Biology subjects

Varsano, N.

Publications and source records attributed to Varsano, N..

8 recordsLinked to original sources

bioRxiv2026

Vesicular pseudopodia define the fusion site on large secretory vesicles of the Drosophila salivary glands

Large secretory vesicles (LSVs) pose a scaling problem for regulated exocytosis. Their micron-scale dimensions greatly increase the vesicular membrane surface area, making productive engagement between the vesicular and target membrane fusion machinery unlikely. Here, we show that vesicular pseudopodia define the fusion sites of LSVs in Drosophila larval salivary glands. Focused ion beam scanning electron microscopy revealed that most LSVs project polarized pseudopodia that interconnect neighboring vesicles and orient toward the apical membrane. Exposed pseudopodia were frequently observed at the apical surface and associated with narrow fusion pores, indicating that fusion occurs at these structures. Three-dimensional correlative light and electron microscopy showed that the I-BAR protein Missing in Metastasis (MIM) selectively localizes to exposed pseudopodia. Proteomic analysis based on a MIM pull-down assay identified exocyst components, including Sec15, which localizes to pseudopodia and persists at fusion sites throughout secretion. Finally, the tetraspanin Tsp42Ee marked complementary apical fusion domains and was required for efficient exocytosis. Our findings support a model in which prepatterned vesicular and apical membrane domains coordinate efficient exocytosis. SummaryRegulated exocytosis of large secretory vesicles is facilitated by vesicular pseudopodia and an apical fusion domain that spatially organizes membrane tethering and fusion during secretion.

cell biology↗

bioRxiv2025

Azimuthal Segment Imaging in cryo-STEM Tomography

Cryo-electron microscopy is transitioning from investigation of isolated macromolecules to in situ studies bridging the realms of structural and cellular biology. Newly available detector technologies enable unconventional contrast modes with particular advantages. Here we demonstrate application of quadrant diode detectors to visualize a range of biological specimens by cryo-Scanning Transmission Electron Tomography (cryo-STET). Theoretically, we decompose coherent contrast by parallax analysis to isolate phase and amplitude contributions in specimens too thick for energy-filtered TEM. We thereby expand the cryo-STEM toolchest to parallax-filtered bright field (pBF) and parallax-filtered integrated differential phase contrast ({pi}DPC) and demonstrate their advantages in tomography using T4-bacteriophages, whole cells, and cryo-lift-out lamellae of cellular multilayers. The results show significant improvements over traditional STEM modalities in a realm where conventional wide-field transmission EM imaging methods are not applicable. The commercial availability of such detectors and the relative ease and speed of image reconstruction should make this realm accessible to the broader community in life science EM and beyond. TeaserParallax-corrected cryo-STET imaging provides coherent phase and amplitude contrast of thick biological specimens.

cell biology↗

bioRxiv2025

Specialized Molecular Pathways Drive the Formation of Light-Scattering Assemblies in Leucophores

Pigmentation plays a vital role in the survival of organisms, supporting functions such as camouflage, communication, and mate attraction. In vertebrates, these functions are mediated by specialized pigment cells known as chromatophores of which, uric acid crystal-forming leucophores remain the least understood, with little known about their molecular mechanisms. A key question in pigment cell biology is whether different crystal chemistries require distinct molecular pathways, or whether similar cellular processes drive the formation of diverse crystals. This study was designed to unravel the uncharacterized process of uric acid crystallization in leucophores and compare them to guanine crystal formation in iridophores and pterin formation in xanthophores. The results of our transcriptomic, ultrastructural, and metabolomic analyses, demonstrate that leucophores share molecular pathways with iridophores, particularly those connected to organelle organization and purine metabolism, but express discrete genes involved in uric acid biosynthesis and storage. Additionally, leucophores share intracellular trafficking and pterin biosynthesis genes with xanthophores, suggesting universally conserved processes. Ultrastructural studies reveal star-like fibrous structures in leucosomes, which likely serve as scaffolds for unique one-dimensional uric acid assemblies that radiate from the core and act as efficient light scatterers. These findings provide new insights into leucophore cell biology and the specialized mechanisms driving molecular crystalline assembly, and reveal that while some cellular processes are conserved, the specific chemistry of each crystal type drives the evolution of distinct molecular pathways.

cell biology↗

bioRxiv2024

Snapshots of Mitochondrial Fission Imaged by Cryo-Scanning Transmission Electron Tomography

Mitochondria undergo constant remodeling via fission, fusion, extension, and degradation. Fission, in particular, depends on the accumulation of the mitochondrial fission factor (MFF) and subsequent recruitment of the dynamin-related protein Drp1. We used cryo-scanning transmission electron tomography (cryo-STET) to investigate mitochondrial morphologies in MFF mutant cells (MFF-/-) in ATP-depleting conditions that normally induce fission. The capability of cryo-STET to image through the cytoplasmic volume to a depth of 1 {micro}m provides visualization of mitochondria and their surroundings intact. We imaged changes in mitochondrial morphology and cristae structure and contacts with endoplasmic reticulum, degradative organelles, and cytoskeleton at stalled fission sites. We found disruption of the outer membrane at contact sites with endoplasmic reticulum and degradative organelles at sites of mitophagy. We identified fission sites where the inner mitochondrial membrane is already separated while the outer is still continuous. While MFF is a general fission factor, these observations demonstrate that mitochondrial fission can proceed to the final stage in its absence. The use of cryo-STET allays concerns about the loss of structures due to sample thinning required for cryo-TEM tomography. Summary StatementImaging the whole cytosol in three dimensions greatly aids in understanding cellular processes. Here, we applied cryo-scanning transmission electron tomography to study stages of mitochondrial fission in the absence of the mitochondrial fusion factor protein.

cell biology↗

bioRxiv2024

Controlled pH Alteration Enables Guanine Accumulation and Drives Crystallization within Iridosomes

Many animals exhibit remarkable colors produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These systems are utilized for various purposes, including vision, camouflage, communication, and thermal regulation. Each guanine crystal forms within a membrane-bound organelle called an iridosome, where precise control over crystal formation occurs. While the presence of guanine crystals in iridosomes is well-documented, the mechanisms facilitating the accumulation of water-insoluble guanine and driving its crystallization remain unclear. Here, we employ advanced imaging and spectroscopy techniques to characterize the maturation of iridosomes in zebrafish iridophores during development. Using cryo-electron microscopy, we found that amorphous guanine accumulates in early-stage iridosomes. Synchrotron-based soft X-ray microscopy studies revealed that, unlike mature crystals, the accumulated guanine is initially in its protonated state. Live imaging with a pH sensor demonstrated that early-stage iridosomes are acidic and that their pH gradually approaches neutrality during maturation. Additionally, the application of a V-ATPase inhibitor reduced the acidity of iridosomes and significantly decreased crystal formation, suggesting the involvement of V-ATPase in regulating the organelle pH. Our findings reveal new insights into the molecular mechanisms facilitating guanine accumulation and crystallization within iridosomes, emphasizing the pivotal role of pH alternations in the precise formation of biogenic crystals.

cell biology↗

bioRxiv2022

Biogenic plate-like guanine crystals form via templated nucleation of thin crystal leaflets on amyloid scaffolds

Controlling the morphology of crystalline materials is challenging, as crystals have a strong tendency towards thermodynamically stable structures. Yet, organisms form crystals with distinct morphologies, such as the plate-like guanine crystals produced by many terrestrial and aquatic species for light manipulation. Regulation of crystal morphogenesis was hypothesized to entail physical growth restriction by the surrounding membrane, combined with fine-tuned interactions between organic molecules and the growing crystal. Using cryo electron tomography of developing zebrafish larvae, we found that guanine crystals form via templated nucleation of thin leaflets on preassembled scaffolds made of 20-nm-thick amyloid fibers. These leaflets then merge and coalesce into a single plate-like crystal. Our findings provide new insights into how organisms control the morphology and, thereby, the optical properties of crystals.

biophysics↗

bioRxiv2022

The Colon Mucosal Sialylglycome Is Redox-Regulated by the Golgi Enzyme QSOX1

Mucus shields the intestinal epithelium from pathogens and provides a supportive environment for commensal bacteria. Mucus is composed of enormous, heavily glycosylated proteins called mucins, which become disulfide crosslinked in a multi-step biosynthetic pathway culminating in the Golgi apparatus and secretory granules of goblet cells. We observed that knockout mice lacking the Golgi-localized disulfide catalyst QSOX1 produced poorly protective colon mucus, were hypersensitive to induced colitis, and had an altered microbiome. The initial hypothesis arising from these observations was that QSOX1 catalyzes disulfide crosslinking of mucins. Contrary to this hypothesis, the disulfide-mediated polymerization of mucins and related glycoproteins proceeded normally without QSOX1. Instead, we found that QSOX1 forms regulatory disulfides in Golgi glycosyltransferases and thereby promotes effective sialylation of the colon glycome. Our findings reveal that enzymatic control of Golgi redox state impacts glycan elaboration in goblet cells, and that this pathway is crucial for maintaining mucosal function.

cell biology↗

bioRxiv2020

Cellular pathways of calcium transport and concentration towards mineral formation in sea urchin larvae

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. The primary mesenchyme cells (PMCs) are the cells that are responsible for spicule formation. PMCs endocytose sea water from the larval internal body cavity into a network of vacuoles and vesicles, where calcium ions are concentrated until they precipitate in the form of amorphous calcium carbonate (ACC). The mineral is subsequently transferred to the syncytium, where the spicule forms. Using cryo-soft X-ray microscopy (cryo-SXM) we imaged intra-cellular calcium-containing particles in the PMCs and acquired Ca-L2,3 X-ray absorption near edge spectra (XANES) of these Ca-particles. Using the pre-peak/main peak (L2/ L2) intensity ratio, which reflects the atomic order in the first Ca coordination shell, we determined the state of the calcium ions in each particle. The concentration of Ca in each of the particles was also determined by the integrated area in the main Ca absorption peak. We observed about 700 Ca-particles with order parameters, L2/ L2, ranging from solution to hydrated and anhydrous ACC, and with concentrations ranging between 1-15 M. We conclude that in each cell the calcium ions exist in a continuum of states. This implies that most, but not all water, is expelled from the particles. This cellular process of calcium concentration may represent a widespread pathway in mineralizing organisms. SignificanceOrganisms form mineralized skeletons, many of which are composed of calcium salts. Marine organisms extract calcium ions from sea water. One of the main unresolved issues is how organisms concentrate calcium by more than 3 orders of magnitude, to achieve mineral deposition in their skeleton. Here we determine the calcium state in each of the calcium-containing vesicles inside the spicule-building cells of sea urchin larvae. We show that within one cell there is a wide range of concentrations and states from solution to solid. We hypothesize that calcium concentration increases gradually in each vesicle, starting from sea water levels and until mineral is deposited. This model might well be relevant to other phyla, thus advancing the understanding of biomineralization processes.

biophysics↗