Gur Lab

Iridosomes, the guanine crystal-forming organelles of pigment-producing iridophores, are among the most versatile, visually striking yet mechanistically uncharacterized organelles in vertebrate biology. Lysosome-related organelles (LROs) support cell type-specific functions by adapting endolysosomal pathways for specialized roles. Here, we show that iridosomes represent a previously unrecognized subtype of LROs. Using transcriptomic profiling of zebrafish iridophores, CRISPR-Cas9-mediated gene disruption, and cryogenic transmission electron microscopy, we define the molecular program underlying iridosome biogenesis. Iridosomes have evolved unique adaptations for crystal growth while retaining core features of other LROs. Key regulators, including RAB32a, AP3M2, and HPS5, are essential for crystal formation, with gene knockouts causing reduced crystal number, altered morphology, and distinct maturation defects. We further identify hallmark LRO features in iridosomes, including intraluminal vesicles and pH-regulated developmental transitions. Cross-species transcriptomic analysis confirms that iridosomes share an LRO signature across vertebrates, including teleost fish and reptiles, suggesting ancient evolutionary origins. These findings establish iridosomes as crystalline LROs and as a model for investigating how cells construct structurally specialized organelles through coordinated trafficking, acid-base regulation, and crystallization, with implications for LRO evolution and human disease.Competing Interest StatementThe authors have declared no competing interest.European Research Council- Starting Grant, 101077470Israel Science Foundation, https://ror.org/04sazxf24, 691/22

Precise control over crystal morphology is a longstanding challenge in materials science, as crystal shape governs optical, mechanical, and electronic properties. In contrast, living organisms achieve remarkable control over crystal morphology, though the underlying mechanisms are not fully understood. Among the most common organic materials are guanine crystals, notable for their plate-like morphology and high anisotropic refractive index, arising from the stacking of hydrogen-bonded molecular layers along the (100) plane. Here we show that simple synthetic polymers not only reproduce this biogenic plate-like form in vitro but also expand the accessible morphology space to prisms and needles by systematically varying i) multivalent interactions, ii) functional-group identity, and iii) polarity and hydrogen-bonding capacity. Using microscopy, spectroscopy, and molecular dynamics simulations, we find that carbonyl-bearing polymers selectively adsorb to the (100) stacking face, cap layer addition along the a-axis, and yield large plates indistinguishable from biogenic crystals. In contrast, reducing carbonyl polarity, increasing steric-bulk, or introducing highly-polar groups redirect adsorption and produces bulkier prisms or slender needles. Simulations corroborate facet selectivity, identify contact motifs involving carbonyl oxygens and adjacent methylenes. Together, these findings provide design principles for sculpting organic crystals and suggest analogous interactions exploited by biological scaffolds to orchestrate guanine assembly.

Many animals produce vivid colors by reflecting and amplifying light with stacked guanine crystals within membrane-bound organelles called iridosomes. 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 used cryo-electron microscopy, live-cell pH imaging, pharmacological perturbations and spectroscopy to study iridosome maturation in zebrafish. Cryo-electron and synchrotron-based soft X-ray microscopies revealed that amorphous guanine initially accumulates in early-stage iridosomes in its protonated state. Live-cell imaging with a pH sensor demonstrated that early iridosomes are acidic, with pH gradually neutralizing during development. Inhibiting V-ATPase disrupted this acidification and significantly reduced crystal formation, indicating its role in pH regulation. Our findings reveal insights into the molecular mechanisms facilitating guanine formation within iridosomes, emphasizing the pivotal role of pH alternations in the precise formation of biogenic crystals.

Multimodal scanning transmission electron microscopy on vitrified frozen-hydrated specimens promises exceptional spatial resolution into the molecular mechanisms underlying the formation of organic crystals in both health and disease. Detection of crystalline volumes is essential for tracking and mapping nucleation and growth. We provide an analytical description of the low-dose detection limit in diffraction for a thin crystal embedded in a thick matrix, focusing on organic crystals and embedding matrices of low-Z elements such as vitrified ice. Numerical calculations refine our description by accounting for the effects of multiple scattering. Often underestimated, wide-angle tails associated with inelastic scattering play a crucial role for the detection of crystalline reflections in a thick ice matrix, common for cryo-electron microscopy. We show that guanine crystals as thin as a few nanometers can be detected with a fluence of just a few thousand electrons if the ice thickness is below one mean free path for inelastic scattering. The required fluence increases non-linearly with the embedding ice thickness, with a pronounced top-bottom effect regarding the location of the crystal in the sample. Energy-filtered recording significantly reduces the fluence needed for thicker samples. The low-dose simulations implemented here validate the analytical description while acknowledging its limitations due to abstraction from multiple scattering and beam spreading.

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 crystalforming 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 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.

Organisms evolve mechanisms that regulate the properties of biogenic crystals to support a wide range of functions, from vision and camouflage to communication and thermal regulation. Yet, the mechanism underlying the formation of diverse intracellular crystals remains enigmatic. Here we unravel the biochemical control over crystal morphogenesis in zebrafish iridophores. We show that the chemical composition of the crystals determines their shape, particularly through the ratio between the nucleobases guanine and hypoxanthine. We reveal that these variations in composition are genetically controlled through tissue-specific expression of specialized paralogs, which exhibit remarkable substrate selectivity. This orchestrated combination grants the organism with the capacity to generate a broad spectrum of crystal morphologies. Overall, our findings suggest a mechanism for the morphological and functional diversity of biogenic crystals and may, thus, inspire the development of genetically designed biomaterials and medical therapeutics. (Figure presented.)