Biology subjects

Wittenberg, N. J.

Publications and source records attributed to Wittenberg, N. J..

4 recordsLinked to original sources

bioRxiv2024

Chaotropic Agent-assisted Supported Lipid Bilayer Formation

Supported lipid bilayers (SLBs) are useful structures for mimicking cellular membranes, and they can be integrated with a variety of sensors. While there are a variety of methods for forming SLBs, many of these methods come with limitations in terms of the lipid compositions that can be employed and the substrates upon which the SLBs can be deposited. Here we demonstrate the use of an all-aqueous chaotropic agent exchange process that can be used to form SLBs on two different substrate materials: SiO2, which is compatible with traditional SLB formation by vesicle fusion and Al2O3, which is not compatible with vesicle fusion. When examined with quartz crystal microbalance with dissipation monitoring, the SLBs generated by chaotropic agent exchange (CASLBs) have similar frequency and dissipation shifts to SLBs formed by the vesicle fusion technique. The CASLBs block nonspecific protein adsorption on the substrate and can be used to sense protein-lipid interactions. Fluorescence microscopy was used to examine the CASLBs, and we observed long-range lateral diffusion of fluorescent probes, which confirmed the CASLBs were composed of a continuous, planar lipid bilayer. Our CASLB method provides another option for forming planar lipid bilayers on a variety of surfaces, including those that are not amenable to the widely used vesicle fusion method.

biophysics↗

bioRxiv2023

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles

Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of gram-negative bacteria limits the efficacy of many existing antibiotics and adds complexity to antibiotic development by limiting transport of antibiotics to the bacterial cytosol. New methods to mimic this barrier would enable high-throughput studies for antibiotic development. In this study, we introduce an innovative approach to modify outer membrane vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, to generate planar supported lipid bilayer membranes. Our method first involves the incorporation of synthetic lipids into OMVs using a rapid freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when in contact with SiO2 surfaces to form a planar outer membrane supported bilayer (OM-SB). We assessed the formation of OM-Hybrids using dynamic light scattering and a fluorescence quenching assay. To analyze the formation of OM-SBs from OM-Hybrids we used quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP). Additionally, we conducted assays to detect surface-associated DNA and proteins on OM-SBs. The interaction of an antimicrobial peptide, polymyxin B, with the OM-SBs was also assessed. These findings emphasize the capability of our platform to produce planar surfaces of bacterial outer membranes, which in turn, could function as a valuable tool for streamlining the development of antibiotics.

bioengineering↗

bioRxiv2023

Identifying size-dependent toxin sorting in bacterial outer membrane vesicles

Gram-negative bacteria produce outer membrane vesicles (OMVs) that play a critical role in cell-cell communication and virulence. Despite being isolated from a single population of bacteria, OMVs can exhibit heterogeneous size and toxin content, which can be obscured by assays that measure ensemble properties. To address this issue, we utilize fluorescence imaging of individual OMVs to reveal size-dependent toxin sorting. Our results showed that the oral bacterium Aggregatibacter actinomycetemcomitans (A.a.) produces OMVs with a bimodal size distribution, where larger OMVs were much more likely to possess leukotoxin (LtxA). Among the smallest OMVs (< 100 nm diameter), the fraction that are toxin positive ranges from 0-30%, while the largest OMVs (> 200 nm diameter) are between 70-100% toxin positive. Our single OMV imaging method provides a non-invasive way to observe OMV surface heterogeneity at the nanoscale level and determine size-based heterogeneities without the need for OMV fraction separation.

biophysics↗

bioRxiv2022

A Luminescent Complex of Europium and Tetracycline Labels Liquid Disordered Membrane Domains and Causes GM1 Redistribution

Microdomains in lipid bilayer membranes are routinely imaged using organic fluorophores that preferentially partition into one of the lipid phases, resulting in fluorescence contrast. Here we show that membrane microdomains in giant unilamellar vesicles (GUVs) can be visualized with europium luminescence using a complex of europium (III) and tetracycline (EuTc). EuTc is unlike typical organic lipid probes in that it is a coordination complex with a unique excitation/emission wavelength combination (396/617 nm), a very large Stokes shift (221 nm), and a very narrow emission bandwidth (8 nm). The probe preferentially interacts with liquid disordered domains in GUVs, which results in intensity contrast across the surface of phase-separated GUVs. Interestingly, EuTc also alters GM1 ganglioside partitioning. GM1 typically partitions into liquid ordered domains, but after labeling phase-separated GUVs with EuTc, cholera toxin B-subunit (CTxB), which binds GM1, labels liquid disordered domains. We also demonstrate that EuTc, but not free Eu3+ or Tc, significantly reduces lipid diffusion coefficients. Finally, we show that EuTc can be used to label cellular membranes similar to a traditional membrane probe. EuTc may find utility as a membrane imaging probe where its large Stokes shift and sharp emission band would enable multicolor imaging.

biophysics↗