Biology subjects

Reinholdt, P.

Publications and source records attributed to Reinholdt, P..

4 recordsLinked to original sources

bioRxiv2026

The orientation of cholesterol's hydroxyl group affects its membrane dynamics and intracellular transport

The brain, though less than 10% of body mass, contains about 25% of total cholesterol (CHL), emphasizing CHLs key role in neuronal function. Many CHL actions are stereospecific, as shown by differences from its 3-hydroxy epimer, epicholesterol (epiCHL). How this minor structural change alters membrane properties and sterol transport remains unclear. Here, we compare fluorescent analogs of CHL (cholestatrienol, CTL) and epiCHL (epicholestatrienol, epiCTL), which closely mimic their natural counterparts. Biophysical membrane properties, such as flip-flop, acyl-chain ordering, and interbilayer transfer, depend on the orientation of the 3-hydroxy group. Similarly, transport by sterol transport proteins (STPs) and intracellular trafficking of the sterols in human astrocytes are stereospecific. Treatment with 25-hydroxycholesterol increases uptake of both epimers, but only CTL shows enhanced esterification and lipid droplet storage. These findings demonstrate that subtle cholesterol structural changes affect cellular homeostasis and establish epiCTL as a useful probe of sterol stereospecificity and trafficking.

biophysics↗

bioRxiv2025

A Novel Model for Proton Transport Mediated by Uncoupling Protein 1

Uncoupling Protein 1 (UCP1) is a mitochondrial protein which drives thermogen-esis in brown adipose tissue. UCP1 facilitates the dissipation of the proton gradient as heat and plays a critical role in energy expenditure and metabolic regulation. We employ advanced molecular simulations and mutagenesis to reveal the mechanism of UCP1-mediated proton and fatty acid (FA) transport. We demonstrate that FAs bind spontaneously to UCP1s central substrate-binding site. In the binding site, a proton transfer to the FA is facilitated by a key aspartate residue (D28) and a coordinating water molecule. The protonated FA exits UCP1 through a well defined pathway, and releases its proton into the mitochondrial matrix. UCP1 then facilitates the return of deprotonated FAs to the intermembrane space. Nucleotide binding disrupts this mechanism by inducing conformational changes in the transmembrane helices and ob-structing the FA return pathway. Our mechanism explains every step of the transport cycle, is supported by simulation and biochemical data, and explains a diverse set of biochemical data about the transport mechanisms in UCP1 and its analogues: ANT, UCP2 and UCP3.

biophysics↗

bioRxiv2024

Ratiometric fluorescence nanoscopy and lifetime imaging of novel Nile Red analogs for analysis of membrane packing in living cells

Subcellular membranes have complex lipid and protein compositions, which give rise to organelle-specific membrane packing, fluidity, and permeability. Due to its exquisite solvent sensitivity, the lipophilic fluorescence dye Nile Red has been used extensively to study membrane packing and polarity. Further improvement of Nile Red can be achieved by introducing electron-donating or withdrawing functional groups. Here, we compare the potential of derivatives of Nile Red with such functional substitutions for super-resolution fluorescence microscopy of lipid packing in model membranes and living cells. All studied Nile Red derivatives exhibit cholesterol-dependent fluorescence changes in model membranes, as shown by spectrally resolved stimulated emission depletion (STED) microscopy. STED imaging of Nile Red probes in cells reveals lower membrane packing in fibroblasts from healthy subjects compared to those from patients suffering from Niemann Pick type C1 (NPC1) disease, a lysosomal storage disorder with accumulation of cholesterol and sphingolipids in late endosomes and lysosomes. We also find small but consistent changes in the fluorescence lifetime of the Nile Red derivatives in NPC1 cells, suggesting altered hydrogen-bonding capacity in their membranes. All Nile Red derivatives are essentially non-fluorescent in water but increase their brightness in membranes, allowing for their use in MIN-FLUX single molecule tracking experiments. Our study uncovers the potential of Nile Red probes with functional substitutions for nanoscopic membrane imaging.

biophysics↗

bioRxiv2023

Synthesis and characterization of novel intrinsically fluorescent analogs of cholesterol with improved photophysical properties.

Live-cell imaging of cholesterol trafficking depends on suitable cholesterol analogs. However, existing fluorescent analogs of cholesterol either show very different physico-chemical properties compared to cholesterol or demand excitation in the ultraviolet spectral region. We present novel intrinsically fluorescent sterols containing four conjugated double bonds in the ring system and either a hydroxy or a keto group in the C3 position. Synthesis of these probes involves dehydrogenation of 7-dehydrocholesterol using mercury(II) acetate, Swern oxidation/dehydrogenation, and stereoselective Luche reduction. Molecular dynamics simulations and nuclear magnetic resonance spectroscopy reveal that the analog with a 3-hydroxy-group like cholesterol can condense fatty acyl chains and form hydrogen bonds to water molecules at the bilayer interface. The emission of both probes is red-shifted by 80-120 nm compared to the widely used sterol analogs dehydroergosterol or cholestatrienol. This allows for their imaging on conventional microscopes, as we here show in giant unilamellar vesicles. These experiments reveal a preferred partitioning of both sterol probes into the biologically relevant liquid-ordered phase. In conclusion, we present a synthesis strategy leading to novel intrinsically fluorescent sterol probes with close resemblance of cholesterol. Their improved photophysical properties will allow for live-cell imaging of sterol transport in the future.

biophysics↗