Marcelle Lefort-Tran

Fixation of phycobiliproteins to photosynthetic membranes by glutaraldehyde

SummaryTreatment of intact cells of two unicellular red algae (Porphyridium spp.) and of an unicellular blue-green alga (Gloeocapsa alpicola) with glutaraldehyde (3% v/v) has only a slight effect on the absorption spectra of their photopigment systems, but stabilizes the normally labile physical association between phycobili-proteins and the chlorophyll-bearing thylacoids. On subsequent breakage of such cells, a substantial fraction of the total phycobiliprotein (30–70%) sediments in a sucrose gradient, in association with the chlorophyll-containing membrane fragments, from which it cannot be dissociated by treatment with detergents.

Aggregation and proton release of purple and white membranes following cleavage of the carboxyl-terminal tail of bacteriorhodopsin.

Our results indicate that the previously reported decrease in proton release by proteolyzed purple membrane sheets was due merely to the aggregation state of these preparations and not to the loss of the carboxyl-terminal tail. Changes in H+/M412 ratios obtained for purple and white membrane preparations correlate with the measured aggregation. White membrane preparations consistently exhibit H+/M412 ratios more than twice those measured for native purple membranes under the same conditions. Quasi-elastic light scattering was used to characterize the size of isolated purple and white membrane sheets before and after proteolysis. The results clearly show that native purple membrane preparations are larger in size than would be expected and that, following trypsin treatment, they are on average more than an order of magnitude larger. Negative staining electron microscopy showed that the purple membrane became aggregated in stacked arrays. Bleaching and reconstitution with retinal also affect aggregation, but iodination or nitration of purple membrane does not affect the measured size. The average size of white membranes is smaller; this is consistent with results of electron microscopy and the size increase is much less than that of purple membranes following trypsin treatment. No size change occurs with retinal reconstitution. In aggregated purple membrane preparations, protons and other cations are unable to exchange freely with the aqueous medium, explaining why proteolysis lowers the proton release from purple membrane sheets in suspension.

Freeze-fracture study of ordered arrays of particles in the plasma membrane ofChlamydobotrys stellata Korsch. (Volvocales)

SummaryUltrarapid cyrofixation procedures revealed the existence of ordered arrays of intramembrane particles on E fracture faces and corresponding ordered imprints on P faces in freeze-fractured plasma membrane of the green algaeChlamydobotrys stellata (Korschikoff). The structure of these arrays is very sensitive to cryofixation conditions and particularly to glutaraldehyde prefixation which leads to the formation of amorphous two-dimensional aggregates. The size of the individual ordered arrays and the ratio of ordered to total surface of the membrane increase with growth temperature from 15°C to 30°C with a corresponding decrease in cell generation time. Above 30°C the size of the individual ordered arrays decreases. At high, but sublethal temperature (above 37°C) the ordered arrays become smaller. In addition to the predominant two-dimensional oblique organization (a=12.0nm, b=12.6nm, γ=80°), square and tetragonal arrangements are also present. The cell wall is composed of many layers, one of which displays a “zipper-like structure” composed of periodic ridges 25 nm distant, sandwiched between two more or less fibrillar layers. The appearance and changes of the organization of ordered arrays are discussed in relation to their eventual physiological role during the life cycle of the cells and in particular to the formation of the cell wall and the median periodic leaflet.

Modulation of fibroblast motility by a cytosolic extract of cyanobacteria

Proliferation and migratory behavior of L929 murine fibroblasts were shown to be modified in the presence of a cytosolic extract of Phormidium sp. (Cyanobacteria). The addition of Phormidium extract to the growth medium (Dulbeccos modified Eagles medium) supplemented with 0.5% newborn calf serum increased cell proliferation. The effect was shown to be cell line specific. A quantitative analysis performed according to De Laat, Tertoolen, and Bluemink (1981, Eur. J. Cell Biol., 23, 273-279), showed that Phormidium extract was a potential aggregative effector for fibroblasts. Heating (100 degrees C, 4 min) inactivated the clustering effect of the extract, but the effect on cell proliferation was retained. A video analysis of cells after divisions showed that the extract activated cell migration in the same way as 5% serum did during the first 24 h of treatment. Between 24 and 48 h of treatment, cell migration in the presence of the extract was inhibited when compared to migration in 0.5 or 5% serum. We have shown that Phormidium extract may contain two or three kinds of effectors which acted as exogenous growth factors (allowing attachment and proliferation) and as modulator(s) of the cell migratory behavior (activator of migration in early times of the growth and inhibitor later).

Stacking of Purple Membranes in Vitro

Negative staining electron microscopy showed that purple membranes isolated from Halobacterium halobium are aggregated in vitro in the form of stacked arrays. This effect is more marked after trypsin treatment. White membranes isolated from mutant strains do not stack and exhibit an average size consistent with previous results of electron microscopy. White membrane fragments also do not exhibit stacking in vitro after retinal reconstitution or trypsin treatment. Quasi-elastic light scattering was also used to characterize the size (hydrodynamic radius) of isolated purple and white membranes before and after proteolysis. These results also show that native purple membrane preparations are larger in size than expected and that, following trypsin treatment, they are on average more than an order of magnitude larger. In stacked purple preparations, cations are unable to exchange freely with the aqueous medium. This explains why proteolysis lowers the efficiency of proton release by illuminated bacteriorhodopsin in purple membranes in vitro. Thus, previously reported decreases in efficiency of proton release by bacteriorhodopsin in proteolyzed purple membranes are due to the stacking effect and not per se to loss of the carboxyl terminus tail.