De Beule, P. | Search bioRxiv

bioRxiv2022

Understanding Hybrid Lipid-Polymer Gene Delivery Nanoparticle Assembly with Fluorescence Cross Correlation Spectroscopy

Hybrid lipid-polymer-nucleic acid nanoparticles (LPNPs) provide unique delivery strategies for nonviral gene therapy. Since LPNPs consist of multiple components that can undergo different pairwise interactions between them, LPNPs are difficult to prepare and characterize. Here we demonstrate that the interaction between the polycation (polylysine) and DNA is robust through an innovative implementation of fluorescence cross-correlation spectroscopy, implying that the polycation is not displaced by cationic liposomes in the formation process. Hence, the polycation-DNA cores (polyplexes) and liposome shells must be oppositely charged to associate. Furthermore, we prove that the liposome:polyplex number ratio ({rho}N) is the primary critical parameter to predict stable LPNP formation. We establish that{rho} N [≥] 1 is required to ensure that every polyplex is enveloped by a liposome, avoiding the coexistence of oppositely charged species and thereby inhibiting aggregation. We expect our observations to be valid for the formation of many other LPNPs and composite nanomaterials.

biophysics↗

bioRxiv2021

Interactions of cytosolic termini of the Jen1 monocarboxylate transporter are critical for trafficking, transport activity and endocytosis

Plasma membrane (PM) transporters of the major facilitator superfamily (MFS) are essential for cell metabolism and growth, as well as for survival in response to stress or cytotoxic drugs, in both prokaryotes and eukaryotes. In the yeast Saccharomyces cerevisiae, Jen1 is a monocarboxylate/H+ symporter that has been used to dissect the molecular details underlying control of cellular expression, transport mechanism and turnover of MFS transporters. Here, we present evidence supporting previously non-described roles of the cytosolic N- and C- termini in Jen1 biogenesis, PM stability and activity, through functional analyses of rationally designed truncations and chimeric constructs with UapA, a S. cerevisiae endocytosis-insensitive purine transporter from Aspergillus nidulans. Our results reveal a cryptic role of the N-terminal region and thus show that both cytosolic N- and C-termini are critical for Jen1 trafficking to the PM, transport activity and endocytosis. In particular, we provide evidence that the N- and the C-cytosolic termini of Jen1 undergo transport-dependent dynamic intra-molecular interactions, which critically affect the mechanism of transport and turnover of Jen1. Our results support an emerging concept where the cytosolic tails of PM transporters control transporter expression and function, through flexible intra-molecular interactions with each other and the transmembrane core of the protein. This idea may be extended to other MFS members providing a deeper understanding of conserved, but also evolving, mechanisms underlying MFS transporter structure-function relationships.

microbiology↗