This review focuses on our studies over the past ten years which reveal that the mitochondrial inner membrane is a fluid-state rather than a solid-state membrane and that all membrane proteins and redox components which catalyze electron transport and ATP synthesis are in constant and independent diffusional motion. The studies reviewed represent the experimental basis for the random collision model of electron transport. We present five fundamental postulates upon which the random collision model of mitochondrial electron transport is founded: All redox components are independent lateral diffusants; Cytochrome c diffuses primarily in three dimensions; Electron transport is a diffusion-coupled kinetic process; Electron transport is a multicollisional, obstructed, long-range diffusional process; The rates of diffusion of the redox components have a direct influence on the overall kinetic process of electron transport and can be rate limiting, as in diffusion control. The experimental rationales and the results obtained in testing each of the five postulates of the random collision model are presented. In addition, we offer the basic concepts, criteria and experimental strategies that we believe are essential in considering the significance of the relationship between diffusion and electron transport. Finally, we critically explore and assess other contemporary studies on the diffusion of inner membrane components related to electron transport including studies on: rotational diffusion, immobile fractions, complex formation, dynamic aggregates, and rates of diffusion. Review of all available data confirms the random collision model and no data appear to exist that contravene it. It is concluded that mitochondrial electron transport is a diffusion-based random collision process and that diffusion has an integral and controlling affect on electron transport.

INTRODUCTION Membrane-potential-dependent dyes such as Rhodamine 123, tetramethylrhodamine methyl ester (TMRM), and tetramethylrhodamine ethyl ester (TMRE) are useful as long as the mitochondrion maintains its negative membrane potential. MitoTracker is a commercially available fluorescent dye (Invitrogen/Molecular Probes) that, like the aforementioned dyes, labels mitochondria within live cells utilizing the mitochondrial membrane potential. However, MitoTracker is chemically reactive, linking to thiol groups in the mitochondria. The dye becomes permanently bound to the mitochondria, and thus remains after the cell dies or is fixed. In addition, it can be used in experiments in which multiple labeling diminishes mitochondrial function. This protocol describes the labeling of mitochondria in live and fixed cells with MitoTracker dyes.

INTRODUCTIONA number of fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells, including Hoechst, 4,6-diamidino-2-phenylindole (DAPI), ethidium bromide, propidium iodide, and acridine orange. Although not as bright as the vital Hoechst stains for DNA, DAPI has greater photostability. It is believed that DAPI associates with the minor groove of double-stranded DNA, with a preference for the adenine-thymine clusters. Cells must be permeabilized and/or fixed for DAPI to enter the cell and to bind DNA. Fluorescence increases approximately 20-fold when DAPI is bound to double-stranded DNA. This protocol describes the use of DAPI to label nuclear DNA of cells grown in culture. RELATED INFORMATIONAutofluorescence from endogenous cellular molecules such as the reduced forms of nicotinamide adenine dinucleotide or flavin adenine dinucleotide can interfere with imaging by reducing the signal-to-noise ratio. This occurs when the excitation and/or emission wavelengths of the probe and the autofluorescing molecules are similar, e.g., frequently with excitation wavelengths <500 nm, and particularly at ultraviolet wavelengths. Autofluorescence can be reduced by careful selection of the excitation and the emission wavelengths used, by treating fixed cells with reducing agents (e.g., a 1% solution of sodium borohydride [NaBH 4 ] for 20 min), and by comparing the experimental images with unlabeled control slides. Avoid fixation with glutaraldehyde, because it can increase interference from cellular autofluorescence, most frequently at wavelengths <500 nm.This protocol assumes that the cells of interest were grown on glass microscope coverslips immersed in small Petri dishes containing culture medium. Generally, labeling conditions vary by cell type, and it might be necessary to alter the protocol for a particular use. To mount cells labeled using the technique described here, see Mounting Live Cells onto Microscope Slides (Chazotte 2011). MATERIALS CAUTIONS AND RECIPES:Please see the end of this protocol for appropriate handling of materials marked with , and recipes for reagents marked with . ReagentsCells of interest, grown on coverslips DAPI (10 mg/mL in H 2 O stock solution; Invitrogen D1306)

INTRODUCTIONThe LysoTracker probes (Invitrogen/Molecular Probes) are used to label lysosomes in live cells, as described in this protocol. They are more selective for acidic organelles than the classically used neutral red or acridine orange dyes. They are freely membrane permeant at neutral pH and effectively label lysosomes at nanomolar concentrations, with ~50 nM being optimal for selectivity. It has been reported that in larger acidic compartments, the staining pattern is preserved even after fixation with aldehydes. LysoTracker dyes can be visualized at different wavelengths, so they are useful when multiple fluorescent dyes are used to label cells simultaneously.

JC-1 (5,5',6,6'-tetrachloro1,1',3,3'-tetramethylbenzimidazolylcarbocyanine iodide) dye has been used to monitor mitochondrial potential. The monomeric form has an emission maximum of ∼529 nm. The dye at higher concentrations or potentials forms red fluorescent J-aggregates with an emission maximum at 590 nm. The ratio of this green/red fluorescence is independent of mitochondrial shape, density, or size, but depends only on the membrane potential. It has been used to study whether all mitochondria in the same cell are at the same potential and whether membrane potential in a single long mitochondrion is uniform. JC-1 has also been useful in flow-cytometry studies, because the membrane potential can be followed without the need for confocal microscopy. This protocol describes the labeling of mitochondria in cultured cells with JC-1.