Absolute optical cross sections of cells and chloroplasts

Mitochondria are the powerhouses of eukaryotic cells as they feed metabolism with its major substrate. Oxidative-phosphorylation relies on the generation, by an electron/proton transfer chain, of an electrochemical transmembrane potential utilized to synthesize ATP. Although these fundamental principles are not a matter of debate, the emerging picture of the respiratory chain diverges from the linear and fluid scheme. Indeed, a growing number of pieces of evidence point to membrane compartments that possibly restrict the diffusion of electron carriers, and to supramolecular assembly of various complexes within various kinds of supercomplexes that modulate the thermodynamic and kinetic properties of the components of the chain. Here, we describe a method that allows the unprecedented time-resolved study of the respiratory chain in intact cells that is aimed at assessing these hypotheses. We show that, in yeast, cytochrome c is not trapped within supercomplexes and encounters no particular restriction to its diffusion which questions the functional relevance of these supramolecular edifices.bioenergetics | electon transfer | respiration | compartmentalization

Section: Resultsmentioning

confidence: 99%

Questioning the functional relevance of mitochondrial supercomplexes by time-resolved analysis of the respiratory chain

Trouillard

Meunier

Rappaport

2011

“…This means that the experimental scattering spectra may contain more than one component. No improved fitting could be expected either, by varying the particle parameters (i.e., by taking chloroplasts as random ellipsoids instead of spheres), because selective scattering of chloroplasts has been shown practically not to be affected by the particle shape (21).…”

Section: Resultsmentioning

confidence: 99%

Selective scattering spectra as an approach to internal structure of granal and agranal chloroplasts.

Bialek¹,

Horváth²,

Garab³

et al. 1977

Selective scattering spectra of granal and agranal chloroplasts were measured in the red spectral region and compared with calculations based on the Mie theory. The spectra were influenced considerably by the intactness and ultrastructural pattern of the chloroplasts. It was demonstrated that the spectra consist of two components: one attributable to grana and the other, to single lamellae. The dependence of the selective scattering spectra on the ultrastructural characteristics offers a convenient method for monitoring the quality of chloroplast preparations by a procedure much faster than electron microscopy. Selective scattering spectra of algal cells such as Chlorella and Anacystis, as well as of spinach chloroplasts and fragments, have been reported in the literature (1)(2)(3)(4)(5)(6)(7)(8). In these studies the photosynthetic lamellar system was considered as a whole with no regard to the ultrastructural pattern characteristic of the chloroplasts. As shown under the electron microscope, chloroplasts consist of two types of lamellae: single lamellae that appear never to fuse with each other, and stacked lamellae that build up the highly ordered piles called "grana." The individual contributions of single and stacked lamellae in the selective scattering spectra have not been distinguished in previous reports.The aim of the present work was to study the selective scattering spectra of chloroplasts containing various amounts of grana and to compare intact class A and class B chloroplasts (9) in order to obtain information on the relationship between ultrastructure and selective scattering. Selective spectra of chloroplasts were shown to be dependent on their intactness and ultrastructure. This phenomenon offers a convenient method for checking the quality of chloroplast preparations. MATERIALS AND METHODSChloroplast preparations with high and low granum contents were obtained from maize leaves (Zea mays L. var. MV SC 660). The plants were grown in a greenhouse for 9-l1 days.Class A chloroplasts were isolated from a suspension of mesophyll protoplasts (10) or bundle sheath cells. Mesophyll protoplasts were prepared in a digestion medium containing 0.6 M d-sorbitol, 0.1 M potassium phosphate, 0.5% (wt/vol) Macerozyme R-10, 2.0% (wt/vol) cellulase Onozuka R-10, and 0.5% (wt/vol) potassium dextran sulfate, pH 5.8. Bundle sheath cells were separated in a maceration medium containing 0.4 M d-sorbitol, 0.1 M potassium phosphate, 0.5% Macerozyme R-10, 1.0% cellulase Onozuka R-10, and 1.0% Helicase, pH 5.8. Macerozyme and cellulase preparations were purchased from Kinki Yakult MFG Co. Ltd., Nishinomiya, Japan; the Helicase was obtained from Industrie Biologique Frangaise, Gennevilliers, France; and potassium dextran sulfate was from Meito Sangyo Co. Ltd., Nagoya, Japan. The incubation was for 90 min at 37°. Protoplasts and cells were washed three times in 0.6 M d-sorbitol and transferred to the medium of Anderson and Boardman (11) containing 0.3 M sucrose, 0.05 M potassium phosphate, and 0.01 M potassium chlor...

“…Large particles of the size of chloroplasts scatter predominantly in the forward direction (Mie scattering) (27) and with a small angular profile (cf. [28][29][30]. However, with chloroplasts we must also consider scattering from structures within the chloroplast, particularly the grana thylakoids (0.3 gum in diameter) where the pigments are predominantly localized, and particles within the thylakoid membranes.…”

Section: Dissymmetry Ratiomentioning

confidence: 99%

Light-dependent absorption and selective scattering changes at 518 nm in chloroplast thylakoid membranes.

Thorne

Horváth

Kahn

et al. 1975

The light-induced absorbance change at 518 nm of isolated chloroplasts consists of a rapid phase, and a slow phase which is complete in about 20 sec. The slow component of the 518 nm absorbance change correlates with the light-induced change in 900 light scattering at 518 nm. Both show a similar time course, similar pH dependence with a maximum at pH 6.0, and similar sensitivity to inhibitors and to treatment of the chloroplasts with a low concentration of glutaraldehyde. Their light minus dark difference spectra are similar with maxima at about 520 nm. It is concluded that they are manifestations of the same phenomenon, and the slow absorbance increase at 518 nm is due to enhanced scattering. It is proposed that the light-induced changes in scattering at 518 nm reflect alterations in selective ispersion, due to proton uptake and conformational changes in the chloroplast thylakoid membrane.Isolated chloroplasts exhibit reversible light-dependent structural changes, which can be monitored by electron microscopy and by changes in packed volume, transmittance, and 900 light scattering (1-8). Similar changes have also been observed with chloroplasts in idvo (5,(9)(10)(11). Electron microscopy has demonstrated a thinning of the chloroplast thylakoid membrane on illumination (4). The light-induced volume decrease (shrinkage) of chloroplasts and the associated increases in absorbance and 900 light scattering are dependent on photosynthetic electron flow. They are inhibited by uncouplers of photophosphorylation (5, 12). The increase in light scattering parallels the proton uptake which is observed on illumination of chloroplasts (13). It was generally supposed that the increase in light scattering reflected the decrease in chloroplast volume, but further studies (14) showed that this was not the case when chloride was the only anion present in the medium. It was suggested that scattering was influenced by protonation and an increased refractive index of the chloroplast thylakoid system (14), or by changes in the optical properties of the stacked thylakoids (7).In the work described in this paper, we examined lightinduced 90°scattering by isolated chloroplasts in relation to the light-induced absorbance change at 518 nm. It appears that the slow kinetic component of the absorbance increase is caused by an increase in selective scattering which is dependent on the refractive index difference between the chromophore-containing particles of the chloroplast and their environment. MATERIALS AND METHODS Intact chloroplasts were isolated from hydroponically grown spinach plants, essentially by the method of Reeves and Hall (15). Detached leaves (20 g) were preilluminated for 15 min at 40C in white light (16) and ground in a Sorvall Omnimixer for 4 sec at 90% of line voltage (230 V) in a medium containing 0.33 M sorbitol, 10 mM NaCl, 5 MM MgCl2, 1 mM MnCl2, 2 mM EDTA, 2 mM isoascorbate, 0.4% bovine serum albumin, and 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes) buffer, pH 6.5. The brei was filtered through four layer...