Abstract
Despite more than 20 years of work since the lipid raft concept was proposed, the existence of these nanostructures remains highly controversial due to the lack of noninvasive methods to investigate their native nanorganization in living unperturbed cells. There is an unmet need for probes for direct imaging of nanoscale membrane dynamics with high spatial and temporal resolution in living cells. In this paper, a bioorthogonal-based cholesterol probe (chol-N3 ) is developed that, combined with nanoscopy, becomes a new powerful method for direct visualization and characterization of lipid raft at unprecedented resolution in living cells. The chol-N3 probe mimics cholesterol in synthetic and cellular membranes without perturbation. When combined with live-cell super-resolution microscopy, chol-N3 demonstrates the existence of cholesterol-rich nanodomains of <50 nm at the plasma membrane of resting living cells. Using this tool, the lipid membrane structure of such subdiffraction limit domains is identified, and the nanoscale spatiotemporal organization of cholesterol in the plasma membrane of living cells reveals multiple cholesterol diffusion modes at different spatial localizations. Finally, imaging across thick organ samples outlines the potential of this new method to address essential biological questions that were previously beyond reach.
Highlights
Biological membranes are poised to laterally self organize, forming defined platforms required for proper cell functioning
To investigate the existence of chol-rich nanodomains in living cells, we undertook the synthesis of a chol chemical probe, cholN3, based on bioorthogonal chemistry that can be used to explore many unknown features of lipid raft in living cell membranes
Considering that the maximum colocalization coefficient obtained for chol imaged in the confocal versus similar within a diffraction-limited (STED) mode was 0.814 (Figure S10D,E, right panels, Supporting Information), the results show that more than 75% of GPI-anchor CD59 protein localize into cholenriched nanodomains in the cell surface of resting living cells
Summary
Biological membranes are poised to laterally self organize, forming defined platforms required for proper cell functioning. Those solvatochromic dyes only inform about membrane order state without giving accurate information about the lipid and protein composition of those domains.[8] Alternatively, secondary ion mass spectro metry has shown to be a valuable technique to visualize the micro- and nanoscale lateral organization of cellular membranes in situ This technique is performed under ultra-high vacuum and requires sample fixation.[13] To date, lipid chemical tools used to study lipid membranes have not addressed the features of lipid raft and chol dynamics in living cells at the nanoscale, and the challenge to develop membrane lipid probes suitable for live-cell SRM remains unachieved. PM model containing yet unknown features of nanoscale chol dynamics in living cell membranes
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