Biology subjects

Lauritsen, L.

Publications and source records attributed to Lauritsen, L..

6 recordsLinked to original sources

bioRxiv2026

Analysis of motor-based transport in primary cilia by dynamic mode decomposition of live-cell imaging data

Kinesin-3 motor proteins are increasingly recognized for their important roles in cilia. The mammalian kinesin-3 motor KIF13B moves bidirectionally in primary cilia and regulates ciliary content, but its relationship to the intraflagellar transport (IFT) machinery is unclear. Here, we combine quantitative live-cell imaging with a new kymograph analysis based on dynamic mode decomposition (DMD) to separate mobile from immobile protein populations in primary cilia. This approach simplifies extraction of molecular velocities from kymographs and reveals that a KIF13B deletion mutant retaining only the motor domain and part of the forkhead-associated domain does not alter steady-state IFT velocity or frequency. However, when retrograde dynein-2 function is inhibited by Ciliobrevin D, both anterograde and retrograde IFT velocities decrease in parental cells, as expected, but remain unchanged in KIF13B mutant cells. Structured illumination, confocal, and STED microscopy further show that KIF13B localizes to the ciliary membrane and concentrates at the periciliary membrane region and the centriolar subdistal appendages, below the distal appendage marker FBF1. Our improved kymograph approach provides new insight into KIF13B ciliary function and simplifies the quantitative analysis of ciliary protein transport.

biophysics↗

bioRxiv2026

Single-cell analysis of sterol-induced Ca2+ signaling in human astrocytes by dynamic mode decomposition

Ca2+ signaling in astrocytes is a central mechanism of intercellular communication in the brain and plays a key role in regulating neuronal excitability, synaptic plasticity, and energy metabolism. Disruption of astrocytic Ca2+ dynamics is a characteristic of neurodegenerative diseases, as are deviations in cholesterol trafficking and metabolism, which are essential for maintaining membrane structure and function. Although recent studies have begun to explore links between Ca2+ signaling and sterol homeostasis in astrocytes, unbiased analytical workflows and mechanistic insight into how cholesterol and related sterols regulate astrocytic Ca2+ dynamics remain limited. Here, we apply dynamic mode decomposition to dissect and classify Ca2+ signals obtained from time-lapse imaging of human astrocytes. Using both synthetic and experimental datasets, we show that delay-embedded dynamic mode decomposition combined with clustering separates heterogeneous Ca2+ activity into distinct dynamical states. This analysis reveals that increasing cholesterol levels shift astrocytes toward more active oscillatory states, whereas acute cholesterol depletion suppresses Ca2+ activity. In addition, pretreatment with the oxysterols 24-, 25-, and 27-hydroxycholesterol impaired cholesterolinduced Ca2+ oscillations. Together, this work presents a general computational framework for decomposing and analyzing complex spatiotemporal Ca2+ signals, with broad applicability to quantitative imaging in cell biology.

biophysics↗

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↗

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↗

bioRxiv2024

Multimodal intrinsic activation of GPCRs in ultrastable plasma membrane nanodomains

G protein-coupled receptors (GPCRs) mediate many physiological functions and are key targets in drug development1-3. A long-held tenet of molecular pharmacology is that GPCRs can spontaneously sample preexisting active conformations. This concept is pivotal to our understanding of ligand pharmacology4, however, direct evidence supporting it has only been obtained with reconstituted receptors5-12. Here, we introduce a method for quantitatively imaging the intrinsic activation probability of GPCRs directly at the plasma membrane of live cells, utilizing fluorescent conformational biosensors13,14. Our findings unveil a remarkable spatial multimodality in intrinsic activation probability, with a significant majority (up to 99%) of plasma membrane-expressed receptors showing negligible spontaneous activation. In contrast, the remaining minority of receptors exhibits spontaneous activation up to 22-fold higher than previously estimated. Experiments and theoretical calculations revealed that receptors diffuse into and out of ultralong-lived ([~]5 minutes) nanodomains where the local membrane curvature allosterically enhances activation in the absence and presence of ligands. Extensive testing across five prototypic GPCRs indicates spatial nanoscale multimodality is ubiquitous, but varying in magnitude depending on the receptor and cell type. Upending conventional wisdom, this study reveals that drug efficacy is not a constant number but a spatiotemporal function {varepsilon} (x, y, z, t) whose properties define and multiplex the signaling potency and efficacy of ternary complexes of GPCRs and likely other plasma membrane-receptors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/582451v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@e4680borg.highwire.dtl.DTLVardef@16aaebcorg.highwire.dtl.DTLVardef@f5df52org.highwire.dtl.DTLVardef@18a5ea_HPS_FORMAT_FIGEXP M_FIG C_FIG GPCR spontaneous activation and intrinsic efficacy are not uniform across the plasma membrane but exhibit ultralong-lived spatial multimodality. Spatial variations in the curvature and composition of the plasma membrane, lead to the emergence of ultralong-lived nanodomains with contrasting physicochemical properties that allosterically regulate GPCR conformations. This results in a multimodal landscape of intrinsic efficacy{epsilon} (x, y, z, t) that ultimately governs cell signaling. XY scalebar: 500 nm. Z-range: 100 nm.

biophysics↗

bioRxiv2023

Ergosterol mediates aggregation of natamycin in the yeast plasma membrane

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability becomes compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and resulted in reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogs of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. We conclude that ergosterol-specific aggregation of natamycin in the yeast PM underlies its antifungal activity, which can be synergistically enhanced by inhibitors of sphingolipid synthesis. SignificanceErgosterol is the major sterol in the membranes of fungi and a major target for antifungal treatments. Polyene macrolides, such as natamycin, are known to target ergosterol but the underlying mechanisms for their preference for this yeast sterol compared to mammalian cholesterol is not understood. This study shows that natamycin forms aggregates when associated with yeast S. cerevisiae in an ergosterol-dependent manner. Cholesterol can only partially substitute for ergosterol with respect to natamycin binding and aggregation. Membrane-associated aggregation of natamycin is not the result of pre-formed sterol domains in the cell membrane, as we show by direct visualization of minimally modified ergosterol and cholesterol analogs. Inhibiting sphingolipid synthesis increased membrane association and antifungal activity of natamycin, suggesting that targeting sphingolipids in combination with polyene macrolides could lead to novel drug treatment approaches against fungal infections.

biophysics↗