Influence of Metal Ions on Model Protoamphiphilic Vesicular Systems: Insights from Laboratory and Analogue Studies

Metal ions strongly affect the self-assembly and stability of membranes composed of prebiotically relevant amphiphiles (protoamphiphiles). Therefore, evaluating the behavior of such amphiphiles in the presence of ions is a crucial step towards assessing their potential as model protocell compartments. We have recently reported vesicle formation by N-acyl amino acids (NAAs), an interesting class of protoamphiphiles containing an amino acid linked to a fatty acid via an amide linkage. Herein, we explore the effect of ions on the self-assembly and stability of model N-oleoyl glycine (NOG)-based membranes. Microscopic analysis showed that the blended membranes of NOG and Glycerol 1-monooleate (GMO) were more stable than pure NOG vesicles, both in the presence of monovalent and divalent cations, with the overall vesicle stability being 100-fold higher in the presence of a monovalent cation. Furthermore, both pure NOG and NOG + GMO mixed systems were able to self-assemble into vesicles in natural water samples containing multiple ions that were collected from active hot spring sites. Our study reveals that several aspects of the metal ion stability of NAA-based membranes are comparable to those of fatty acid-based systems, while also confirming the robustness of compositionally heterogeneous membranes towards high metal ion concentrations. Pertinently, the vesicle formation by NAA-based systems in terrestrial hot spring samples indicates the conduciveness of these low ionic strength freshwater systems for facilitating prebiotic membrane-assembly processes. This further highlights their potential to serve as a plausible niche for the emergence of cellular life on the early Earth.

Effect of Mutations in GvpJ and GvpM on Gas Vesicle Formation of Halobacterium salinarum

The two haloarchaeal proteins, GvpM and GvpJ, are homologous to GvpA, the major gas vesicle structural protein. All three are hydrophobic and essential for gas vesicle formation. The effect of mutations in GvpJ and GvpM was studied in Haloferax volcanii transformants by complementing the respective mutated gene with the remaining gvp genes and inspecting the cells for the presence of gas vesicles (Vac+). In case of GvpJ, 56 of 66 substitutions analyzed yielded Vac– ΔJ + Jmut transformants, indicating that GvpJ is very sensitive to alterations, whereas ten of the 38 GvpM variants resulted in Vac– ΔM + Mmut transformants. The variants were also tested by split-GFP for their ability to interact with their partner protein GvpL. Some of the alterations leading to a Vac– phenotype affected the J/L or M/L interaction. Also, the interactions J/A and J/M were studied using fragments to exclude an unspecific aggregation of these hydrophobic proteins. Both fragments of GvpJ interacted with the M1–25 and M60–84 fragments of GvpM, and fragment J1–56 of GvpJ interacted with the N-terminal fragment A1–22 of GvpA. A comparison of the results on the three homologous proteins indicates that despite their relatedness, GvpA, GvpJ, and GvpM have unique features and cannot substitute each other.

Dehydration Enhances Prebiotic Lipid Remodeling and Vesicle Formation in Acidic Environments

The encapsulation of genetic polymers inside lipid bilayer compartments is a vital step in the emergence of cell-based life. However, even though acidic conditions promote many reactions required for generating prebiotic building blocks, prebiotically-relevant lipids tend to form denser aggregates at acidic pHs rather than prebiotically useful vesicles that exhibit sufficient solute encapsulation. Here we describe how dehydration/rehydration (DR) events, a prebiotically-relevant physicochemical process known to promote polymerization reactions, can remodel dense lipid aggregates into thin-walled vesicles capable of RNA encapsulation even at acidic pHs. Furthermore, DR events appears to favor the encapsulation of RNA within thin-walled vesicles over more lipid-rich vesicles, thus conferring such vesicles a selective advantage.

Membrane Trafficking Proteins: A New Target to Identify Resistance to Viruses in Plants

Replication cycles from most simple-stranded positive RNA viruses infecting plants involve endomembrane deformations. Recent published data revealed several interactions between viral proteins and plant proteins associated with vesicle formation and movement. These plant proteins belong to the COPI/II, SNARE, clathrin and ESCRT endomembrane trafficking mechanisms. In a few cases, variations of these plant proteins leading to virus resistance have been identified. In this review, we summarize all known interactions between these plant cell mechanisms and viruses and highlight strategies allowing fast identification of variant alleles for membrane-associated proteins.

Functions of Intracellular Alpha-Synuclein in Microglia: Implications for Parkinson’s Disease Risk

Alpha-synuclein accumulation in dopaminergic neurons is one of the primary features of Parkinson’s disease (PD). Despite its toxic properties during PD, alpha-synuclein has some important physiological functions. Although the activity of the protein has been extensively studied in neurons, the protein is also expressed in other cell types including immune cells and glia. Genetic studies show that mutations in synuclein alpha (SNCA), the gene that encodes alpha-synuclein, and alterations in its expression levels are a significant risk factor for PD, which likely impact the functions of a broad range of cell types. The consequences of altered SNCA expression in other cell types is beginning to be explored. Microglia, the primary macrophage population in the Central Nervous System (CNS), for example, are affected by variations in alpha-synuclein levels and functions. Studies suggest that deviations of alpha-synuclein’s normal activity influence hematopoiesis, the process that gives rise to microglia, and microglia’s immune functions. Alpha-synuclein levels also dictate the efficiency of SNARE-mediated vesicle formation, which could influence autophagy and cytokine release in microglia. Starting from the time of conception, these effects could impact one’s risk for developing PD. Further studies are needed to determine the physiological role of alpha-synuclein and how the protein is affected during PD in non-neuronal cells such as microglia. In this review we will discuss the known roles of alpha-synuclein in differentiation, immune responses, and vesicle formation, with insights into how abnormal alpha-synuclein expression and activity are linked to altered functions of microglia during PD.

Potential and Whole-genome Sequence-based Mechanism of Elongated-prismatic Magnetite Magnetosome Formation in Acidithiobacillus Ferrooxidans BYM

A magnetosome-producing bacterium Acidithiobacillus ferrooxidans BYM (At. ferrooxidans BYM) was isolated and magnetically screened. The magnetosome yield from 0.5896 to 13.1291 mg/g was achieved under different aeration rates, ferrous sulfate, ammonium sulfate, and gluconic acid concentrations at 30 ℃. TEM observed 6–9 magnetosomes in size of 20–80 nm irregularly dispersed in a cell. STEM-EDXS and HRTEM-FFT implied that the elongated-prismatic magnetite magnetosomes with {110} crystal faces grown along the [111] direction. Whole-genome sequencing and annotation of BYM showed that 3.2 Mb chromosome and 47.11 kb plasmid coexisted, and 322 genes associated with iron metabolism were discovered. Ten genes shared high similarity with magnetosome genes were predicted, providing sufficient evidence for the magnetosome-producing potential of BYM. Accordingly, we first proposed a hypothetic model of magnetosome formation including vesicle formation, iron uptake and mineralization, and magnetite crystal maturation in At. ferrooxidans. These indicated that At. ferrooxidans BYM would be used as a commercial magnetosome-producing microorganism.

Abstract P419: Revealing The Dynamics Of Vesicle Formation During Clathrin-mediated Endocytosis In Living Endothelial Cells

Clathrin-mediated endocytosis (CME) is an essential cellular process for internalizing nutrients and therapeutics at endothelial cell barriers. Studying the formation of cargo containing endocytic vesicles in living cells is challenging due to the limited resolution of fluorescence microscopy and the highly dynamic nature of CME. Moreover, it is currently unknown how the physiological conditions present in vasculature affect CME in endothelial cells. To address this challenge we used a novel microscopy approach, Simultaneous Two-wavelength Axial Ratiometry (STAR), to image vesicle formation dynamics with nanometer axial resolution in living cells. High-throughput analysis revealed that 80% of de novo clathrin accumulations contributed to endocytosis while 20% remained flat, consistent in both human umbilical vein endothelial cells (HUVECs) and our test-bed model green monkey kidney fibroblast-like (Cos-7) cells. We next investigated the interplay between coat curvature and clathrin accumulation in vesicle initiation to identify the mechanism of vesicle formation. Our results support the flexible model of vesicle formation with curvature and clathrin accumulation initiating together at shorter-lived vesicles (<20s) and through a flat-to-curved transition of clathrin lattices at longer-lived vesicles (>20s). Finally, we addressed if physiological conditions present in vasculature alter the dynamics of vesicle formation. We show that increasing osmotic pressure decreased the total number of internalizations but had no impact on the number of flat clathrin accumulations or the mechanism of vesicle formation in Cos-7 cells. In future research, we will test the hypothesis that HUVECs have distinct mechanisms to retain vesicle formation under osmotic pressure or shear stress conditions similar to their native environment. Additionally targeted drug delivery to vascular endothelial cells, for example nanocarriers binding to flat lattices or CCVs leading to different therapeutic outcomes or bioavailability, can potentially be informed by identifying clathrin morphology and dynamics and the mechanisms of endocytosis using STAR microscopy.