Shab Ladha

The membrane-permeabilizing effect of avenacin A-1 involves the reorganization of bilayer cholesterol.

Avenacin A-1 is a member of a group of naturally occurring compounds called saponins. It is found in oat plants, where it protects against fungal pathogens. A combined electrical and optical chamber was used to determine the interaction of avenacin A-1 with Montal-Mueller planar lipid bilayers. This system allowed simultaneous measurement of the effect of avenacin A-1 on the fluorescence and lateral diffusion of a fluorescent lipid probe and permeability of the planar lipid bilayer. As expected, cholesterol was required for avenacin A-1-induced bilayer permeabilization. The planar lipid bilayers were also challenged with monodeglucosyl, bis-deglucosyl, and aglycone derivatives of avenacin A-1. The results show that the permeabilizing activity of the native avenacin A-1 was completely abolished after one, two, or all three sugar residues are hydrolyzed (monodeglucosyl, bis-deglucosyl, and aglycone derivatives, respectively). Fluorescence recovery after photobleaching (FRAP) measurements on cholesterol-containing planar lipid bilayers revealed that avenacin A-1 caused a small but significant reduction in the lateral diffusion of the phospholipid probe N-(7-nitrobenzoyl-2-oxa-1,3-diazol-4-yl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). Similarly, with the sterol probe (22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-Chol), avenacin A-1, but not its derivatives, caused a more pronounced reduction in the lateral diffusion than that observed with the phospholipid probe. The data indicate that an intact sugar moiety of avenacin A-1 is required to reorganize membrane cholesterol into pores.

Lipid heterogeneity and membrane fluidity in a highly polarized cell, the mammalian spermatozoon.

The mammalian spermatozoon is a highly polarized cell consisting of two main sections, the head and the tail. These sections are enclosed by a differentiated plasma membrane to give five specialized regions: acrosome (AC), equatorial region (EQ), postacrosome (PA), midpiece (MP) and the principle piece (PP) (Fig. 1) (Bedford & Hoskins, 1990). Each region has a specialized function; its associated membrane has a lipid and protein composition that is tailored to provide a suitable environment for that function (Holt, 1984; Peterson & Russell, 1985). The membrane lipid infrastructure and fluidity develops during maturation to ensure that the genetic material carried by the sperm cell is joined with that of the ovum for fertilization. Mammalian spermatozoa, upon leaving the testis, pass through the epididymis, where they undergo a process of maturation. The sperm transits from the caput to the cauda via the corpus, during which it undergoes various morphological, physiological and biochemical changes (Bedford & Hoskins, 1990). The plasma membrane especially undergoes considerable reorganization to provide the potential for capacitation (Cooper, 1986; Fraser, 1995; Harrison & Gadella, 1995). The process of capacitation prepares the plasma membrane for the acrosome reaction, and is essential if the spermatozoon is to fertilize an egg (Fraser, 1995). The acrosome is a membrane limited vesicle that is located in the anterior head region, between the nuclear envelope and the plasma membrane. It consists of an inner and outer membrane, which in the equatorial region, form a stable parallel arrangement. During the acrosome reaction enzymes are released from this vesicle. The outer acrosomal membane fuses with the overlaying plasma membrane of the anterior head region. The hybrid membrane is shed, before the spermatozoon can penetrate the egg. The inner membrane and the equatorial region of the acrosome remain intact after completion of the reaction (Bedford & Hoskins, 1990). Energy for the cell is provided by mitochondria that are located in the midpiece region. The highest concentration of membranous structures is therefore located in the anterior head (AC) and the midpiece regions. The literature regarding lipid composition and fluidity of mammalian spermatozoan membranes is fragmentary and often species-specific. However, this review summarizes current knowledge of how the membrane organization of the mammalian spermatozoon is determined by its complex mixture of lipids. Where necessary aspects such as the role of the lipids in cryopreservation (Parks & Graham, 1992), peroxidation (Aitken, 1995; Lenzi et al., 1996) and protein regionalization (Cowan & Myles, 1993; Cowan et al., 1997) will be mentioned but have been adequately dealt with by the reviews cited.

Measurement of the lateral diffusion of human MHC class I molecules on HeLa cells by fluorescence recovery after photobleaching using a phycoerythrin probe.

The mobility of cell surface MHC class I molecules on HeLa cells was measured by fluorescence recovery after photobleaching (FRAP). The probe used for these studies was the phycobiliprotein R-phycoerythrin coupled to Fab fragments of a monoclonal antibody specific for human monomorphic MHC class I molecules. It was found that the recovery curves could be equally well fitted by either a random diffusion model with an immobile component or by an anomalous diffusion model. In the latter case, the anomalous diffusion exponent was consistent with that previously determined by single-particle tracking (SPT) experiments using the same probe (P. R. Smith, I. E. G. Morrison, K. M. Wilson, N. Fernandez, and R. J. Cherry. 1999. Biophys. J. 76:3331-3344). The FRAP experiments, however, yielded a considerably higher value of D(0), the diffusion coefficient for a time interval of 1 s. To determine whether the results were probe dependent, FRAP measurements were also performed with the same monoclonal antibody labeled with Oregon Green. These experiments gave similar results to those obtained with the phycoerythrin probe. FRAP experiments with the lipid probe 5-N-(octadecanoyl) aminofluoroscein (ODAF) bound to HeLa cells gave typical results for lipid diffusion. Overall, our observations and analysis are consistent with anomalous diffusion of MHC class I diffusion on HeLa cells, but quantitative differences between FRAP and SPT data remain to be explained.

INTERACTION OF NISIN WITH PLANAR LIPID BILAYERS MONITORED BY FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING

Abstract.Nisin, a prominent member of the lantibiotic family of antimicrobial agents, has wide application as a food preservative despite poor understanding of its mode of action. Fluorescence recovery after photobleaching has been used with planar lipid bilayers as a model membrane system to examine how nisin might interact with the surface of bacterial cells. Nisin associates with planar lipid bilayers in the absence of an applied membrane potential causing an array of effects consistent with adsorption of nisin onto the membrane surface which involves inhibition of the lateral diffusion and fluorescence of the lipid probe N-(7--1,2,3-benzoxadiazol-4-yl) phosphatidylethanolamine (NBD-PE) and a reduction of the capacitance of the bilayer. Nisin adsorption is dependent on phospholipid composition. In the presence of dioleoylphosphatidylcholine (PC): cardiolipin (CL) 4:1, the rate of lateral mobility of phospholipid is reduced to 61% of the control level which decreases to a value of 46% when CL is replaced by 1-palmitoyl-2-oleoylphosphatidylserine (PS). These effects on bilayer parameters are transient, and with time the values return to near original levels. High electrical conductivity is observed on application of a voltage ramp suggesting that insertion into the membrane follows surface association. Results have been interpreted in terms of a model in which nisin initially binds to the surface of the membrane causing a modulation of bilayer properties.

LATERAL DIFFUSION AND CONDUCTANCE PROPERTIES OF A FLUORESCEIN-LABELLED ALAMETHICIN IN PLANAR LIPID BILAYERS

In order to follow alamethicin diffusion within membranes under conditions of pore-formation, a fluorescein isothiocyanate (FITC) analogue was synthesized. To test the influence of the fluorescent probe addition on the pore-forming activity of the new analogue, macroscopic and single-channel experiments into planar lipid bilayers were performed. Although the apparent mean number of monomers per conducting aggregate was equivalent, the voltage-dependence of the new analogue was slightly reduced and hysteresses were broader, in agreement with the much longer duration of the open single-channels. Thus, the conducting aggregates seem to be stabilized by the introduction of the probe, presumably through the interaction of the conjugated cycles with the lipid headgroups, while the added steric hindrance may account for the slightly higher conductances of the open substates. Lateral diffusion of the labelled peptide associated with the bilayer was then investigated by the fluorescence recovery after photobleaching technique. Under applied voltage, associated with high conductance, D, the lateral diffusion coefficient, was reduced by 50% when compared to peptide at rest. These results provide new independent experimental evidence for a voltage-driven insertion of the highly mobile surface-associated peptide into the bilayer as a prominent step in pore formation.

Diffusion barriers in ram and boar sperm plasma membranes : Directionality of lipid diffusion across the posterior ring

Abstract The plasma membrane of mammalian spermatozoa, like that of other differentiated cells, is compartmentalized into discrete regions or domains that are biochemically and functionally distinct from one another. Physical structures within the membrane, such as the posterior ring at the juncture of the sperm head and tail, have long been thought to act as diffusion barriers to help segregate important molecules required for fertilization within specific domains and to regulate migration of molecules between domains. In this investigation, we used a quantitative photobleaching technique (video-FRAP) to assess the efficacy of the posterior ring as a barrier to exchange of lipids between the postacrosomal and midpiece plasma membranes. A lipid reporter probe (1,1′-diduodecyl-3,3,3′,3′-tetramethylindocarbocyanine; DiIC12) was incorporated into the plasma membrane of live ram and boar spermatozoa, and the directionality of its diffusion across the posterior ring was measured by line-profile analysis. Results showed that DiIC12 was able to traverse the posterior ring from the direction of the postacrosomal plasma membrane and to diffuse onto the midpiece plasma membrane. These results suggest that the posterior ring is not an immutable barrier to lipid exchange in mature spermatozoa and that there are other mechanisms for maintaining in-plane lipid asymmetry, such as differential phase behavior and interaction with the submembranous cytoskeleton.