Mechanical Properties of Giant Liposomes Compressed between Two Parallel Plates: Impact of Artificial Actin Shells

The mechanical response of giant liposomes to compression between two parallel plates is investigated in the context of an artificial actin cortex adjacent to the inner leaflet of the bilayer. We found that nonlinear membrane theory neglecting the impact of bending sufficiently describes the mechanical response of liposomes consisting of fluid lipids to compression whereas the formation of an actin cortex or the use of gel-phase lipids generally leads to substantial stiffening of the shell. Giant vesicles are gently adsorbed on glassy surfaces and are compressed with tipless cantilevers using an atomic force microscope. Force–compression curves display a nonlinear response that allows us to determine the membrane tension σ₀ and the area compressibility modulus <i>K</i>_A by computing the contour of the vesicle as a function of the compression depth. The values for <i>K</i>_A of fluid membranes correspond well to what is known from micropipet-suction experiments and computed from monitoring membrane undulations. The presence of a thick actin shell adjacent to the inner leaflet of the liposome membrane stiffens the system considerably, as mirrored in a significantly higher apparent area compressibility modulus

Combining Reflectometry and Fluorescence Microscopy: An Assay for the Investigation of Leakage Processes across Lipid Membranes

The passage of solutes across a lipid membrane plays a central role in many cellular processes. However, the investigation of transport processes remains a serious challenge in pharmaceutical research, particularly the transport of uncharged cargo. While translocation reactions of ions across cell membranes is commonly measured with the patch-clamp, an equally powerful screening method for the transport of uncharged compounds is still lacking. A combined setup for reflectometric interference spectroscopy (RIfS) and fluorescence microscopy measurements is presented that allows one to investigate the passive exchange of uncharged compounds across a free-standing membrane. Pore-spanning lipid membranes were prepared by spreading giant 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine (DOPC) vesicles on porous anodic aluminum oxide (AAO) membranes, creating sealed attoliter-sized compartments. The time-resolved leakage of different dye molecules (pyranine and crystal violet) as well as avidin through melittin induced membrane pores and defects was investigated

Summary of the results obtained in this study.

<p>¹ growth rescue at 39°C.</p><p>‘n.a.’ = not assayed’.</p

Reflectometric Interference Spectroscopy (RIfS) and Surface plasmon resonance (SPR) measurements.

<p>Affinity constants of TRC40 and TRC40/RAMP4 complex for rough microsomes, WRBcc and CAMLcyt. Values represent the average calculated from three independent measurements.</p

In combination, WRB and CAML rescue Get3 localization at the ER membrane and TA protein targeting.

<p>(A) <i>get1/get2</i> yeast cells carrying a genomically GFP-tagged version of Get3 were transformed with combinations of WRB, CAML, Get1 and Get2 encoding constructs. Subcellular Get3-GFP localization was analyzed by fluorescence microscopy. (B) <i>get1/get2</i> yeast cells were transformed with a plasmid containing the coding sequence of GFP-tagged Sed5 and combinations of WRB, CAML, Get1 and Get2 encoding constructs. Subcellular GFP-Sed5 localization was analyzed by fluorescence microscopy. (C) Images taken in (B) were quantified to determine the distribution of fluorescence across bins of different pixel intensity for each strain. A minimum of 41 cells was analyzed per strain.</p

WRB and CAML rescue the growth phenotypes of <i>get1/get2</i> yeast cells.

<p><i>get1/get2</i> yeast cells were transformed with combinations of WRB, CAML, Get1 and Get2 encoding constructs and serial dilutions spotted on different conditions: HC plates incubated at 30°C (control), 37°C+CuSO₄, 39°C, H₂O₂, hydroxyurea, tunicamycin, hygromycin.</p

The transmembrane domains of CAML are essential for a functional WRB/CAML receptor complex.

<p>(A) Schematic representation of CAML-Get2 chimeras. Position of transmembrane domains (TMD) are indicated. (B) <i>get1/get2</i> yeast cells carrying a genomically GFP-tagged version of Get3 were transformed with a plasmid containing the coding sequence of WRB in combination with CAML or CAML-Get2 chimeras. Subcellular Get3-GFP localization was analyzed by fluorescence microscopy. (C) <i>get1/get2</i> yeast cells were transformed with a plasmid containing the coding sequence of GFP-tagged Sed5 and Get1/Get2 or WRB in combination with CAML or CAML-Get2 chimeras. Subcellular GFP-Sed5 localization was analyzed by fluorescence microscopy. (D) Images taken in (C) were quantified to determine the distribution of fluorescence across bins of different pixel intensity for each strain. A minimum of 42 cells was analyzed per strain.</p

The Nonbilayer Lipid MGDG and the Major Light-Harvesting Complex (LHCII) Promote Membrane Stacking in Supported Lipid Bilayers

The thylakoid membrane of algae and land plants is characterized by its intricate architecture, comprising tightly appressed membrane stacks termed grana. The contributions of individual components to grana stack formation are not yet fully elucidated. As an <i>in vitro</i> model, we use supported lipid bilayers made of thylakoid lipid mixtures to study the effect of major light-harvesting complex (LHCII), different lipids, and ions on membrane stacking, seen as elevated structures forming on top of the planar membrane surface in the presence of LHCII protein. These structures were examined by confocal laser scanning microscopy, atomic force microscopy, and fluorescence recovery after photobleaching, revealing multilamellar LHCII–membrane stacks composed of connected lipid bilayers. Both native-like and non-native interactions between the LHCII complexes may contribute to membrane appression in the supported bilayers. However, applying <i>in vivo</i>-like salt conditions to uncharged glycolipid membranes drastically increased the level of stack formation due to enforced LHCII–LHCII interactions, which is in line with recent crystallographic and cryo-electron microscopic data [Wan, T., et al. (2014) <i>Mol. Plant 7</i>, 916–919; Albanese, P., et al. (2017) <i>Sci. Rep. 7</i>, 10067–10083]. Furthermore, we observed the nonbilayer lipid MGDG to strongly promote membrane stacking, pointing to the long-term proposed function of MGDG in stabilizing the inner membrane leaflet of highly curved margins in the periphery of each grana disc because of its negative intrinsic curvature [Murphy, D. J. (1982) <i>FEBS Lett. 150</i>, 19–26]

Single Particle Plasmon Sensors as Label-Free Technique To Monitor MinDE Protein Wave Propagation on Membranes

We use individual gold nanorods as pointlike detectors for the intrinsic dynamics of an oscillating biological system. We chose the pattern forming MinDE protein system from <i>Escherichia coli</i> (<i>E. coli</i>), a prominent example for self-organized chemical oscillations of membrane-associated proteins that are involved in the bacterial cell division process. Similar to surface plasmon resonance (SPR), the gold nanorods report changes in their protein surface coverage without the need for fluorescence labeling, a technique we refer to as NanoSPR. Comparing the dynamics for fluorescence labeled and unlabeled proteins, we find a reduction of the oscillation period by about 20%. The absence of photobleaching allows us to investigate Min proteins attaching and detaching from lipid coated gold nanorods with an unprecedented bandwidth of 100 ms time resolution and 1 h observation time. The long observation reveals small changes of the oscillation period over time. Averaging many cycles yields the precise wave profile that exhibits the four phases suggested in previous reports. Unexpected from previous fluorescence-based studies, we found an immobile static protein layer not dissociating during the oscillation cycle. Hence, NanoSPR is an attractive label-free real-time technique for the local investigation of molecular dynamics with high observation bandwidth. It gives access to systems, which cannot be fluorescently labeled, and resolves local dynamics that would average out over the sensor area used in conventional SPR

Colocalization of the Ganglioside G_M1 and Cholesterol Detected by Secondary Ion Mass Spectrometry

The characterization of the lateral organization of components in biological membranes and the evolution of this arrangement in response to external triggers remain a major challenge. The concept of lipid rafts is widely invoked; however, direct evidence of the existence of these ephemeral entities remains elusive. We report here the use of secondary ion mass spectrometry (SIMS) to image the cholesterol-dependent cohesive phase separation of the ganglioside G_M1 into nano- and microscale assemblies in a canonical lipid raft composition of lipids. This assembly of domains was interrogated in a model membrane system composed of palmitoyl sphingomyelin (PSM), cholesterol, and an unsaturated lipid (dioleoylphosphatidylcholine, DOPC). Orthogonal isotopic labeling of every lipid bilayer component and monofluorination of G_M1 allowed generation of molecule specific images using a NanoSIMS. Simultaneous detection of six different ion species in SIMS, including secondary electrons, was used to generate ion ratio images whose signal intensity values could be correlated to composition through the use of calibration curves from standard samples. Images of this system provide the first direct, molecule specific, visual evidence for the colocalization of cholesterol and G_M1 in supported lipid bilayers and further indicate the presence of three compositionally distinct phases: (1) the interdomain region; (2) micrometer-scale domains (<i>d</i> > 3 μm); (3) nanometer-scale domains (<i>d</i> = 100 nm to 1 μm) localized within the micrometer-scale domains and the interdomain region. PSM-rich, nanometer-scale domains prefer to partition within the more ordered, cholesterol-rich/DOPC-poor/G_M1-rich micrometer-scale phase, while G_M1-rich, nanometer-scale domains prefer to partition within the surrounding, disordered, cholesterol-poor/PSM-rich/DOPC-rich interdomain phase