Karakus, C. | bioRxiv Science

bioRxiv2026

Application of D4 Fluorescent Probes for Quantitative and Spatial Analysis of Cholesterol in Cells

Cholesterol is a key component of cellular membranes, regulating membrane organization, fluidity, and signaling. However, cholesterol analysis remains technically challenging, as no single method currently allows both accurate quantification and spatially resolved visualization. Biochemical assays provide accurate quantification but lack spatial resolution, whereas imaging strategies can perturb membrane organization or cholesterol accessibility. Here, we describe optimized protocols using fluorescent D4 probes derived from the cholesterol-binding domain of perfringolysin O (D4-mCherry and D4-GFP) to detect, visualize, and quantify cholesterol in biological samples. We detail procedures for probe production, purification, and application, and establish conditions that ensure robust and reproducible labeling of membrane-accessible cholesterol. By combining fluorescence-based imaging with quantitative analysis, this approach enables the assessment of cholesterol distribution while preserving its native membrane environment. The proposed methodology provides a versatile and reliable framework for studying cholesterol in a wide range of experimental systems.

biochemistry↗

bioRxiv2026

Ganglioside GM1-enriched rafts regulate the neuronal chloride co-transporter 1 KCC2.

During brain development, dynamic remodeling of membrane lipid composition accompanies the maturation of inhibitory neurotransmission and the progressive establishment of low intracellular chloride levels. Central to this developmental transition is the neuronal K-Cl- cotransporter KCC2, whose stabilization at the plasma membrane enables the emergence of hyperpolarizing GABAergic signaling. Although KCC2 regulation by protein partners has been extensively characterized, whether lipid remodeling actively contributes to its membrane organization and chloride transport remains unclear. Here we identify the ganglioside GM1, a complex lipid abundant in plasma membrane of neurons, as a developmentally regulated lipid determinant of KCC2 membrane localization and function. We show that KCC2 interacts with GM1 within plasma membrane lipid rafts and that this interaction increases during postnatal brain maturation. Molecular modeling identified a conserved ganglioside-binding domain (GBD) in KCC2 centered on tryptophan 318 (W318). Biophysical analyses revealed a specific and saturable interaction between this domain and GM1 that is abolished by the epilepsy-associated W318S mutation. Disruption of KCC2-GM1 interactions, either by W318S mutation or by pharmacological depletion of GM1, excludes KCC2 from lipid rafts, alters its membrane diffusion and clustering, and reduces its surface stability. Functionally, these perturbations impair KCC2-mediated chloride extrusion and disrupt the somato-dendritic chloride gradient in hippocampal neurons. Consistent with these cellular effects, GM1-deficient (St3gal5-/-) mice exhibit selective reduced hippocampal KCC2 expression. Together, these findings reveal a lipid-protein mechanism that links developmental membrane remodeling to KCC2 stabilization and chloride homeostasis, highlighting membrane lipids as active regulators of transporter maturation and inhibitory circuit development.

neuroscience↗