Methods, compositions and kits for assaying mitochondrial function

Abstract

The invention provides methods, compositions, devices, and kits relating to the use of cholesterol-dependent cytolysins (e.g., PFOs) for measuring intracellular mitochondrial activity.

Description

RELATED APPLICATION[0001]This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No. 61 / 473,730, filed Apr. 8, 2011, and entitled “Methods, Compositions and Kits for Assaying Mitochondrial Function,” which is incorporated herein by reference in its entirety for all purposes.GOVERNMENT SUPPORT[0002]This work was supported under grant number 5R21NS057224-03, awarded by National Institutes of Health (NIH). The U.S. Government has certain rights in the invention.FIELD OF THE INVENTION[0003]The invention relates to methods for evaluating intracellular functions as well as cell health and viability.BACKGROUND OF THE INVENTION[0004]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 a...

AI Technical Summary

Benefits of technology

[0007]Aspects of the invention relate to methods, compositions, and devices that are useful for measuring mitochondrial functions accurately and reproducibly under intracellular conditions. In some embodiments, 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. In some embodiments, cholesterol-dependent cytolysins (CDCs) (e.g., perfringolysin O (PFO)) are used to permeabilize plasma membranes and not internal cellular membranes such as mitochondrial membranes. 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.

[0009]Cholesterol-dependent cytolysin-based (e.g., PFO-based) methods provide effective permeabilization of cellular membranes without disrupting the mitochondrial membranes. Thus, 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. Moreover, cholesterol-dependent cytolysins are surprisingly effective at creating conditions suitable for measuring mitochondrial activity in cells. In some embodiments, 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.

[0010]Therefore, 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). Accordingly, cholesterol-dependent cytolysin-based (e.g., PFO-based) assay results can provide an accurate assessment of mitochondrial activity in a natural cellular environment. In some embodiments, 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.

[0011]In some embodiments, 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. In some embodiments, cholesterol-dependent cytolysins (e.g., PFOs) facilitate the use of exogenous agents such as dyes, fluorescent proteins, markers or other agents to probe one or more intracellular functions. For example, cholesterol-dependent cytolysin-based (e.g., PFOs) 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. In some embodiments, cholesterol-dependent cytolysin-based (e.g., PFOs) may be used to facilitate transfection of cells with exogenous nucleic acids (e.g., expression vectors).

[0014]In some embodiments, 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. In some embodiments, 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.

Problems solved by technology

Detergents, for example, 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.

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