Biology subjects

Scott, H. L.

Publications and source records attributed to Scott, H. L..

5 recordsLinked to original sources

bioRxiv2022

Optimization of cryo-electron microscopy for quantitative analysis of lipid bilayers

Cryogenic electron microscopy (cryo-EM) is among the most powerful tools available for interrogating nanoscale structure of biological structures. We recently showed that cryo-EM can be used to measure the bilayer thickness of lipid vesicles and biological membranes with sub-angstrom precision, resulting in the direct visualization of nanoscopic domains of different thickness in multicomponent lipid mixtures and giant plasma membrane vesicles. Despite the great potential of cryo-EM for revealing the lateral organization of biomembranes, a large parameter space of experimental conditions remains to be optimized. Here, we systematically investigate the influence of instrument parameters and image post-processing steps on the ability to accurately measure bilayer thickness and discriminate regions of different thickness within unilamellar liposomes. We also demonstrate a spatial autocorrelation analysis to extract additional information about lateral heterogeneity. SignificanceRaft domains in unstimulated cells have proven difficult to directly visualize owing to their nanoscopic size and fleeting existence. The few techniques capable of nanoscopic spatial resolution typically rely on interpretation of indirect spectroscopic or scattering signals or require stabilizing the membrane on a solid support. In contrast, cryo-EM yields direct images of nanoscale domains in probe-free, unsupported membranes. Here, we systematically optimize key steps in the experimental and analysis workflow for this new and specialized application. Our findings represent an important step toward developing cryo-EM into a robust method for investigating phase behavior of membranes at length scales relevant to lipid rafts.

biophysics↗

bioRxiv2021

Lactoferricins access the cytosol of Escherichia coli within few seconds

We report the real-time response of E. coli to lactoferricin-derived antimicrobial peptides (AMPs) on length-scales bridging microscopic cell-sizes to nanoscopic lipid packing using millisecond time-resolved synchrotron small-angle X-ray scattering. Coupling a multi-scale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly saturate the bacterial envelope and reach the cytosol within less than three seconds--much faster than previously considered. Final cytosolic AMP concentrations of ~ 100 mM suggest an efficient shut-down of metabolism as primary cause for bacterial killing. On the other hand, the damage of the cell envelope is a collateral effect of AMP activity that does not kill the bacteria. This implies that the impairment of the membrane barrier is a necessary but not sufficient condition for microbial killing by lactoferricins. The most efficient AMP studied exceeds others in both speed of reaching cytoplasm and lowest cytosolic peptide concentration.

biophysics↗

bioRxiv2020

Direct label-free imaging of nanodomains in biomimetic and biological membranes by cryogenic electron microscopy

The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron microscopy (cryoEM) can be used to directly image coexisting nanoscopic domains in synthetic and bio-derived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying lengths, we demonstrate that cryoEM can distinguish bilayer thickness differences as small as 0.5 [A], comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryoEM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolated mammalian plasma membranes, these methods reveal similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization. SIGNIFICANCEWe have used cryoEM to achieve direct, probe-free imaging of lateral domains in biomimetic lipid membranes under native conditions and to characterize differences in their structures. First, measurements of membrane thickness in laterally uniform single-component membranes show that cryoEM is capable of sub-angstrom resolution of interleaflet membrane thickness. All-atom simulations are used to predict the cryo-EM appearance of submicron domains in vesicles with coexisting liquid domains and these are quantitatively validated by direct imaging of phase separated membranes. We then extend this approach to observe nanoscopic domains in isolated cellular membranes, comprising the first direct imaging of nanodomains in biomembranes.

biophysics↗

bioRxiv2019

On the mechanism of bilayer separation by extrusion; or, why your large unilamellar vesicles are not really unilamellar

Extrusion through porous filters is a widely used method for preparing biomimetic model membranes. Of primary importance in this approach is the efficient production of single bilayer (unilamellar) vesicles that eliminate the influence of interlamellar interactions and strictly define the bilayer surface area available to external reagents such as proteins. Sub-microscopic vesicles produced using extrusion are widely assumed to be unilamellar, and large deviations from this assumption would dramatically impact interpretations from many model membrane experiments. Using three probe-free methods--small-angle X-ray and neutron scattering (SAXS and SANS) and cryogenic electron microscopy (cryoEM)--we report unambiguous evidence of extensive multilamellarity in extruded vesicles composed of neutral phosphatidylcholine lipids, including for the common case of neutral lipids dispersed in physiological buffer and extruded through 100 nm diameter pores. In such preparations, only ~35% of lipids are externally accessible, and this fraction is highly dependent on preparation conditions. Charged lipids promote unilamellarity, as does decreasing solvent ionic strength, indicating the importance of electrostatic interactions in determining the lamellarity of extruded vesicles. Smaller extrusion pore sizes also robustly increase the fraction of unilamellar vesicles, suggesting a role for membrane bending. Taken together, these observations suggest a mechanistic model for extrusion, wherein formation of unilamellar vesicles involves competition between bilayer bending and adhesion energies. The findings presented here have wide-ranging implications for the design and interpretation of model membrane studies, especially ensemble-averaged observations relying on the assumption of unilamellarity.\n\nSTATEMENT OF SIGNIFICANCEExtruded vesicles are a ubiquitous tool in membrane research. It is widely presumed that extrusion produces unilamellar (i.e., single bilayer) vesicles, an assumption that is often crucial for data analysis and interpretation. Using X-ray and neutron scattering and cryogenic electron microscopy, we show that a substantial amount of lipid remains inaccessible after extrusion due to an abundance of multilamellar vesicles (MLVs). While this is a general phenomenon for neutral lipids, MLV contamination can be reduced by several complementary approaches such as including charged lipids in the mixture, reducing the ionic strength of the aqueous medium, and reducing the extrusion pore size. These observations together suggest a mechanism by which extrusion strips MLVs of their layers.

biophysics↗

bioRxiv2018

PS membrane asymmetry influences the folding and insertion of a transmembrane helix

The plasma membrane (PM) contains an asymmetric distribution of lipids between the inner and outer leaflets of its bilayer. A lipid of special interest in eukaryotic cells is the negatively charged phosphatidylserine (PS). In healthy cells, PS is actively sequestered to the inner leaflet of the PM but can redistribute to the outer leaflet when the cell is damaged or at the onset of apoptosis. The influence of PS asymmetry and its loss on membrane protein structure and organization have not been widely addressed. Marginally hydrophobic membrane proteins contain acidic residues in their transmembrane sequence, which can enable topological transitions after membrane association. The pH low insertion peptide (pHLIP), which undergoes a topological reorientation and inserts into the membrane at acidic pH - as its name implies, is a useful and well-characterized model for studying these transitions. Although it is known that the inclusion of PS in symmetric vesicles affects the membrane insertion process of pHLIP by lowering the pH midpoint of insertion, it is unclear how PS asymmetry influences these topological transitions. Here, we studied pHLIPs topology using freely-floating asymmetric phosphatidylcholine (PC)/PS vesicles with PS enriched in the inner leaflet. We developed a modified protocol to create asymmetric vesicles containing PS and employed Annexin V labeled with an Alexa 568 fluorophore as a new probe to quantifying PS asymmetry. For pHLIP, membrane insertion was affected by the surface charge difference between bilayer leaflets caused by the asymmetric distribution of charged lipids between the leaflets. We thus conclude that lipid asymmetry can have consequences for the behavior of membrane-associated proteins. A corollary is that model studies using symmetric bilayers to mimic the PM may fail to capture important aspects of protein-membrane interactions.

biophysics↗