Biology subjects

Zoccoler, M. L.

Publications and source records attributed to Zoccoler, M. L..

4 recordsLinked to original sources

bioRxiv2026

pH-dependent trapping of cationic amphiphilic drugs perturbs insulin granule homeostasis

Pancreatic {beta}-cells store insulin in acidic secretory granules (SGs), specialized organelles that also contain monoamine neurotransmitters such as serotonin. Many neuroactive drugs with monoaminergic activity are cationic amphiphilic drugs (CADs) that accumulate in acidic compartments by pH-dependent trapping. Yet, whether insulin SGs represent a site of CAD accumulation and if this affects their properties such as monoamine storage and pH remain unclear. Here, we show that Slc18a1/VMAT1 is required for vesicular monoamine uptake and maintenance of cellular serotonin levels in insulinoma INS-1 cells. In contrast, neuroactive CADs accumulate via pH-dependent trapping at luminal pH values characteristic of insulin SGs. CADs inhibit VMAT-mediated uptake of the fluorescent monoamine probe FFN206 and induce its efflux to the extracellular space without detectable changes in SG luminal pH. Conversely, natural VMAT substrates such as serotonin and dopamine increase SG pH in a VMAT-dependent manner. These findings identify insulin SGs as acidic organelles susceptible to CAD accumulation and uncover distinct mechanisms regulating secretory granule homeostasis.

cell biology↗

bioRxiv2025

Fluorescence Lifetime Unmixing: A New Workflow for FLIM Live-Cell Imaging

Fluorescence lifetime imaging microscopy (FLIM) translates the duration of excited states of fluorophores into lifetime information as additional source of contrast in images of biological samples. This offers the possibility to separate fluorophores particularly beneficial in case of similar excitation spectra. Here, we demonstrate the distinction of fluorescent molecules based on FLIM phasor analysis, called lifetime unmixing, in live-cell imaging using open-source software for analysis. We showcase two applications using Caenorhabditis elegans as a model system. First, we unmixed the highly spectrally overlapping fluorophores mCherry and mKate2 to distinctively track tagged proteins in six-dimensional datasets to investigate cell division in the developing early embryo. Second, we unmixed fluorescence of tagged proteins of interest from masking natural autofluorescence in adult hermaphrodites. For FLIM data handling and workflow implementation, we developed the open-source plugin napari-FLIM-phasor-plotter to implement conversion, visualization, analysis and reuse of FLIM data of different formats. Our work thus advances technical applications and bioimage data management and analysis in FLIM microscopy for life science research.

cell biology↗

bioRxiv2025

Fluorescence Lifetime Imaging Microscopy (FLIM) visualizes internalization and biological impact of nanoplastics in live intestinal organoids

The increasing micro- and nanoplastic (MNP) pollution poses significant risks to human and animal health, yet the mechanisms of their accumulation and effects on absorptive tissues such as the gastrointestinal tract remain poorly understood. Addressing these knowledge gaps requires tractable models coupled to dynamic live cell imaging methods, to enable multi-parameter analysis at single cell resolution. Here we report a new method combining adult stem cell-derived small intestinal organoid cultures with multi-parameter live Fluorescence Lifetime Imaging Microscopy (FLIM) to study MNP interactions with gut epithelium. To facilitate this, we optimized live imaging of porcine and mouse small intestinal organoids with an apical-out topology. Subsequently, we produced a set of pristine MNPs based on PMMA and PS (<200 nm, doped with deep-red fluorescent dye) exhibiting different surface charges, and evaluated their interaction with organoids displaying controlled epithelial polarity. We found that nanoparticles differently interacted with apical and basal membranes of the organoids and even showed a species-specific pattern of cellular uptake. Using a phasor-FLIM approach, we demonstrate better sensitivity of FLIM over conventional intensity-based microscopy. The fluorescence lifetime barcoding enabled distinguishing different types of MNP and their interaction sites within organoids. Finally, we studied short (1 day)- and long (3 days)-term exposure effects of PMMA and PS-based MNPs on mitochondrial function, total energy budget and epithelial inflammation and found that even pristine MNPs could disrupt chemokine production and mitochondrial membrane potential in intestinal epithelial cells. The presented FLIM approach will advance the study of MNP toxicity, their biological impacts on gastrointestinal tissue and help tracing other types of fluorescent nanoparticles in live organoid and 3D ex vivo systems.

pharmacology and toxicology↗

bioRxiv2024

Gliding motility of the diatom Craspedostauros australis correlates with the intracellular movement of raphid-specific myosins

Raphid diatoms are one of the few eukaryotes capable of gliding motility, which is remarkably fast and allows for quasi-instantaneous directional reversals. Besides other mechanistic models, it has been suggested that an actomyosin system provides the force for diatom gliding. However, in vivo data on the dynamics of actin and myosin in diatoms are lacking. In this study we demonstrate that the raphe-associated actin bundles required for diatom movement do not exhibit a directional turnover of subunits and thus their dynamics do not contribute directly to force generation. By phylogenomic analysis we identified four raphid diatom-specific myosins in Craspedostauros australis (CaMyoA-D) and investigated their in vivo localization and dynamics through GFP-tagging. Only CaMyoB-D but not CaMyoA exhibited coordinated movement during gliding, consistent with a role in force generation. The characterization of raphid diatom-specific myosins lays the foundation for unraveling the molecular mechanisms that underlie the gliding motility of diatoms.

cell biology↗