Showing papers by "Daniel Wüstner published in 2021"

Substituted 9-Diethylaminobenzo[a]phenoxazin-5-ones (Nile Red Analogues): Synthesis and Photophysical Properties.

Abstract: Nile Red is a benzo[a]phenoxazone dye containing a diethylamino substituent at the 9-position. In recent years, it has become a popular histological stain for cellular membranes and lipid droplets due to its unrivaled fluorescent properties in lipophilic environments. This makes it an attractive lead for chemical decoration to tweak its attributes and optimize it for more specialized microscopy techniques, e.g., fluorescence lifetime imaging or two-photon excited fluorescence microscopy, to which Nile Red has never been optimized. Herein, we present synthesis approaches to a series of monosubstituted Nile Red derivatives (9-diethylbenzo[a]phenoxazin-5-ones) starting from 1-naphthols or 1,3-naphthalenediols. The solvatochromic responsiveness of these fluorophores is reported with focus on how the substituents affect the absorption and emission spectra, luminosity, fluorescence lifetimes, and two-photon absorptivity. Several of the analogues emerge as strong candidates for reporting the polarity of their local environment. Specifically, the one- and two-photon excited fluorescence of Nile Red turns out to be very responsive to substitution, and the spectroscopic features can be finely tuned by judiciously introducing substituents of distinct electronic character at specific positions. This new toolkit of 9-diethylbenzo[a]phenoxazine-5-ones constitutes a step toward the next generation of optical molecular probes for advancing the understanding of lipid structures and cellular processes.

Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells

Abstract: Mitochondria receive cholesterol from late endosomes and lysosomes (LE/LYSs) or from the plasma membrane for production of oxysterols and steroid hormones. This process depends on the endo-lysosomal sterol transfer protein Niemann Pick C2 (NPC2). Using the intrinsically fluorescent cholesterol analog, cholestatrienol, we directly observe sterol transport to mitochondria in fibroblasts upon treating NPC2 deficient human fibroblasts with NPC2 protein. Soft X-ray tomography reveals the ultrastructure of mitochondria and discloses close contact to endosome-like organelles. Using fluorescence microscopy, we localize endo-lysosomes containing NPC2 relative to mitochondria based on the Euclidian distance transform and use statistical inference to show that about 30% of such LE/LYSs are in contact to mitochondria in human fibroblasts. Using Markov Chain Monte Carlo image simulations, we show that interaction between both organelle types, a defining feature of membrane contact sites (MCSs) can give rise to the observed spatial organelle distribution. We devise a protocol to determine the surface fraction of endo-lysosomes in contact with mitochondria and show that this fraction does not depend on functional NPC1 or NPC2 proteins. Finally, we localize MCSs between LE/LYSs containing NPC2 and mitochondria in time-lapse image sequences and show that they either form transiently or remain stable for tens of seconds. Lasting MCSs between endo-lysosomes containing NPC2 and mitochondria move by slow anomalous sub-diffusion, providing location and time for sterol transport between both organelles. Our quantitative imaging strategy will be of high value for characterizing the dynamics and function of MCSs between various organelles in living cells.

Transient accumulation and bidirectional movement of KIF13B in primary cilia

Abstract: Primary cilia are microtubule-based sensory organelles whose assembly and function rely on the conserved bidirectional intraflagellar transport (IFT) system, which is powered by anterograde kinesin-2 and retrograde cytoplasmic dynein 2 motors. Nematodes additionally employ a male-specific kinesin-3 motor, KLP-6, which regulates ciliary content and function by promoting release of bioactive extracellular vesicles (EVs) from cilia. Here we show by live cell imaging that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured mammalian cells at 0.64 {+/-} 0.07 m/s in the anterograde direction and at 0.39 {+/-} 0.06 m/s in the retrograde direction, reminiscent of conventional IFT. In addition, we found that KIF13B undergoes EV-like release from the ciliary tip whereas a ciliary membrane marker, SMO-tRFP, remains stably associated with cilia during such EV release. Our results suggest that KIF13B, similar to KLP-6, regulates ciliary membrane content by promoting ciliary EV release, possibly in coordination with conventional IFT.

Transient accumulation, bidirectional movement and tip release of KIF13B in primary cilia

Abstract: Primary cilia are microtubule-based sensory organelles whose assembly and function rely on the conserved bidirectional intraflagellar transport (IFT) system, which is powered by anterograde kinesin-2 and retrograde cytoplasmic dynein 2 motors. Nematodes additionally employ a male-specific kinesin-3 motor, KLP-6, which regulates ciliary content and function by promoting release of bioactive extracellular vesicles (EVs) from cilia. Here we provide evidence that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured mammalian cells, as well as occasional EV-like release from the ciliary tip. KIF13B intraciliary movement and tip release required its own motor domain, and a ciliary membrane marker, SMO-tRFP, was not co-released with KIF13B from cilia. Our work provides the first demonstration of intraciliary movement by a vertebrate kinesin other than IFT kinesin-2 motors and suggests that KIF13B regulates ciliary membrane content by promoting EV release at specific ciliary membrane subdomains.

Design, Synthesis, and Evaluation of a Luminescent Cholesterol Mimic

Abstract: The development of new chemical tools with improved properties is essential to chemical and cell biology. Of particular interest is the development of mimics of small molecules with important cellu...

Photophysical and Structural Characterization of Intrinsically Fluorescent Sterol Aggregates.

Abstract: Self-association of cholesterol into aggregates and crystals is a hallmark of developing atherosclerosis Intrinsically fluorescent sterols, such as dehydroergosterol (DHE), can be used to study sterol aggregation by fluorescence spectroscopy and microscopy, but a thorough understanding of DHE's photophysical and structural properties in the aggregated state is missing Here, we show that DHE forms submicron fluorescent aggregates when evaporated from an ethanol solution Using atomic force microscopy, we find that DHE, like cholesterol, forms compact oblate-shape aggregates of <100 nm in diameter DHE's fluorescence is lowered in the aggregate compared to the monomeric form, and characteristic spectral changes accompany the aggregation process Electronic structure calculations of DHE dimers in water indicate that Frenkel-type exciton coupling contributes to the lowered DHE fluorescence in the aggregates Using molecular dynamics (MD) simulations, we show that DHE forms compact aggregates on the nanosecond scale and with strong intermolecular attraction, in which a broad range of orientations, and therefore electronic couplings, will take place Tight packing of DHE in aggregates also lowers the apparent absorption cross section, further reducing the molecular brightness of the aggregates Our results pave the way for systematic solubility studies of intrinsically fluorescent analogues of biologically relevant sterols