Flagellar membrane association via interaction with lipid rafts

The eukaryotic flagellar membrane has a distinct composition from other domains of the plasmalemma. Our work shows that the specialized composition of the trypanosome flagellar membrane reflects increased concentrations of sterols and saturated fatty acids, correlating with direct observation of high liquid order by laurdan fluorescence microscopy. These findings indicate that the trypanosome flagellar membrane possesses high concentrations of lipid rafts: discrete regions of lateral heterogeneity in plasma membranes that serve to sequester and organize specialized protein complexes. Consistent with this, a dually acylated Ca(2+) sensor that is concentrated in the flagellum is found in detergent-resistant membranes and mislocalizes if the lipid rafts are disrupted. Detergent-extracted cells have discrete membrane patches localized on the surface of the flagellar axoneme, suggestive of intraflagellar transport particles. Together, these results provide biophysical and biochemical evidence to indicate that lipid rafts are enriched in the trypanosome flagellar membrane, providing a unique mechanism for flagellar protein localization and illustrating a novel means by which specialized cellular functions may be partitioned to discrete membrane domains

Surface-exposed Tryptophan Residues Are Essential for O-Acetylserine Sulfhydrylase Structure, Function, and Stability

O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism. The subunit is composed of two domains, each containing one tryptophan residue, Trp50 in the N-terminal domain and Trp161 in the C-terminal domain. Only Trp161 is highly conserved in eucaryotes and bacteria. The coenzyme pyridoxal 5'-phosphate is bound in a cleft between the two domains. The enzyme undergoes an open to closed conformational transition upon substrate binding. The effect of single Trp to Tyr mutations on O-acetylserine sulfhydrylase structure, function, and stability was investigated with a variety of spectroscopic techniques. The mutations do not significantly alter the enzyme secondary structure but affect the catalysis, with a predominant influence on the second half reaction. The W50Y mutation strongly affects the unfolding pathway due to the destabilization of the intersubunit interface. The W161Y mutation, occurring in the C-terminal domain, produces a reduction of the accessibility of the active site to acrylamide and stabilizes thermodynamically the N-terminal domain, a result consistent with stronger interdomain interactions

Visualization and analysis of apolipoprotein A-I interaction with binary phospholipid bilayers

Apolipoprotein A-I (apoA-I) interaction with specific cell lipid domains was suggested to trigger cholesterol and phospholipid efflux. We analyzed here apoA-I interaction with dimyristoylphosphatidylcholine/ distearoylphosphatidylcholine (DMPC/DSPC) bilayers at a temperature showing phase coexistence. Solid and liquid-crystalline domains were visualized by two-photon fluorescence microscopy on giant unilamellar vesicles (GUVs) labeled with 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan). A decrease of vesicle size was detected as long as they were incubated with lipid-free apoA-I, together with a shape deformation and a relative enrichment in DSPC. Selective lipid removal mediated by apoA-I from different domains was followed in real time by changes in the Laurdan generalized polarization. The data show a selective interaction of apoA-I with liquid-crystalline domains, from which it removes lipids, at a molar ratio similar to the domain compositions. Next, apoA-I was incubated with DMPC/DSPC small unilamellar vesicles, and products were isolated and quantified. Protein solubilized both lipids but formed complexes relatively enriched in the liquid component. We also show changes in the GUV morphology when cooling down. Our results suggest that the most efficient reaction between apoA-I and DMPC/DSPC occurs in particular bilayer conditions, probably when small fluid domains are nucleated within a continuous gel phase and interfacial packing defects are maximal.Instituto de Investigaciones Bioquímicas de La Plat

Visualization and analysis of apolipoprotein A-I interaction with binary phospholipid bilayers

Apolipoprotein A-I (apoA-I) interaction with specific cell lipid domains was suggested to trigger cholesterol and phospholipid efflux. We analyzed here apoA-I interaction with dimyristoylphosphatidylcholine/ distearoylphosphatidylcholine (DMPC/DSPC) bilayers at a temperature showing phase coexistence. Solid and liquid-crystalline domains were visualized by two-photon fluorescence microscopy on giant unilamellar vesicles (GUVs) labeled with 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan). A decrease of vesicle size was detected as long as they were incubated with lipid-free apoA-I, together with a shape deformation and a relative enrichment in DSPC. Selective lipid removal mediated by apoA-I from different domains was followed in real time by changes in the Laurdan generalized polarization. The data show a selective interaction of apoA-I with liquid-crystalline domains, from which it removes lipids, at a molar ratio similar to the domain compositions. Next, apoA-I was incubated with DMPC/DSPC small unilamellar vesicles, and products were isolated and quantified. Protein solubilized both lipids but formed complexes relatively enriched in the liquid component. We also show changes in the GUV morphology when cooling down. Our results suggest that the most efficient reaction between apoA-I and DMPC/DSPC occurs in particular bilayer conditions, probably when small fluid domains are nucleated within a continuous gel phase and interfacial packing defects are maximal.Instituto de Investigaciones Bioquímicas de La Plat

Visualization and analysis of apolipoprotein A-I interaction with binary phospholipid bilayers

Apolipoprotein A-I (apoA-I) interaction with specific cell lipid domains was suggested to trigger cholesterol and phospholipid efflux. We analyzed here apoA-I interaction with dimyristoylphosphatidylcholine/ distearoylphosphatidylcholine (DMPC/DSPC) bilayers at a temperature showing phase coexistence. Solid and liquid-crystalline domains were visualized by two-photon fluorescence microscopy on giant unilamellar vesicles (GUVs) labeled with 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan). A decrease of vesicle size was detected as long as they were incubated with lipid-free apoA-I, together with a shape deformation and a relative enrichment in DSPC. Selective lipid removal mediated by apoA-I from different domains was followed in real time by changes in the Laurdan generalized polarization. The data show a selective interaction of apoA-I with liquid-crystalline domains, from which it removes lipids, at a molar ratio similar to the domain compositions. Next, apoA-I was incubated with DMPC/DSPC small unilamellar vesicles, and products were isolated and quantified. Protein solubilized both lipids but formed complexes relatively enriched in the liquid component. We also show changes in the GUV morphology when cooling down. Our results suggest that the most efficient reaction between apoA-I and DMPC/DSPC occurs in particular bilayer conditions, probably when small fluid domains are nucleated within a continuous gel phase and interfacial packing defects are maximal.Instituto de Investigaciones Bioquímicas de La Plat

Factors influencing island floras in northern Lake Michigan.

The floras of four islands in northern Lake Michigan (South Manitou (21.3 km\sp2), North Manitou (59.7 km\sp2), South Fox (13.2 km\sp2), and North Fox (3.4 km\sp2)) and a mainland portion of Sleeping Bear Dunes National Lakeshore (93.8 km\sp2) were analyzed to determine the factors influencing their floristic composition. The major influence on the composition of the Island floras is the availability of appropriate habitat. The number of native species shows a closer relationship to the number of soil series (r\sp2 =.764) than to the island area (r\sp2 =.535). The number of alien species on the islands shows the closest relationship with island area (r\sp2 =.792). The small number of islands (four) precludes a determination of the best species-area model. Probable dispersal types of the Mainland and Island floras were analyzed based on diaspore morphology. All islands except North Manitou showed significant differences in the proportion of fleshy-fruited species (sarchochores) compared to the flora of the entire study area. Preston similarity coefficients among the Islands are greater than between Islands and the Mainland suggesting that the Manitou Passage is a barrier to many species. The survival of several species on the Islands may be enhanced by island climate. Temperature records from Coast Guard Stations on North Manitou, South Manitou, and the Mainland (Sleeping Bear Point) show that most temperature differences between the Islands and the Mainland occur during the summer months with June showing the greatest difference. The advent of spring on South Manitou, based on growing degree-days, is delayed by almost two weeks. The introduced deer population on North Manitou has had a profound impact on the structure of the island's vegetation. The apparent absence of several species easily found on the other Islands can be attributed to the deer. The presence of postglacial landbridges between the Mainland and the Manitou Islands is of little importance in the development of the flora on those islands since the Fox Islands, which were never connected to the mainland, show just as many heavy-fruited species as the Manitous.Ph.D.Biological SciencesBotanyEcologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/128218/2/8821581.pd

Giant vesicles, Laurdan, and two-photon fluorescence microscopy: evidence of lipid lateral separation in bilayers.

This chapter describes giant vesicles, Laurdan, and two-photon fluorescence microscopy. The combination of Laurdan, giant unilamellar vesicles (GUVs), and two-photon fluorescence microscopy has been extremely useful in producing a microscopic picture of lipid-phase coexistence in the GUV bilayer model system. Laurdan is a unique probe, giving simultaneous information about morphology and phase state of lipid domains from fluorescence images. The critical issues in membrane biophysics today are centered on the molecular dynamics of the bilayer structure. The interplay between lipids that results in the formation of domains on a bilayer surface and the interactions among these domains and relevant membrane-associated biomolecules is of particular interest. The advantages of using a microscope as the optical arrangement are clear. The light collection efficiency of a well-designed microscope is greatly enhanced over other optical arrangements. In addition, the flexibility of fluorescence microscopes creates for the spectroscopist a malleable optical compartment that can be designed and readily redesigned as needed.Fil: Bagatolli, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Sanchez, Susana A.. No especifíca;Fil: Hazlett, Theodore. No especifíca;Fil: Gratton, Enrico. No especifíca

Giant vesicles, laurdan, and two-photon fluorescence microscopy: Evidence of lipid lateral separation in bilayers

This chapter describes giant vesicles, Laurdan, and two-photon fluorescence microscopy. The combination of Laurdan, giant unilamellar vesicles (GUVs), and two-photon fluorescence microscopy has been extremely useful in producing a microscopic picture of lipid-phase coexistence in the GUV bilayer model system. Laurdan is a unique probe, giving simultaneous information about morphology and phase state of lipid domains from fluorescence images. The critical issues in membrane biophysics today are centered on the molecular dynamics of the bilayer structure. The interplay between lipids that results in the formation of domains on a bilayer surface and the interactions among these domains and relevant membrane-associated biomolecules is of particular interest. The advantages of using a microscope as the optical arrangement are clear. The light collection efficiency of a well-designed microscope is greatly enhanced over other optical arrangements. In addition, the flexibility of fluorescence microscopes creates for the spectroscopist a malleable optical compartment that can be designed and readily redesigned as needed.Fil: Bagatolli, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Sanchez, Susana A.. No especifíca;Fil: Hazlett, Theodore. No especifíca;Fil: Gratton, Enrico. No especifíca

A two-photon view of an enzyme at work: Crotalus atrox venom PLA2 interaction with single-lipid and mixed-lipid giant unilamellar vesicles.

We describe the interaction of Crotalus atrox-secreted phospholipase A2 (sPLA2) with giant unilamellar vesicles (GUVs) composed of single and binary phospholipid mixtures visualized through two-photon excitation fluorescent microscopy. The GUV lipid compositions that we examined included 1-palmitoyl-2-oleoyl-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (above their gel-liquid crystal transition temperatures) and two well characterized lipid mixtures, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE):DMPC (7:3) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC) (1:1) equilibrated at their phase-coexistence temperature regime. The membrane fluorescence probes, 6-lauroyl-2-(dimethylamino) napthalene, 6-propionyl-2-(dimethylamino) naphthalene, and rhodamine-phosphatidylethanolamine, were used to assess the state of the membrane and specifically mark the phospholipid domains. Independent of their lipid composition, all GUVs were reduced in size as sPLA2-dependent lipid hydrolysis proceeded. The binding of sPLA2 was monitored using a fluorescein-sPLA2 conjugate. The sPLA2 was observed to associate with the entire surface of the liquid phase in the single phospholipid GUVs. In the mixed-lipid GUV's, at temperatures promoting domain coexistence, a preferential binding of the enzyme to the liquid regions was also found. The lipid phase of the GUV protein binding region was verified by the introduction of 6-propionyl-2-(dimethylamino) naphthalene, which partitions quickly into the lipid fluid phase. Preferential hydrolysis of the liquid domains supported the conclusions based on the binding studies. sPLA2 hydrolyzes the liquid domains in the binary lipid mixtures DLPC:DAPC and DMPC:DMPE, indicating that the solid-phase packing of DAPC and DMPE interferes with sPLA2 binding, irrespective of the phospholipid headgroup. These studies emphasize the importance of lateral packing of the lipids in C. atrox sPLA2 enzymatic hydrolysis of a membrane surface