Biology subjects

Shimoni, E.

Publications and source records attributed to Shimoni, E..

7 recordsLinked to original sources

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

Metal Ions Guide the Production of Silkworm Fibers

Silk fibers unique mechanical properties have made them a desirable material for various applications, from medical to optical materials and even in sensing. Yet, to date, no synthetic method has come close to reproducing this remarkably strong biomaterial due to the complexity and insufficient understanding of the mechanism of silk fiber formation. While ions are known to play a key role in the production of natural silk fiber, how they do so has thus far eluded discovery. Here we report that a broad composition of metal ions guides structural transformations in the silk fibroin protein inside the silkworm silk gland. By using a combination of cryo-electron microscopy techniques coupled with elemental analysis, we followed the changes in the composition and spatial localization of metal ions inside the silk gland. We observed that ions are homogenously dispersed during the initial stages of silk secretion and storage inside the silk gland, but once the fibers are spun, the ions delocalize from the silk fibroin fiber core to the sericin coating gum layer. This shift in ion localization is accompanied by the alignment of protein chains and an increase in silk feedstock viscosity inside the silk gland - changes that make the protein more sensitive to shear and enable the initiation of the liquid-to-solid transition in the silk. Moreover, the selective doping of the spun silk fibers with metal ions modifies their mechanical performance. These findings highlight the importance and the dynamic role of metal ions in the evolution of silk fibers mechanical properties, enhance our understanding of the mechanism of silk fiber formation, and lay the foundations for developing new concepts in biomaterial design.

biochemistry↗

bioRxiv2023

Rim aperture of autophagic membranesbalances cargo inclusion with vesicle maturation

Autophagy eliminates cytoplasmic material by engulfment in membranous vesicles targeted for lysosome degradation. Nonselective autophagy coordinates the sequestration of bulk cargo with the growth of the isolation membrane (IM) in a yet-unknown manner. Here we show the yeast IM expands while maintaining a rim sufficiently wide for sequestration of large cargo but tight enough to mature in due time. An obligate complex of Atg24/Snx4 with Atg20 or Snx41 assembles locally at the rim in a spatially-extended manner that specifically depends on autophagic PI(3)P. This assembly stabilizes the open rim to promote autophagic sequestration of large cargo in correlation with vesicle inflation. Moreover, constriction of the rim by the PI(3)P-dependent Atg2-Atg18 complex and clearance of PI(3)P by Ymr1 antagonize the rim opening to promote autophagic maturation and consumption of small cargo. Tight regulation of membrane rim aperture by PI(3)P thus couples the mechanism and physiology of nonselective autophagy.

cell biology↗

bioRxiv2020

Designer protein assemblies with tunable phase diagrams in living cells

The self-organization of proteins into specific assemblies is a hallmark of biological systems. Principles governing protein-protein interactions have long been known. However, principles by which such nanoscale interactions generate diverse phenotypes of mesoscale assemblies, including phase-separated compartments, remains challenging to characterize and understand. To illuminate such principles, we create a system of two proteins designed to interact and form mesh-like assemblies in living cells. We devise a novel strategy to map high-resolution phase diagrams in vivo, which provide mesoscale self-assembly signatures of our system. The structural modularity of the two protein components allows straightforward modification of their molecular properties, enabling us to characterize how point mutations that change their interaction affinity impact the phase diagram and material state of the assemblies in vivo. Both, the phase diagrams and their dependence on interaction affinity were captured by theory and simulations, including out-of-equilibrium effects seen in growing cells. Applying our system to interrogate biological mechanisms of self-assembly, we find that co-translational protein binding suffices to recruit an mRNA to the designed micron-scale structures.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2019

ERM-Dependent Assembly of T-Cell Receptor Signaling and Co-stimulatory Molecules on Microvilli Prior to Activation

SummaryT-cell surfaces are covered with microvilli, actin-rich and flexible protrusions. We use super-resolution microscopy to show that [≥]90% T-cell receptor (TCR) complex molecules TCR{beta} and TCR{zeta}, as well as the co-receptor CD4 and the co-stimulatory molecule CD2 reside on microvilli of human T cells. Furthermore, TCR proximal signaling molecules involved in the initial stages of the immune response, such as the protein tyrosine kinase Lck and the key adaptor molecule LAT, are also enriched on microvilli. Notably, phosphorylated proteins of the ERM (ezrin, radixin, moesin) family colocalize with these heterodimers as well as with actin filaments within the microvilli of resting T cells. This finding implies a role for one or more phosphorylated ERMs in linking the TCR complex to the actin cytoskeleton within microvilli. Indeed, expression of a dominant-negative ezrin fragment effectively redistributes TCR molecules over the whole T cell surface. Our results establish microvilli as key signaling hubs, in which the TCR complex and its proximal signaling molecules and adaptors are pre-assembled prior to activation in an ERM-dependent manner. The preformed positioning of these actin-binding TCR assemblies on individual microvilli can facilitate the local transmission of TCR signals seconds after TCR occupancy and impacts the slower subsequent events that lead to the assembly of immunological synapses.

immunology↗

bioRxiv2019

Fenestrae-associated protein Plvap regulates the rate of blood-borne proteins passage into the hypophysis

To maintain body homeostasis, endocrine systems must detect and integrate a multitude of blood-borne peripheral signals. This is mediated by specialized permeable pores in the endothelial membrane, dubbed fenestrae. Plasmalemma vesicles-associated protein (Plvap) is located in the fenestral diaphragm and is thought play a role in the selective passage of proteins through the fenestrae. However, this suggested function has yet to be demonstrated directly. Here, we studied the development of fenestrated capillaries in a major neuroendocrine interface between the blood and brain, namely the hypophysis. Using a transgenic permeability biosensor to visualize the vascular excretion of a genetically tagged plasma protein (DBP-EGFP), we show that the developmental acquisition of vascular permeability is associated with differential expression of zebrafish plvap orthologs in the hypophysis versus brain. Ultrastructural analysis of the hypophyseal vasculature revealed that plvapb mutants display deficiencies in fenestral and stomatal diaphragms as well as increased density of fenestrae, but not of caveolae. Measurements of DBP-EGFP dynamics in live plvapb mutant larvae provided a direct proof that Plvap limits the rate of blood-borne protein passage through fenestrated endothelia. Overall, we present the regulatory role of Plvap in the development of blood-borne protein detection machinery in a major neuroendocrine interface between the brain and the general circulation.

developmental biology↗

bioRxiv2017

Large-scale rewiring in a yeast hybrid

The merging of genomes in inter-specific hybrids can result in novel phenotypes, including increased growth rate and biomass yield, a phenomenon known as heterosis. We describe a budding yeast hybrid that grows faster than its parents under different environments. Phenotypically, the hybrid progresses more rapidly through cell cycle checkpoints, relieves the repression of respiration in fast growing conditions, does not slow down its growth when presented with ethanol stress, and shows increasing signs of DNA damage. A systematic genetic screen identified hundreds of alleles affecting hybrid growth whose identity vastly differed between the hybrid and its parent and between growth conditions. This large-scale rewiring of allele effects suggests that despite showing clear heterosis, the hybrid is perturbed in multiple regulatory processes. We discuss the possibility that incompatibilities contribute to hybrid vigor by perturbing safeguard mechanisms that limit growth in the parental background.

systems biology↗