Structure and Dynamics of Cell Membranes and Lipid Nanoparticles Frédéric PRZYBILLA and Toshihide KOBAYASHI

Our body contains thousands of different lipid species. These lipids are not randomly distributed in different organs and different organelles. Even in one membrane, the lipid composition of the outer and inner leaflets of the lipid bilayer is different. In addition, lipid distribution is heterogeneous in each leaflet. However, how this lipid organization is built, maintained and regulated remains poorly understood as is the physiological significance of lipid organization. We have been developing a wide range of innovative tools (probes, protocols, equipment) that allows the characterization of these lipid organizations in situ and at nanometer scale. In particular, a series of lipid binding proteins (LBPs) were designed and optimized to visualize the distribution of endogenous lipids at the nanoscale.^1,2 Our objective is to use these LBPs to study the molecular mechanisms regulating the distribution of lipids as well as their dynamics. To follow lipid dynamics, we will couple our LBPs with the upconversion nanoparticles (UCNPs) approaches recently developed in the team to monitor the long-term dynamics of lipid nanodomains (NDLs). In addition, we will characterize the structure and intracellular fate of lipid nanoparticles encapsulating mRNAs (LNPs for lipid nano-particles) used in vaccine strategies.

Uncovering how HIV-1 assembles its lipid coat, which allows the virus to enter cells

Human immunodeficiency virus type 1 (HIV-1) lipid envelope is obtained during budding from the plasma membrane of infected host cells. Interestingly, HIV-1 selects specific set of lipids such as sphingomyelin (SM) and cholesterol (Chol) that are important for virus activity. Viral Gag protein plays a central role in virus budding. Different quantitative and super-resolution microscopy techniques in combination with SM-specific and Chol-specific LBPs revealed that the expressed Gag recruits SM and Chol and induces fusion of SM-rich and Chol-rich domains.³ SM is distributed in the outer leaflet of the plasma membrane whereas Gag binds to the inner leaflet lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). Thus, communication between outer and inner leaflet lipids is crucial for lipid recruitment by Gag. However, little is known about the interbilayer lipid communication in the biomembranes. Using molecular genetics and various LBPs, we will study molecular mechanisms that regulate interbilayer lipid communication. Our study will uncover how HIV-1 assembles its lipid coat. Our study will also provide essential information on membrane-mediated signal transduction.

Upconversion nanoparticles functionalized with lipid specific peptides for long-term studies of the dynamics of lipid domains in living cells

Mechanisms driving and regulating lateral membrane heterogeneity are poorly understood, due to difficulties to visualize and monitor these small and highly dynamic lipid domains. Single particle tracking (SPT) is a powerful technique to study membrane dynamics and organization, but suffers from the lack of well-validated probes allowing direct observation of endogenous lipids and lipid domains with high spatial and temporal resolution over extended timescales and large areas. In order to address these challenges we are developing innovative SPT probes based on the unique combination of protein-sized upconversion nanoparticles (UCNPs) and well validated lipid binding proteins (LBPs) able to recognize specifically LNDs. The key property of UCNPs is their anti-Stokes emission that converts near infra-red excitation to UV/Visible emission, enabling their detection with a high signal-to-noise ratio (SNR), even in a complex environment. UCNPs exhibit all properties of an ideal label for long-term SMT studies due to their (i) high SNR, (ii) non-blinking and (iii) photostable emission, (iv) small size, and (v) low photo toxicity.^4–6 To achieve this goal, we will employ recently-developed phospholipid coated UCNPs that are bright and stable in cell culture media and show only marginal unspecific binding to cell membranes. UCNP-LBPs probes will allows us to track lipids and lipid domains at the plasma membrane of living cells. Studies of lipid dynamics in different pathological cells with altered membrane composition will provide additional information on the roles of SM and Chol in the formation and dynamics of lipid domains and disease-related alterations of the plasma membrane.

Monitoring the intracellular fate of mRNA-loaded lipid nanoparticles (LNPs)

mRNA-based therapies hold great promise for treating a wide range of diseases. To deliver mRNAs into cells, LNPs are the most advanced platforms, but lipid organizations and their evolution during cell trafficking (endocytosis, endosomal escape, unpacking and packaging into extracellular vesicles) remain unclear. With our toolbox dedicated to lipid studies, we propose to follow the evolution of their structural characteristics during the different stages of their intracellular life. With environmentally sensitive fluorescent dyes, we are able to follow the physical properties of LNPs and with LBPs, we will be able to measure the distribution of lipids in LNPs in the cytoplasm. In addition, we will also fluorescently label mRNAs with the nuclear analog (thG- and tzG developed in axis 1). These probes are be able to use without affecting the structure of LNP and to characterize the intracellular fate of mRNA. Simultaneous monitoring of mRNA and lipid organization at all stages, from cell internalization to translation into mRNA-encoded protein (typically luciferase or eGFP) or leakage as EVs, has never been done before. This set of experiences should allow to easily evaluating the impact of changes in the composition of the LNPs, and external factors, such as storage conditions to the efficient of mRNA delivery and help to design optimized vaccines.

Caption : Characterization and monitoring of the intracellular fate of lipid nanoparticles (LNPs) delivering mRNA.

1.            Tomishige, N., Takahashi, K., Pollet, B., Richert, L., Mély, Y. & Kobayashi, T. in Methods in Enzymology700, 217–234 (Elsevier, 2024).

2.            Kobayashi, T., Tomishige, N., Inaba, T., Makino, A., Murata, M., Yamaji-Hasegawa, A. & Murate, M. Impact of Intrinsic and Extrinsic Factors on Cellular Sphingomyelin Imaging with Specific Reporter Proteins. Contact4, 251525642110424 (2021).

3.            Tomishige, N., Bin Nasim, M., Murate, M., Pollet, B., Didier, P., Godet, J., Richert, L., Sako, Y., Mély, Y. & Kobayashi, T. HIV-1 Gag targeting to the plasma membrane reorganizes sphingomyelin-rich and cholesterol-rich lipid domains. Nat Commun14, 7353 (2023).

4.            Dukhno, O., Przybilla, F., Collot, M., Klymchenko, A., Pivovarenko, V., Buchner, M., Muhr, V., Hirsch, T. & Mély, Y. Quantitative assessment of energy transfer in upconverting nanoparticles grafted with organic dyes. Nanoscale9, 11994–12004 (2017).

5.            Dukhno, O., Przybilla, F., Muhr, V., Buchner, M., Hirsch, T. & Mély, Y. Time-dependent luminescence loss for individual upconversion nanoparticles upon dilution in aqueous solution. Nanoscale10, 15904–15910 (2018).

6.            Dukhno, O., Ghosh, S., Greiner, V., Bou, S., Godet, J., Muhr, V., Buchner, M., Hirsch, T., Mély, Y. & Przybilla, F. Targeted Single Particle Tracking with Upconverting Nanoparticles. ACS Appl. Mater. Interfaces16, 11217–11227 (2024).