Embryonic and adult isoforms of XLAP2 form microdomains associated with chromatin and the nuclear envelope

Laminin-associated polypeptide 2 (LAP2) proteins are alternatively spliced products of a single gene; they belong to the LEM domain family and, in mammals, locate to the nuclear envelope (NE) and nuclear lamina. Isoforms lacking the transmembrane domain also locate to the nucleoplasm. We used new specific antibodies against the N-terminal domain of Xenopus LAP2 to perform immunoprecipitation, identification and localization studies during Xenopus development. By immunoprecipitation and mass spectrometry (LC/MS/MS), we identified the embryonic isoform XLAP2??, which was downregulated during development similarly to XLAP2??. Embryonic isoforms XLAP2?? and XLAP2?? were located in close association with chromatin up to the blastula stage. Later in development, both embryonic isoforms and the adult isoform XLAP2?? were localized in a similar way at the NE. All isoforms colocalized with lamin B2/B3 during development, whereas XLAP2?? was colocalized with lamin B2 and apparently with the F/G repeat nucleoporins throughout the cell cycle in adult tissues and culture cells. XLAP2?? was localized in clusters on chromatin, both at the NE and inside the nucleus. Embryonic isoforms were also localized in clusters at the NE of oocytes. Our results suggest that XLAP2 isoforms participate in the maintenance and anchoring of chromatin domains to the NE and in the formation of lamin B microdomains.     

Cholesterol affects spectrin-phospholipid interactions in a manner different from changes resulting from alterations in membrane fluidity due to fatty acyl chain composition

We previously showed that erythrocyte and brain spectrins bind phospholipid vesicles and monolayers prepared from phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (Review: A.F. Sikorski, B. Hanus-Lorenz, A. Jezierski, A. R. Dluzewski, Interaction of membrane skeletal proteins with membrane lipid domain, Acta Biochim. Polon. 47 (2000) 565). Here, we show how changes in the fluidity of the phospholipid monolayer affect spectrin-phospholipid interaction. The presence of up to 10%-20% cholesterol in the PE/PC monolayer facilitates the penetration of the monolayer by both types of spectrin. For monolayers constructed from mixtures of PI/PC and cholesterol, the effect of spectrins was characterised by the presence of two maxima (at 5 and 30% cholesterol) of surface pressure for erythroid spectrin, and a single maximum (at 20% cholesterol) for brain spectrin. The binding assay results indicated a small but easily detectable decrease in the affinity of erythrocyte spectrin for FAT-liposomes prepared from a PE/PC mixture containing cholesterol, and a 2- to 5-fold increase in maximal binding capacity (B_max) depending on the cholesterol content. On the other hand, the results from experiments with a monolayer constructed from homogenous synthetic phospholipids indicated an increase in Δπ change with the increase in the fatty acyl chain length of the phospholipids used to prepare the monolayer. This was confirmed by the results of a pelleting experiment. Adding spectrins into the subphase of raft-like monolayers constructed from DOPC, SM and cholesterol (1/1/1) induced an increase in surface pressure. The Δπ change values were, however, much smaller than those observed in the case of a natural PE/PC (6/4) monolayer. An increased binding capacity for spectrins of liposomes prepared from a “raft-like” mixture of lipids could also be concluded from the pelleting assay. In conclusion, we suggest that the effect of membrane lipid fluidity on spectrin-phospholipid interactions is not simple but depends on how it is regulated, i.e., by cholesterol content or by the chemical structure of the membrane lipids.     

Laser interferometry analysis of ciprofloxacin and ampicillin diffusion from liposomal solutions to water phase

The paper presents experimental investigations of diffusion of antibiotics (ciprofloxacin or ampicillin) into the water phase from mixtures of neutral or negatively charged liposomes, and antibiotic–liposome interactions. Using the laser interferometry technique, the amounts and fluxes of released antibiotics, concentration field evolution, and the velocity of the concentration boundary layer’s “growth” were determined. To avoid the limitations of membranes, a measurement system without the artificial boundary of phases with a free water–solution interface has been proposed. It was found that the diffusion of anionic and neutral liposomes into the water phase was insignificant and mainly the diffusion of antibiotics was measured. Differences in the diffusion kinetics of ciprofloxacin and ampicillin from liposomal solutions to the water phase were observed. Ampicillin diffused more efficiently than ciprofloxacin regardless of the liposomal solution type. Moreover, the amount of ampicillin and ciprofloxacin released from the anionic liposomal phase was higher than that from the neutral one. Our results confirm that ciprofloxacin at neutral pH shows little tendency to bind neutral liposomes. Additionally, it was also observed that ciprofloxacin disrupts negatively charged liposomes as a final effect of antibiotic–lipid interactions.     

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC).In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the β-subunit, possibly in, or in close proximity of, the ankyrin-binding site.In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.     

Comparative studies of the biological activities of lysosomotropic aminoesters and quaternary ammonium salts on the yeast Saccharomyces cerevisiae.

The biological activity of lysosomotropic n-alkyl N,N-dimethylglycinates (DMG-n) was compared with that of a quaternary ammonium salt IM (methochloride of DMG-12). The activity of the glycinates appeared to be carbon chain length dependent and was similar at pH 6 and pH 8. Nutritional auxotrophy and respiratory deficiencies have no influence on DMG-n sensitivity. Both IM and DMG-n inhibit plasma membrane H+-ATPase activity while mitochondrial ATPase is relatively non-sensitive to glycinates. No cross-resistance to IM and DMG-n was observed.     

Phospholipid-induced structural changes to an erythroid beta spectrin ankyrin-dependent lipid-binding site

The region of beta-spectrin that is responsible for interactions with ankyrin was shown to comprise an ankyrin-sensitive lipid-binding site. Structural studies indicate that it exhibits a mixed 3(10)/alpha helical conformation and is highly amphipathic. These features together with the distinctively conserved sequence of the lipid-binding site motivated us to explore the mechanism of its interactions with biological membranes. A series of singly and doubly spin-labeled erythroid beta-spectrin-derived peptides was constructed, and the spin-label mobility and spin-spin distances were analyzed via electron paramagnetic resonance spectroscopy and two different calculation methods. The results indicate that in beta-spectrin, the lipid-binding domain, which is part of the 14(th) segment, has the topology of typical triple-helical spectrin repeat. However, it undergoes significant changes when interacting with phospholipids or detergents. A mechanism for these interactions is proposed in this paper.     

Organelle evolution: Paulinella breaks a paradigm

It is commonly assumed that transformations of endosymbionts into organelles are exceptionally rare evolutionary events because of hypothetical difficulties in the origin of an import apparatus for nuclear-encoded, organelle-targeted proteins along with their targeting signals. A challenge to this view comes from recent studies of protein import into the cyanobacterial endosymbionts/organelles of Paulinella chromatophora.