GUV preparation and imaging: Minimizing artifacts

Morales-Penningston, Nelson F.; Wu, Jing; Farkas, Elaine R.; Goh, Shih Lin; Konyakhina, Tatyana M.; Zheng, Judy Y.; Webb, Watt W.; Feigenson, Gerald W.

doi:10.1016/j.bbamem.2010.03.011

Cited by 181 publications

(196 citation statements)

References 33 publications

Supporting

Mentioning

186

Contrasting

Order By: Relevance

“…Feigenson indeed noted that lipids can be hydrolyzed during electroformation on ITO glass slides. 39 The serious problem encountered with the electroformation protocol led us to have a closer look at the phenomenon. Indeed, a GUV formulated from Pad-PC-Pad (3) via electroformation showed the typical domain structure often encountered in recent literature (see Figure 4A).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Bilayer Properties of 1,3-Diamidophospholipids

et al. 2015

View full text Add to dashboard Cite

A series of 1,3-diamido phosphocholines was synthesized, and their potential to form stable bilayers was investigated. Large and giant unilamellar vesicles produced from these new lipids form a wide variety of faceted liposomes. Factors such as cooling rates and the careful choice of the liposome preparation method influence the formation of facets. Interdigitation was hypothesized as a main factor for the stabilization of facets and effectively monitored by small-angle X-ray scattering measurements.

show abstract

Section: ■ Results and Discussionmentioning

confidence: 99%

Bilayer Properties of 1,3-Diamidophospholipids

et al. 2015

View full text Add to dashboard Cite

show abstract

“…The maximum and minimum values of tensor A (A max and A min ) were determined from each spectrum, and the order parameter was calculated according to Schorn and Marsh (18) using the hyperfine tensor (A xx , A yy , A zz ) ϭ (5, 5, 32.8 G). Giant unilamellar vesicles (GUVs) containing the lipid dye Lissamine rhodamine-conjugated dioleoyl-phosphatidylethanolamine (DOPE) were prepared by gentle hydration (19) in 100 mM sugar buffer (sucrose inside, glucose outside) and imaged on a Nikon eclipse Ti-E microscope.…”

Section: Gagmentioning

confidence: 99%

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

Dick

Datta

Nanda

et al. 2015

J Virol

View full text Add to dashboard Cite

Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. IMPORTANCERetroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs. Many of the principles underlying retrovirus assembly are understood. For example, the retrovirus structural protein, Gag, is able to assemble into morphologically normal virus-like particles (VLPs) in cells (1) and as a purified protein in vitro (2). The structures and functions of the multiple Gag domains are known. Thus, the MA domain mediates binding to the plasma membrane (PM), where for most retroviruses the Gag lattice is formed and budding occurs, the CA domain and immediately adjoining sequences engage in critical contacts to form the hexagonal Gag lattice, t...

show abstract

“…Especially, raft formation can be regarded as the phase separation between saturated lipid and cholesterol-rich and unsaturated lipid-rich phases in a model membrane system, so from a biological aspect, it is important to discuss phase behavior of model membrane. 7 In this study, we observed the phase separation in lipid membranes containing typical LAs, lidocaine and tetracaine, by optical microscopy. In order to understand the phase separation, the membrane fluidity is estimated by Laurdan generalized polarization measurement.…”

mentioning

confidence: 99%

Destabilization of Phase-separated Structures in Local Anesthetic-containing Model Biomembranes

2015

View full text Add to dashboard Cite

We observed the phase behaviors, and the changes of membrane fluidity in lipid membranes containing local anesthetics (LAs), lidocaine and tetracaine. The phase separation in DOPC/DPPC binary membranes had not been changed significantly by addition of LAs, whereas that in DOPC/DPPC/ Chol ternary membranes was suppressed by LAs. In order to evaluate the membrane fluidity, Laurdan generalized polarization values of membranes were measured. The fluidity of DOPC-rich phase in the ternary membrane became lower as LAs concentration had increased. Our results indicated the correlation between physical property change on membrane and pharmacological functions of LAs.Local anesthetics (LAs) are essential drugs for modern medicine that are often used to suppress patients' pain. LAs are known to target functional proteins, voltage-dependent sodium channels, which are located on cellular membranes. The mechanism how LAs shield pain signals was believed to be that LAs interact with the channel protein directly, and deactivate it.1 However, binding sites on the channel proteins had not been elucidated completely. In addition, according to past reports, LAs exhibit toxicity against human cells. The presence of LAs sometimes induces fatal side effects such as allergy, liver malfunction, and analgesia. 2 The mechanisms by which these side effects are induced by LAs are also not fully understood. Recently, however, some groups are expecting that these mechanisms can be explained by the change of the physical properties of lipid membranes where the proteins are buried induced by addition of LAs. 3Cell membranes, which are composed of lipid bilayer structure, are known to relate with signal transductions via membrane dynamics such as vesicular formation. In fact, some studies are providing correlation between physical property changes on biomembrane and behavior of channel proteins. 4 Moreover, a "lipid raft model" was proposed by Simons and Ikonen in 1997. 5,6 Lipid raft is a region where a large amount of saturated lipids and cholesterols are included and some functional molecules such as ion channels and membrane receptors can be localized, and lipid raft is speculated as a platform for signal transduction.In order to reveal the mechanism of raft formation, liposome, which is mainly composed of phospholipids, is used as a mimetic model of a biomembrane system. Liposome has similar structure to actual biomembranes, so we can directly observe similar phenomena on liposome with optical microscopy. Especially, raft formation can be regarded as the phase separation between saturated lipid and cholesterol-rich and unsaturated lipid-rich phases in a model membrane system, so from a biological aspect, it is important to discuss phase behavior of model membrane. 7In this study, we observed the phase separation in lipid membranes containing typical LAs, lidocaine and tetracaine, by optical microscopy. In order to understand the phase separation, the membrane fluidity is estimated by Laurdan generalized polarization measurement. Finally, w...

show abstract

GUV preparation and imaging: Minimizing artifacts

Cited by 181 publications

References 33 publications

Bilayer Properties of 1,3-Diamidophospholipids

Bilayer Properties of 1,3-Diamidophospholipids

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

Destabilization of Phase-separated Structures in Local Anesthetic-containing Model Biomembranes

Contact Info

Product

Resources

About