Biology subjects

Rechav, K.

Publications and source records attributed to Rechav, K..

6 recordsLinked to original sources

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↗

bioRxiv2024

The Natural Material Evolution and Stage-wise Assembly of Silk Along the Silk Gland

Silk fibers, with their highly ordered structure and mechanically superb properties, are produced in arthropod glands at minimal energy input and ambient conditions, a remarkable feat yet to be achieved synthetically. Due to the high instability and shear sensitivity of the silk protein feedstock, understanding silk fiber formation has been largely limited to in-vitro studies of certain gland sections, offering only a fragmented view of this process. Here, we monitor the whole silk feedstock processing in-situ, at the nano- to micron-scales, through imaging its progressive macromolecular assemblies and phase transitions along the entire Bombyx mori silkworm silk gland. This is done by combining state-of-the-art microscopy techniques, such as cryogenic sample preparation, fixation, and imaging. Our work reveals that fibroin assembles into micron-sized spherical storage "compartments" in the posterior and middle gland sections, a state that ensures its stability and avoids premature fibrillation. These compartments undergo several structural transformations along the gland and eventually disassemble at the entry to the anterior section, before the silk feedstock spinning begins. The spinning itself commences via a series of structural transitions, from the alignment of protein chains in liquid feedstock, through the formation of several fibrillated nano-structures and, in the final stage, a network of cross-linked nano-bundles, which determines the structure and properties of the final microfiber. Importantly, the length of the anterior section of the silk gland enables such gradual and balanced structural transitions. This direct imaging of silks natural formation process can help formulate a template for the transformation of fibrillar proteins into synthetic bio-fibers. DedicationThis work is dedicated to the memory of Dr. Eyal Shimoni, who was a valued colleague and a dear friend. Eyal was a vital part of this research and was essential in shaping its direction. He will be deeply missed for his intellect, mindfulness, creativity, and unwavering dedication to scientific development. Though he is no longer with us, his influence and spirit continue to inspire us in our scientific pursuits. May his passion for discovery and commitment to excellence live on through this work.

biochemistry↗

bioRxiv2023

Cell-mediated cholesterol crystal processing and clearance observed by 3D cryo-imaging in human atherosclerotic plaques

Atherosclerosis is a pathology affecting the arteries, characterized by the buildup of plaques in the blood vessel walls. Atherosclerosis is the main cause of cardiovascular diseases, which constitute the leading cause of death in the world. Cholesterol crystals are the main components of the plaques, which actively participate in plaque growth and rupture and do not dissolve in aqueous environments. Employing novel cryo-scanning electron microscopy techniques, we examined human atherosclerotic plaques at high resolution, in 3D, and in close to native conditions. We show that cholesterol crystal clearance occurs in advanced human plaques through the activity of cells. We suggest that this occurs by enzymatic esterification of cholesterol to cholesteryl ester, which aggregates into intra- and extra-cellular pools. This discovery provides further understanding of the disease process in atherosclerosis, and may inspire new therapeutic approaches.

pathology↗

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

Diffraction contrast in cryo-scanning transmission electron tomography reveals the boundary of hemozoin crystals in situ

Malaria is a potentially fatal infectious disease caused by the obligate intracellular parasite Plasmodium falciparum. The parasite infects human red blood cells (RBC) and derives nutrition by catabolism of hemoglobin. As amino acids are assimilated from the protein component, the toxic heme is released. Molecular heme is detoxified by rapid sequestration to physiologically insoluble hemozoin crystals within the parasites digestive vacuole (DV). Common antimalarial drugs interfere with this crystallization process, leaving the parasites vulnerable to the by-product of their own metabolism. A fundamental debate with important implications on drug mechanism regards the chemical environment of crystallization in vivo, whether aqueous or lipid. This issue had been addressed previously by cryogenic soft X-ray tomography. We employ cryo-scanning transmission electron tomography (CSTET) to probe parasite cells throughout the life cycle in a fully hydrated, vitrified state at higher resolution. During the acquisition of CSTET data, Bragg diffraction from the hemozoin provides a uniquely clear view of the crystal boundary at nanometer resolution. No intermediate medium, such as a lipid coating or shroud, could be detected surrounding the crystals. The present study describes a unique application of CSTET in the study of malaria. The findings can be extended to evaluate new drug candidates affecting hemozoin crystal growth.

biophysics↗

bioRxiv2021

In-situ fiducial markers for 3D correlative cryo- fluorescence and FIB-SEM imaging

1.Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), which offer high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples. However, correlative light and electron microscopy (CLEM) workflows combining cryo-fluorescence microscopy (cryo-FM) and FIB-SEM are not yet commonly available. Here, we demonstrate that fluorescently labeled lipid droplets can serve as in-situ fiducial markers for correlating cryo-FM and FIB-SEM datasets, and that this approach can be used to target the acquisition of large FIB-SEM stacks spanning tens of microns under cryogenic conditions. We also show that cryo-FIB-SEM imaging is particularly informative for questions related to organelle structure and inter-organellar contacts, nuclear organization and mineral deposits in cells.

cell biology↗