A study of rapid mitochondrial structural changes in vitro by spray-freeze-etching

Abstract

The spray-freeze-etching technique has been used to study energy-linked mitochondrial structural changes in rat liver mitochondria incubated in vitro. The technique involves spraying the suspension of mitochondria into liquid propane at -190~and does not require the use of cryoprotectants or chemical fixatives. The results confirmed that freshly isolated mitochondria have a condensed matrix and that this expands at the expense of the outer compartment to give the orthodox configuration when the mitochondria are incubated in a K + medium in the presence of substrate and phosphate. Addition of adenosine diphosphate (ADP) caused a rapid shrinkage of the matrix compartment, and the time-course and extent of this shrinkage has been measured quantitatively by coupling a rapid sampling device to the spray-freezing apparatus. These data show that for orthodox mitochondria the onset of phosphorylation is accompanied by a reduction of 30% in the matrix volume in 20 s, and there is no evidence that the decrease in matrical volume affects the phosphorylation efficiency. These results suggest that natural ionophores in the mitochondrial inner membrane make it permeable enough to permit a rapid readjustment of matrix volume after the addition of ADP, and that the associated ion movement does not cause uncoupling of oxidative phosphorylation.KEY WORDS spray-freeze-etching 9 mitochondrial structural changes mitochondria 9 oxidative phosphorylation valinomycin effects mitochondrial ion movements Energy-linked mitochondrial structural changes occurring in vitro were first examined in the electron microscope by Hackenbrock (7, 8) and Green et al. (6). Following the work of Packer et al. (23)~ there has been fairly general acceptance that there is a qualitative correlation between changes in the volume of the mitochondrial matrix observed with the electron microscope and changes in light scattering by mitochondrial suspensions. More recently, Hackenbrock (9, 10) and others (28, 29) have used standard freezeetching techniques to show that the volume of the mitochondrial matrix can vary as a consequence of alterations in the extent to which the mitochondria are energized. However, these results were obtained in the presence of relatively high concentrations of cryoprotectants to prevent ice crystal damage on freezing, and Vail et al. (29) found that glutaraidehyde fixation was necessary to prevent structural damage caused by glycerol when it was used as a cryoprotectant. Hochli and Hackenbrock (11) claim that, if properly used, glycerol will not affect phosphorylation efficiency or damage mitochondrial structure, but the latter point has been disputed by Neidermeyer and Moor (22).The development of spray-freeze-etching by Bachmann and Schmitt (1) in 1971 suggested that it might be possible to examine changes in mitochondrial structure by spray-freezing without the use of fixatives or cryoprotectants. This new tech- 134J. CELL BIOLOGY 9 The Rockefeller University Press 9

Preparation of cryofixed cells for improved 3D ultrastructure with scanning transmission electron tomography

“…1). This structure is diVerent from the generally accepted model with a wide intermembrane space (Sun et al 2007;Rabl et al 2009), but in agreement with old cryoWxation and freeze fracturing studies of mitochondria (Lang and Bronk 1978;Knoll and Brdiczka 1983), where it is stated that the membranes in "energized" mitochondria are in close apposition, whereas they separate when the mitochondria are "de-energized". It also Wts well with our earlier studies using freeze substitution (Walther and Ziegler 2002) as well as using high-pressure freezing and freeze fracturing (Walther et al 2009).…”

K+-dependent regulation of matrix volume improves mitochondrial function under conditions mimicking ischemia-reperfusion

“…In sucrose buffer, isolated mitochondria are known to have a contracted matrix and expanded intermembrane space (12,15,28). It is also known that increase in external osmolarity from 210 to 310 mosM (using sucrose) results in parallel decreases of mitochondrial O 2 consumption in state 3 (7).…”

“…We used light scattering to track matrix volume changes, based on previous observations that light scattering correlates with matrix volume shifts with no obvious effect on total mitochondrial volume (15,28). In the original studies, a single 250 M ADP pulse was added to a relatively large amount of mitochondria (Ͼ0.5 mg/ml), and ADP-induced changes were determined in parallel by electron microscopy and correlated with light scattering.…”

A histological study of mineralised tissue formation around implants with 3D culture of HMS0014 cells in Cellmatrix Type I-A collagen gel scaffold <i>in vitro</i>

“…A previous study has elucidated that mitochondria with matrix of slow metabolite diffusion rate in the condensed state were dominant in a tissue with high level of oxidative phosphorylation 55) . However, it has been noted that the onset of phosphorylation was accompanied by a reduction of the matrix volume of orthodox mitochondria, but there is no evidence that the decrease in matrix volume affected the phosphorylation efficiency 56) . Nevertheless, other studies have reported that volume-dependent regulation of matrix protein packing may modulate metabolite diffusion and, in turn, mitochondrial metabolism of cells [57][58][59] .…”