Heimo Wolinski

Lipid droplets are central hubs of cellular lipid metabolism and play crucial roles in health and disease. The yeast Saccharomyces cerevisiae serves as a powerful model for studying lipid droplet biology at the cellular level. Estimation of the number and size distribution of lipid droplets in a cell population is critical for functional studies. However, accurate segmentation and quantification of lipid droplets through image-based methods present significant challenges. Organelle motion, point-spread function (PSF) overlap of closely associated organelles and heterogeneous fluorescence labeling often compromise histogram-, shape- or machine-learning-based methods. Here, we present an alternative, seeding-based radial raytracing approach that is independent of image histograms and considers the PSF-blurred nature of imaged organelles. To improve lipid droplet resolution and contrast, we applied custom 3D deconvolution and integrated a supervised machine-learning step that corrected typical deconvolution artifacts. An integrated, extensible toolset including deep-learning-based cell registration enables detailed lipid droplet statistics, including user-defined size classes. As a proof of principle, we applied our approach to analyze lipid droplets in wild-type and mutant cells lacking the Sei1-Ldb16 seipin complex required for regular lipid droplet biogenesis.

White adipose tissue (WAT) comprises a plethora of cell types beyond adipocytes forming a regulatory network that ensures systemic energy homeostasis. Intertissue communication is facilitated by metabolites and signaling molecules that are spread by vasculature and nerves. Previous works have indicated that WAT responds to environmental cues by adapting the abundance of these ‘communication routes’; however, the high intra-tissue heterogeneity questions the informative value of bulk or single-cell analyses and underscores the necessity of whole-mount imaging. The applicability of whole-mount WAT-imaging is currently limited by two factors – (1) methanol-based tissue clearing protocols restrict the usable antibody portfolio to methanol-resistant antibodies and (2) the vast amounts of data resulting from 3D imaging of whole-tissue samples require high computational expertise and advanced equipment. Here, we present a protocol for whole-mount WAT clearing, overcoming the constraints of antibody-methanol sensitivity. Additionally, we introduce TiNeQuant (for ‘tissue network quantifier’) a Fiji tool for automated 3D quantification of neuron or vascular network density, which we have made freely available. Given TiNeQuants versatility beyond WAT, it simplifies future efforts studying neuronal or vascular alterations in numerous pathologies.

The one‐pot synthesis of multicomponent hydrogen‐bonded organic framework (HOF) biocomposites is reported. The co‐immoblization of enzymes and magnetic nanoparticles (MNPs) into the HOF crystals yielded biocatalysts (MNPs‐enzyme@BioHOF‐1) with dynamic localization properties. Using a permanent magnet, it is possible to separate the MNPs‐enzyme@BioHOF‐1 particles from a solution. Catalase (CAT) and glucose oxidase (GOx) show increased retention of their activity when coimmobilized with MNPs. MNPs‐GOx@BioHOF‐1 biocomposites are used to prepare a proof‐of‐concept glucose microfluidic biosensor, where a magnet allow to position and keep in place the biocomposite inside a microfluidic chip. The magnetic response of these biocatalysts can pave the way for new applications for the emerging HOF biocomposites.

We report the one-pot synthesis of multicomponent hydrogen-bonded organic framework (HOF) biocomposites. The coimmoblization of magnetic nanoparticles (MNPs) and enzymes into the HOF crystals yielded a nano-composite biocatalytic material (MNPs-enzyme@BioHOF-1) that posses dynamic localization properties. Using a permanent magnet, it is possible to separate the MNPs-enzyme@BioHOF-1 particles from a solution. When coimmobilized with MNPs, Catalase (CAT) and glucose oxidase (GOx) showed an increased retention of their activity when coimmobilized with MNPs. MNPs-GOx@BioHOF-1 biocomposites were used to prepare a proof-of-concept glucose microfluidic biosensor, where a magnet allowed to position and keep in place the biocomposite inside a microfluidic chip. The magnetic response of these biocatalysts can pave the way to new applications for the emerging HOF biocomposites

Films and patterns of 3D‐oriented metal‐organic frameworks (MOFs) afford well‐ordered pore structures extending across centimeter‐scale areas. These macroscopic domains of aligned pores are pivotal to enhance diffusion along specific pathways and orient functional guests. The anisotropic properties emerging from this alignment are beneficial for applications in ion conductivity and photonics. However, the structure of 3D‐oriented MOF films and patterns can rapidly degrade under humid and acidic conditions. Thus, more durable 3D‐ordered porous systems are desired for practical applications. Here, oriented porous polymer films and patterns are prepared by using heteroepitaxially oriented N₃‐functionalized MOF films as precursor materials. The film fabrication protocol utilizes an azide–alkyne cycloaddition on the Cu₂(AzBPDC)₂DABCO MOF. The micropatterning protocol exploits the X‐ray sensitivity of azide groups in Cu₂(AzBPDC)₂DABCO, enabling selective degradation in the irradiated areas. The masked regions of the MOF film retain their N₃‐functionality, allowing for subsequent cross‐linking through azide‐alkyne coupling. Subsequent acidic treatment removes the Cu ions from the MOF, yielding porous polymer micro‐patterns. The polymer has high chemical stability and shows an anisotropic fluorescent response. The use of 3D‐oriented MOF systems as precursors for the fabrication of oriented porous polymers will facilitate the progress of optical components for photonic applications.

While aggregated alpha-Synuclein (αSyn) is commonly associated with Parkinson’s disease, its physiological function as a membrane-binding protein is poorly understood. Here, we show that endogenous αSyn binds lipid droplets (LDs) in multiple human cell lines and in stem cell-derived dopaminergic neurons. LD-binding encompasses αSyn residues 1-100, which masks their detection by immunofluorescence microscopy, probably explaining the scarcity of similar observations in earlier studies. αSyn-LD interactions are highly temperature-sensitive and selective for cholesteryl-ester-rich LDs. They promote the formation of αSyn multimers that dissociate from LDs at non-permissive temperatures. αSyn remains LD-bound throughout starvation-induced lipolysis, whereas siRNA-knockdown diminishes LD abundance and compromises cell viability upon nutrient depletion, without affecting LD biosynthesis. Reciprocally, excess αSyn stimulates LD accumulation in dependence of lipid availability, restricts organelle size and ensures intracellular LD organization, which strictly depends on functional membrane-binding. Supporting a general role of αSyn in cellular lipid and cholesterol metabolism, our results point to additional loss-of-function similarities between Parkinson’s, Alzheimer’s and Gaucher’s disease.

Bis(monoacylglycero)phosphate (BMP) is a major phospholipid constituent of intralumenal membranes in late endosomes/lysosomes, where it regulates the degradation and sorting of lipid cargo. Recent observations suggest that the Batten disease - associated protein CLN5 functions as lysosomal BMP synthase. Here, we show that transacylation reactions catalyzed by cytosolic and secreted enzymes enhance BMP synthesis independently of CLN5. The transacylases identified in this study are capable of acylating the precursor lipid phosphatidylglycerol (PG), generating acyl-PG, which is subsequently hydrolyzed to BMP. Extracellularly, acyl-PG and BMP are generated by endothelial lipase in cooperation with other serum enzymes of the pancreatic lipase family. The intracellular acylation of PG is catalyzed by several members of the cytosolic phospholipase A2 group IV (PLA2G4) family. Overexpression of secreted or cytosolic transacylases was sufficient to correct BMP deficiency in HEK293 cells lacking CLN5 . Collectively, our observations suggest that functionally overlapping pathways promote BMP synthesis in mammalian cells.

Films and patterns of 3D-oriented metal-organic frameworks (MOFs) afford well-ordered pore structures extending across centimeter-scale areas. These macroscopic domains of aligned pores are pivotal to enhance diffusion along specific pathways and orient functional guests. The anisotropic properties emerging from this alignment are beneficial for applications in ion conductivity and photonics. However, the structure of 3D-oriented MOF films and patterns can rapidly degrade under humid and acidic conditions. Thus, more durable 3D-ordered porous systems are desired for practical applications. Here, oriented porous polymer films and patterns are prepared by using heteroepitaxially oriented N3-functionalized MOF films as precursor materials. The film fabrication protocol utilizes an azide–alkyne cycloaddition on the Cu2(AzBPDC)2DABCO MOF. The micropatterning protocol exploits the X-ray sensitivity of azide groups in Cu2(AzBPDC)2DABCO, enabling selective degradation in the irradiated areas. The masked regions of the MOF film retain their N3-functionality, allowing for subsequent cross-linking through azide-alkyne coupling. Subsequent acidic treatment removes the Cu ions from the MOF, yielding porous polymer micro-patterns. The polymer has high chemical stability and shows an anisotropic fluorescent response. The use of 3D-oriented MOF systems as precursors for the fabrication of oriented porous polymers will facilitate the progress of optical components for photonic applications.

Sterols exert a profound influence on numerous cellular processes, playing a crucial role in both health and disease. However, comprehending the effects of sterol dysfunction on cellular physiology is challenging. Consequently, numerous processes affected by impaired sterol biosynthesis still elude our complete understanding. In this study, we made use of yeast strains that produce cholesterol instead of ergosterol and investigated the cellular response mechanisms on the transcriptome as well as the lipid level. The exchange of ergosterol for cholesterol caused the downregulation of phosphatidylethanolamine and phosphatidylserine and upregulation of phosphatidylinositol and phosphatidylcholine biosynthesis. Additionally, a shift towards polyunsaturated fatty acids was observed. While the sphingolipid levels dropped, the total amounts of sterols and triacylglycerol increased, which resulted in 1.7-fold enlarged lipid droplets in cholesterol-producing yeast cells. In addition to internal storage, cholesterol and its precursors were excreted into the culture supernatant, most likely by the action of ABC transporters Snq2, Pdr12 and Pdr15. Overall, our results demonstrate that, similarly to mammalian cells, the production of non-native sterols and sterol precursors causes lipotoxicity in K. phaffii, mainly due to upregulated sterol biosynthesis, and they highlight the different survival and stress response mechanisms on multiple, integrative levels.

Micropatterning crystalline materials with oriented pores is necessary for the fabrication of devices with anisotropic properties. Crystalline and porous metal–organic frameworks (MOFs) are ideal materials as their chemical and structural mutability enables precise tuning of functional properties for applications ranging from microelectronics to photonics. Herein, a patternable oriented MOF film is designed: by using a photomask under X‐ray exposure, the MOF film decomposes in the irradiated areas, remaining intact in the unexposed regions. The MOF film acts simultaneously as a resist and as functional porous material. While the heteroepitaxial growth from aligned Cu(OH)₂ nanobelts is used to deposit oriented MOF films, the sensitivity to radiation is achieved by integrating a brominated dicarboxylate ligand (Br₂BDC) into a copper‐based MOF Cu₂L₂DABCO (DABCO = 1,4‐diazabicyclo[2.2.2]octane; L = BDC/Br₂BDC). The lithographed samples act as diffraction gratings upon irradiation with a laser, thus confirming the quality of the extended MOF micropattern. Furthermore, the oriented MOF patterns are functionalized with fluorescent dyes. As a result, by rotating the polarization angle of the laser excitation, the alignment of the dye in the MOF is demonstrated. By controlling the functional response to light, this MOF patterning protocol can be used for the microfabrication of optical components for photonic devices.