Fusion of small unilamellar vesicles with viable EDTA-treated Escherichia coli cells.

Fusion characteristics of EDTA-treated Escherichia coli cells with small unilamellar vesicles were investigated, using a membrane fusion assay based on resonance energy transfer. Ca2+-EDTA treatments of Escherichia coli O111:B4 (wild type), E. coli C600 (rough), and E. coli D21f2 (deep rough) which permeabilize the outer membrane by inducing the release of lipopolysaccharide and outer membrane proteins resulted in fusion activity of the intact and viable bacteria with small unilamellar vesicles. No fusion activity was observed when the EDTA treatment was omitted. Fusion could be elicited at low pH and by a combination of a higher pH and Ca2+. The low-pH-induced fusion was composed of a fast and a slow reaction. The latter and the Ca2+-induced fusion could be completely inhibited by trypsin treatments of the EDTA-treated cells, which also resulted in the simultaneous disappearance of two outer membrane protein bands (50 and 58 kilodaltons) and the appearance of proteins banding at 22, 52, and 54 kilodaltons. The most efficient fusion was obtained with negatively charged liposomes composed of cardiolipin. In contrast to the Ca2+-induced fusion, fusion was observed at low pH with small unilamellar vesicles containing lipids with decreased negative charge (phosphatidylserine). Fluorescent and phase-contrast microscopy revealed that essentially all bacteria were engaged in fusion. We propose that a Ca2+-EDTA treatment of E. coli cells results in the appearance of phospholipids and the exposure of a protein(s) in the outer leaflet of the outer membrane, both of which could mediate fusion with liposomes.     

Why do organisms produce gametes of only two different sizes? Some theoretical aspects of the evolution of anisogamy

This paper extends the population genetic model of (the evolution of) anisogamy of Charlesworth (1978), which is based on the model of Parker, Baker & Smith (1972). The effect of parthenogenesis on the evolution of anisogamy is examined; this effect turns out to be only quantitative. Furthermore, the problem of the occurrence of only two different gamete sizes is considered. It is shown that a stable polymorphism with three different gamete sizes cannot exist. This result is robust to changes in the mating structure (random or disassortative gamete fusion) and to changes in the mode of reproduction (only sexual or partially parthenogenetic).     

Phase transitions and permeability changes in dry membranes during rehydration

Dry phospholipid bilayers are known to undergo transient changes in permeability during rehydration. In this review, we present evidence from which we suggest that this permeability change is due to a gel to liquid-crystaline phase transition accompanying rehydration. If the transition is avoided, as in lipids that remain in gel phase whether dry or rehydrated, the problem of leakage during rehydration is obviated, at least in part. Further, the evidence that the transition temperature for dry bilayers can be depressed by certain sugars is discussed. Finally, we show that these principles can be extended to intact cells. Using pollen grains as a model, we have measured the transition temperature for membrane phospholipids and show that the transition is correlated with physiological measurements including permeability changes and subsequent germination. From theT _m values taken from pollen grains at different water contents, we have constructed a phase diagram for the intact pollen that has high predictive value for physiological properties.     

Ca2+-independent,protein-mediated fusion of chromaffin granule ghosts with liposomes

We have investigated the interaction between isolated membrane vesicles from chromaffin granules and large unilamellar phospholipid vesicles (liposomes). Mixing of membrane lipids has been monitored continuously, utilizing the fluorescence resonance energy transfer assay described by Struck et al. ((1982) Biochemistry 20, 4093–4099). To demonstrate coalescence of the internal vesicle volumes the transfer of colloidal gold from the liposomes to the interior of the granule membrane vesicles has been examined. Efficient fusion of the liposomes with the granule membranes was observed. Significant fusion occurred in the absence of Ca²⁺, although the extent of interaction was enhanced in its presence. The sensitivity of the interaction to pretreatment of the granule membranes with trypsin showed the fusion reaction to be a protein-mediated process.     

Immunocytochemical analysis of P-fimbrial structure: localization of minor subunits and the influence of the minor subunit FsoE on the biogenesis of the adhesin

Antibodies recognizing the non-adhesive minor P-fimbral subunit protein E and the P-fimbrial adhesin were used in an immunocytochemical analysis of P-fimbrial structure. It was demonstrated that P-fimbriae of the serotypes F71, F72 and F11 carry their adhesin in a complex with protein E. These complexes are commonly found at the tip of the fimbrial structure. In P-fimbriae of serotype F9, expressed by the uropathogenic Escherichia coli strain 21086, adhesin-protein E complexes are localized at the tips as well as along the shafts of the fimbriae. Protein E of F71 fimbriae (FsoE) plays a catalysing role in the biogenesis of the adhesin, but has no effect on the eventual localization of the adhesin.     

Relationship between the proteins encoded by the exclusion determining locus of the IncI plasmid R144 and the cellular localization of these proteins in Escherichia coli K-12

Summary A region of the IncI plasmid R144, determining and controlling exclusion (exc), codes for two proteins, designated 13K and 19K after their apparent molecular weights (respectively 13,000 and 19,000). Both proteins were simultaneously affected by various mutations that resulted in exclusion deficiency. In this paper the relationship between these proteins as well as their cellular location is reported. We found no indications that the 19K protein is a precursor form of the 13K protein. Analysis of gene products of recombinant plasmids carrying exc as well as of several derivatives, however, provided a strong indication that the proteins result from overlapping genes. Besides, evidence was obtained that the 19K protein is essential for exclusion. Localization studies revealed that this protein exists in a membrane-bound form, associated at the periplasmic side of the inner membrane, and in a soluble form residing in the cytoplasm.     

Properties of proteins and the glassy matrix in maturation-defective mutant seeds of Arabidopsis thaliana

In situ Fourier transform infrared microspectroscopy was used to study the heat stability of proteins and hydrogen bonding interactions in dry maturation-defective mutant seeds of Arabidopsis thaliana . α-Helical, turn and β-sheet conformations were the major protein secondary structures in all of these seeds. On heating, intermolecular extended β-sheet structures, typical of protein denaturation, were formed in abscisic acid-insensitive ( abi3 ) and leafy cotyledon ( lec ) mutant seeds. Proteins in dry wild-type seeds did not denature up to 150°C, but those in dry desiccation-sensitive, lec1–1 , lec1–3 and abi3–5 seeds did at 68, 89 and 87°C, respectively. In the desiccation-tolerant abi3–7 and abi3–1 seeds, denaturation commenced above 120 and 135°C, respectively. Seeds of the aba1–1 abi3–1 double mutant showed signs of denaturation already upon drying. The molecular packing in the seeds was studied by observing the shift in the position of the OH-stretching vibration band with temperature. The maximal rate of change of this band with temperature was much higher in the desiccation-sensitive abi3–5 , aba1–1 abi3–1 , lec1–1 , and lec1–3 mutant seeds than in the desiccation-tolerant wild-type, abi3–1 , abi3–7 , and lec2–1 seeds. We interpret this to mean that the molecular packing density is higher in dry desiccation-tolerant than in dry desiccation-sensitive seeds, which is associated with a higher or lower protein denaturation temperature, respectively. The results are discussed in relation to the physiological and biochemical characteristics of these mutant seeds.     

Meiotic recombination in sexual diploid and apomictic triploid dandelions (Taraxacum officinale L.).

Taraxacum officinale L. (dandelion) is a vigorous weed in Europe with diploid sexual populations in the southern regions and partially overlapping populations of diploid sexuals and triploid or tetraploid apomicts in the central and northern regions. Previous studies have demonstrated unexpectedly high levels of genetic variation in the apomictic populations, suggesting the occurrence of genetic segregation in the apomicts and (or) hybridization between sexual and apomictic individuals. In this study we analysed meiosis in both sexual diploid and apomictic triploid plants to find mechanisms that could account for the high levels of genetic variation in the apomicts. Microscopic study of microsporocytes in the triploid apomicts revealed that the levels of chromosome pairing and chiasma formation at meiotic prophase I were lower than in that of the sexual diploids, but still sufficient to assume recombination between the homologues. Nomarski DIC (differential interference contrast) microscopy of optically cleared megasporocytes in the apomicts demonstrated incidental formation of tetrads, which suggests that hybridization can occur in triploid apomicts.     

Effect of aniosmotic media on the volume of the T-lymphocyte nucleus.

Time-resolved measurements of the nuclear volume response of human peripheral T-lymphocytes, under aniosmotic conditions, are presented. In the experiments slit scanning FlowCytometry methods were used. We propose an extension to the standard solid viscoelastic model to interpret the observed dynamical behavior of the nucleus. It is shown that experimental and theoretical evidence indicates a passive nuclear response merely induced by a mechanical link (i.e., the cytoskeleton) between the cell membrane and the nuclear envelope. Implications of this work to the field of cellular mechanics and cytoskeletal rheology are surveyed.