer (FIG. 31 A,B).
  • MEFs Mouse embryonic fibroblasts (MEFs) derived from a Complex I deficient mouse model (Ndufal S55A) were used to assess the extent to which genetically encoded partial respiratory chain deficiencies could be detected using PFO-based assays. Intact MEFs from the mutant mice showed reduced cellular respiration on glucose+pyruvate or pyruvate alone ( FIG. 32A , B). Observed differences in pyruvate containing respiration buffer confirmed the deficiency in oxidative metabolism, and indicated lack of impaired glycolysis.
  • the Ca2+-free buffers contained no added CaCl 2
  • the regular LKB contained 1.3 mM CaCl2 and no added EGTA.
  • Glucose was added at indicated concentrations in the respiration buffers as described in the text and/or figure legends for specific assays.

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