Publications

2025

In-cell structure and variability of pyrenoid Rubisco
Elad N., Hou Z., Dumoux M., Ramezani A., Perilla J. R. & Zhang P. (2025) Nature Communications. 16, 7763. Abstract
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is central to global CO₂ fixation. In eukaryotic algae, its catalytic efficiency is enhanced through the pyrenoid - a protein-dense organelle within the chloroplast that concentrates CO₂. Although Rubisco structure has been extensively studied in vitro, its native structure, dynamics and interactions within the pyrenoid remain elusive. Here, we present the native Rubisco structure inside the green alga Chlamydomonas reinhardtii determined by cryo-electron tomography and subtomogram averaging of cryo-focused ion beam milled cells. Multiple structural subsets of Rubisco are identified, stochastically distributed throughout the pyrenoid. While Rubisco adopts an active conformation in the best-resolved map, comparison among the subsets reveals significant local variations at the active site, at the large subunit dimer interfaces, and at binding protein contact regions. These findings offer a comprehensive understanding of the structure, dynamics, and functional organization of native Rubisco within the pyrenoid, providing valuable insights into its critical role in CO₂ fixation.
A cryo-electron microscopy structure of yeast Pex5 in complex with a cargo uncovers a novel binding interface
Peer L., Dym O., Elad N., Tirosh A., Jacobovitch J., Sivan E., Angel M., Albeck S., Schuldiner M., Peleg Y., Zalckvar E. et al. (2025) Journal of Cell Science. 138, 12, jcs263890. Abstract
Proper protein targeting to organelles is crucial for maintaining eukaryotic cellular function and homeostasis. This necessity has driven the evolution of specific targeting signals on proteins and the targeting factors that recognize them. A prominent example is peroxisomal matrix proteins, most of which depend on the targeting factor Pex5 to localize and function correctly. Although most Pex5 cargoes contain a peroxisomal targeting signal type 1 (PTS1), they are not all targeted similarly. Some undergo priority targeting, facilitated either by stronger binding to specific subsets of PTS1 signals or by additional interaction interfaces. These observations highlight the extensive complexity of Pex5-mediated targeting. In this study, we reveal that the Saccharomyces cerevisiae (yeast) matrix protein Eci1 can reach peroxisomes and bind Pex5 in the absence of PTS1. By solving the structure of the yeast Pex5-Eci1 complex using cryo-electron microscopy, we identified additional binding interfaces. Our findings provide new insights into the versatile interactions between Pex5 and its cargo, Eci1. More broadly, this work highlights the intricate, dynamic nature of the interactions between cargo factors and their cargoes to meet the complex environment within eukaryotic cells.

[All authors]
Inhibition of tau aggregation by the CCT3 and CCT7 apical domains
Ben-Maimon M., Elad N., Naveh-Tassa S., Levy Y. & Horovitz A. (2025) Protein Science. 34, 6, e70162. Abstract
The eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) is a molecular chaperone that assists protein folding in an ATP-driven manner. It consists of two stacked identical rings that are each made up of eight distinct subunits. Here, we show that the apical domains of subunits CCT3 and CCT7 from humans are strong inhibitors of tau aggregation, which is associated with several neurological disorders such as Alzheimer's and Parkinson's diseases. Kinetic analyses and negative-stain electron microscopy indicate that the mechanism of inhibition of tau aggregation by the apical domains of subunits CCT3 and CCT7 differ. Aggregation of tau alone, or in the presence of the apical domain of subunit CCT7, can be described by a fragmentation model whereas in the presence of the apical domain of subunit CCT3, it fits a saturating elongation and fragmentation mechanism. Coarse-grained molecular dynamics simulations show that tau interacts with different regions in the apical domains of subunits CCT3 and CCT7, in agreement with their different inhibition mechanisms.

2024

A pH-Dependent Phase Separation Drives Polyamine-Mediated Silicification from Undersaturated Solutions
Biswas P., Livni N., Paul D., Aram L., Safadi R., Varsano N., Elad N., Kamyshinsky R., Leskes M., Gal A. et al. (2024) ACS Nano. 18, 50, p. 33998-34006 Abstract
Silica polymerization from its soluble monomers is fundamental to many chemical processes. Although industrial methods require harsh conditions and concentrated precursors, biological silica precipitation occurs under ambient conditions from dilute solutions. The hallmark of biosilica is the presence of amine-rich organic macromolecules, but their functional role remains elusive. Here, we show a pH-dependent stimulatory effect of such polyamines on silica polymerization. Notably, this process is decoupled from the saturation degree, allowing the synthesis of polymer-silica hybrid products with controlled network morphologies from undersaturated solutions. The data suggest a two-step phase separation process. First, an associative liquid-liquid phase separation forms a micrometer-size dense phase. Second, silica undergoes a liquid-to-solid transition in the supersaturated condensates to form a bicontinuous silica structure. This study can inspire \u201csoft chemistry\u201d routes to design organic-inorganic nanomaterials with regulatory principles optimized by evolution.

[All authors]
The Role of Macromolecular Condensates in the Regulation of Intracellular Calcium Transport for Coccolith Formation
Bino E., Aram L., Paul D., Kadan Y., Clare D., Gilchrist J. B., Elad N. & Gal A. (2024) Advanced Functional Materials. 35, 7, 2415344. Abstract
Inorganic minerals that form via regulated biological processes exhibit remarkable properties. This is due to the involvement of macromolecules that control biomineralization. Even though the interactions of these biopolymers with solid mineral phases are intensely studied, not much is known about their involvement in the preceding steps of intracellular transport of the mineral building blocks. In this work, the model system of coccolith calcite crystallization is utilized to address the role of mineral-associated polysaccharides in the transport of calcium ions. State-of-the-art cryo-electron tomography is used to image in situ ion-rich dense phases in the wild-type and in two mutant strains, defected in coccolith production. The results show that the abundance and solubility of the calcium-rich condensates need to be finely tuned for proper crystallization. When the native macromolecular assemblage is compromised, calcium is still present in the calcifying fluid as a solute, but this is not sufficient for coccolith development. These results suggest that biomineralizing systems achieve superior regulation of crystallization due to the use of dense macromolecule-rich phases.
Specialized Listeria monocytogenes produce tailocins to provide a population-level competitive growth advantage
Sigal N., Lichtenstein-Wolfheim R., Schlussel S., Azulay G., Borovok I., Holdengraber V., Elad N., Wolf S. G., Zalk R., Zarivach R., Frank G. A., Herskovits A. A. et al. (2024) Nature Microbiology. 9, 10, p. 2727-2737 Abstract
Tailocins are phage tail-like bacteriocins produced by various bacterial species to kill kin competitors. Given that tailocin release is dependent upon cell lysis, regulation of tailocin production at the single-cell and population level remains unclear. Here we used flow cytometry, competition assays and structural characterization of tailocin production in a human bacterial pathogen, Listeria monocytogenes. We revealed that a specialized subpopulation, constituting less than 1% of the total bacterial population, differentiates to produce, assemble and store thousands of tailocin particles. Tailocins are packed in a highly ordered manner, clustered in a liquid crystalline phase that occupies a substantial volume of the cell. Tailocin production confers a competitive growth advantage for the rest of the population. This study provides molecular insights into tailocin production as a form of altruism, showing how cell specialization within bacterial populations can confer competitive advantages at the population level.

[All authors]
Intracellular morphogenesis of diatom silica is guided by local variations in membrane curvature
Aram L., de Haan D., Varsano N., Gilchrist J. B., Heintze C., Rotkopf R., Rechav K., Elad N., Kröger N., Gal A. et al. (2024) Nature Communications. 15, 7888. Abstract
Silica cell-wall formation in diatoms is a showcase for the ability of organisms to control inorganic mineralization. The process of silicification by these unicellular algae is tightly regulated within a membrane-bound organelle, the silica deposition vesicle (SDV). Two opposing scenarios were proposed to explain the tight regulation of this intracellular process: a template-mediated process that relies on preformed scaffolds, or a template-independent self-assembly process. The present work points to a third scenario, where the SDV membrane is a dynamic mold that shapes the forming silica. We use in-cell cryo-electron tomography to visualize the silicification process in situ, in its native-state, and with a nanometer-scale resolution. This reveals that the plasma membrane interacts with the SDV membrane via physical tethering at membrane contact sites, where the curvature of the tethered side of the SDV membrane mirrors the intricate silica topography. We propose that silica growth and morphogenesis result from the biophysical properties of the SDV and plasma membranes.

[All authors]
Altering the structures of 3D supramolecular assemblies from melamine and cyanuric acid derivatives in water
Orekhov N., Bukhtiiarova N., Brushevich Z. A., Muravev A. A., Nadav E., Tsarfati Y., Kossoy A., Feldman I., Zelenina A., Rubekina A. A., Semenov S. N., Skorb E. V. et al. (2024) Chemical Communications. 60, 77, p. 10680-10683 Abstract
Herein, we obtained two supramolecular assemblies with layered structures from melamine, N-methylmelamine, and hexynyl-cyanuric acid in water. By combination of X-ray diffraction, electron microscopy, and molecular dynamics studies, we found that introducing one methyl group in melamine alters the arrangement of the layers in these structures.

[All authors]

2023

Exocytosis of the silicified cell wall of diatoms involves extensive membrane disintegration
de Haan D., Aram L., Peled-Zehavi H., Addadi Y., Ben-Joseph O., Rotkopf R., Elad N., Rechav K. & Gal A. (2023) Nature Communications. 14, 1, 480. Abstract
Diatoms are unicellular algae characterized by silica cell walls. These silica elements are known to be formed intracellularly in membrane-bound silica deposition vesicles and exocytosed after completion. How diatoms maintain membrane homeostasis during the exocytosis of these large and rigid silica elements remains unknown. Here we study the membrane dynamics during cell wall formation and exocytosis in two model diatom species, using live-cell confocal microscopy, transmission electron microscopy and cryo-electron tomography. Our results show that during its formation, the mineral phase is in tight association with the silica deposition vesicle membranes, which form a precise mold of the delicate geometrical patterns. We find that during exocytosis, the distal silica deposition vesicle membrane and the plasma membrane gradually detach from the mineral and disintegrate in the extracellular space, without any noticeable endocytic retrieval or extracellular repurposing. We demonstrate that within the cell, the proximal silica deposition vesicle membrane becomes the new barrier between the cell and its environment, and assumes the role of a new plasma membrane. These results provide direct structural observations of diatom silica exocytosis, and point to an extraordinary mechanism in which membrane homeostasis is maintained by discarding, rather than recycling, significant membrane patches.

2022

Structure and receptor recognition by the Lassa virus spike complex
Katz M., Weinstein J., Eilon-Ashkenazy M., Gehring K., Cohen-Dvashi H., Elad N., Fleishman S. J. & Diskin R. (2022) Nature. 603, 7899, p. 174-179 Abstract
Lassa virus (LASV) is a human pathogen, causing substantial morbidity and mortality1,2. Similar to other Arenaviridae, it presents a class-I spike complex on its surface that facilitates cell entry. The viruss cellular receptor is matriglycan, a linear carbohydrate that is present on α-dystroglycan3,4, but the molecular mechanism that LASV uses to recognize this glycan is unknown. In addition, LASV and other arenaviruses have a unique signal peptide that forms an integral and functionally important part of the mature spike5,6,7,8; yet the structure, function and topology of the signal peptide in the membrane remain uncertain9,10,11. Here we solve the structure of a complete native LASV spike complex, finding that the signal peptide crosses the membrane once and that its amino terminus is located in the extracellular region. Together with a double-sided domain-switching mechanism, the signal peptide helps to stabilize the spike complex in its native conformation. This structure reveals that the LASV spike complex is preloaded with matriglycan, suggesting the mechanism of binding and rationalizing receptor recognition by α-dystroglycan-tropic arenaviruses. This discovery further informs us about the mechanism of viral egress and may facilitate the rational design of novel therapeutics that exploit this binding site.

Accepted version
A counter-enzyme complex regulates glutamate metabolism in Bacillus subtilis
Jayaraman V., Lee D. J., Elad N., Vimer S., Sharon M., Fraser J. S. & Tawfik D. S. (2022) Nature Chemical Biology. 18, 2, p. 161-170 Abstract
Multi-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from Bacillus subtilis composed of two enzymes catalyzing opposite (counter-enzymes) rather than sequential reactions: glutamate synthase (GltAB) and glutamate dehydrogenase (GudB), which make and break glutamate, respectively. In vivo and in vitro studies show that the primary role of complex formation is to inhibit the activity of GudB. Using cryo-electron microscopy, we elucidated the structure of the complex and the molecular basis of inhibition of GudB by GltAB. The complex exhibits unusual oscillatory progress curves and is necessary for both planktonic growth, in glutamate-limiting conditions, and for biofilm growth, in glutamate-rich media. The regulation of a key metabolic enzyme by complexing with its counter enzyme may thus enable cell growth under fluctuating glutamate concentrations. [Figure not available: see fulltext.]

2021

Allostery through DNA drives phenotype switching
Rosenblum G., Elad N., Rozenberg H., Wiggers F., Jungwirth J. & Hofmann H. (2021) Nature Communications. 12, 2967. Abstract
Allostery is a pervasive principle to regulate protein function. Growing evidence suggests that also DNA is capable of transmitting allosteric signals. Yet, whether and how DNA-mediated allostery plays a regulatory role in gene expression remained unclear. Here, we show that DNA indeed transmits allosteric signals over long distances to boost the binding cooperativity of transcription factors. Phenotype switching in Bacillus subtilis requires an all-or-none promoter binding of multiple ComK proteins. We use single-molecule FRET to demonstrate that ComK-binding at one promoter site increases affinity at a distant site. Cryo-EM structures of the complex between ComK and its promoter demonstrate that this coupling is due to mechanical forces that alter DNA curvature. Modifications of the spacer between sites tune cooperativity and show how to control allostery, which allows a fine-tuning of the dynamic properties of genetic circuits.
Electrostatic co-assembly of nanoparticles with oppositely charged small molecules into static and dynamic superstructures
Bian T., Gardin A., Gemen J., Houben L., Perego C., Lee B., Elad N., Chu Z., Pavan G. M., Klajn R. et al. (2021) Nature Chemistry. 13, 10, p. 940-949 Abstract
Coulombic interactions can be used to assemble charged nanoparticles into higher-order structures, but the process requires oppositely charged partners that are similarly sized. The ability to mediate the assembly of such charged nanoparticles using structurally simple small molecules would greatly facilitate the fabrication of nanostructured materials and harnessing their applications in catalysis, sensing and photonics. Here we show that small molecules with as few as three electric charges can effectively induce attractive interactions between oppositely charged nanoparticles in water. These interactions can guide the assembly of charged nanoparticles into colloidal crystals of a quality previously only thought to result from their co-crystallization with oppositely charged nanoparticles of a similar size. Transient nanoparticle assemblies can be generated using positively charged nanoparticles and multiply charged anions that are enzymatically hydrolysed into mono- and/or dianions. Our findings demonstrate an approach for the facile fabrication, manipulation and further investigation of static and dynamic nanostructured materials in aqueous environments.

Accepted version
[All authors]
SARS-CoV-2 variant prediction and antiviral drug design are enabled by RBD in vitro evolution
Zahradník J., Marciano S., Shemesh M., Zoler E., Harari D., Chiaravalli J., Meyer B., Rudich Y., Li C., Marton I., Dym O., Elad N., Lewis M., Andersen H., Gagne M., Seder R., Douek D., Schreiber G. et al. (2021) Nature Microbiology. 6, 9, p. 1188-1198 Abstract
SARS-CoV-2 variants of interest and concern will continue to emerge for the duration of the COVID-19 pandemic. To map mutations in the receptor-binding domain (RBD) of the spike protein that affect binding to angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, we applied in vitro evolution to affinity-mature the RBD. Multiple rounds of random mutagenic libraries of the RBD were sorted against decreasing concentrations of ACE2, resulting in the selection of higher affinity RBD binders. We found that mutations present in more transmissible viruses (S477N, E484K and N501Y) were preferentially selected in our high-throughput screen. Evolved RBD mutants include prominently the amino acid substitutions found in the RBDs of B.1.620, B.1.1.7 (Alpha), B1.351 (Beta) and P.1 (Gamma) variants. Moreover, the incidence of RBD mutations in the population as presented in the GISAID database (April 2021) is positively correlated with increased binding affinity to ACE2. Further in vitro evolution increased binding by 1,000-fold and identified mutations that may be more infectious if they evolve in the circulating viral population, for example, Q498R is epistatic to N501Y. We show that our high-affinity variant RBD-62 can be used as a drug to inhibit infection with SARS-CoV-2 and variants Alpha, Beta and Gamma in vitro. In a model of SARS-CoV-2 challenge in hamster, RBD-62 significantly reduced clinical disease when administered before or after infection. A 2.9 Å cryo-electron microscopy structure of the high-affinity complex of RBD-62 and ACE2, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.

[All authors]

2020

Maintaining Context in Ice: Cryo-EM/ET Workflow Optimizations
Elad N. & Wolf S. G. (2020) Structure. 28, 11, p. 1179-1181 Abstract
In this issue of Structure, breakthroughs in cryo-EM/ET research are presented. Klebl et al. (2020) demonstrate how speed in sample vitrification impacts the quality of macromolecular particles in resultant cryo-EM grids. Wu et al. (2020) combine fluorescence, ion beam milling, and tomography to unravel unique features in vitrified yeast cells.
Assembly Mechanism of Mucin and von Willebrand Factor Polymers
Javitt G., Khmelnitsky L., Albert L., Bigman L. S., Elad N., Morgenstern D., Ilani T., Levy Y., Diskin R., Fass D. et al. (2020) Cell. 183, 3, p. 717-729.e16 Abstract
The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here, we present an integrated structural analysis of the intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi apparatus. Remarkably, a densely O-glycosylated mucin domain performs an organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation.

[All authors]
Comparative Structural Analysis of 20S Proteasome Ortholog Protein Complexes by Native Mass Spectrometry
Vimer S., Ben-Nissan G., Morgenstern D., Kumar-Deshmukh F., Polkinghorn C., Quintyn R. S., Vasil'ev Y. V., Beckman J. S., Elad N., Wysocki V. H., Sharon M. et al. (2020) ACS Central Science. 6, 4, p. 573-588 Abstract
Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multilevel experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the Thermoplasma acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (Saccharomyces cerevisiae) and mammals (rat - Rattus norvegicus, rabbit - Oryctolagus cuniculus and human - HEK293 cells), the proteasome increased both in size and stability. Native MS structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution, three-dimensional structures, highly resembled that of the human complex. Using cryoelectron microscopy single-particle analysis, we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the PSMA7 subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies.

[All authors]
Structural Basis for a Convergent Immune Response against Ebola Virus
Cohen-Dvashi H., Zehner M., Ehrhardt S., Katz M., Elad N., Klein F. & Diskin R. (2020) Cell Host & Microbe. 27, 3, p. 418-427.e4 Abstract
Ebola virus disease is a severe health problem in Africa. Vaccines that display the Zaire ebolavirus glycoprotein spike complex are a prime component for the effort to combat it. The V(H)3-15/V(lambda)1-40-based class of antibodies was recently discovered to be a common response in individuals who received the Ebola virus vaccines. These antibodies display attractive properties, and thus likely contribute to the efficacy of the vaccines. Here, we use cryo-EM to elucidate how three V(H)3-15/V(lambda)1-40 antibodies from different individuals target the virus and found a convergent mechanism against a partially conserved site on the spike complex. Our study rationalizes the selection of the V(H)3-15/V(lambda)1-40 germline genes for specifically targeting this site and highlights Ebolavirus species-specific sequence divergences that may restrict breadth of V(H)3-15/V(lambda)1-40-based humoral response. The results from this study could help develop improved immunization schemes and further enable the design of immunogens that would be efficacious against a broader set of Ebolavirus species.
A highly reflective biogenic photonic material from core-shell birefringent nanoparticles
Palmer B. A., Yallapragada V. J., Schiffmann N., Wormser E. M., Elad N., Aflalo E. D., Sagi A., Weiner S., Addadi L., Oron D. et al. (2020) Nature Nanotechnology. 15, 2, p. 138-144 Abstract
The birefringence of isoxanthopterin crystalline spherulites enhances the reflectivity of a biological photonic crystal.Spectacular natural optical phenomena are produced by highly reflective assemblies of organic crystals. Here we show how the tapetum reflector in a shrimp eye is constructed from arrays of spherical isoxanthopterin nanoparticles and relate the particle properties to their optical function. The nanoparticles are composed of single-crystal isoxanthopterin nanoplates arranged in concentric lamellae around a hollow core. The spherulitic birefringence of the nanoparticles, which originates from the radial alignment of the plates, results in a significant enhancement of the back-scattering. This enables the organism to maximize the reflectivity of the ultrathin tapetum, which functions to increase the eye's sensitivity and preserve visual acuity. The particle size, core/shell ratio and packing are also controlled to optimize the intensity and spectral properties of the tapetum back-scattering. This system offers inspiration for the design of photonic crystals constructed from spherically symmetric birefringent particles for use in ultrathin reflectors and as non-iridescent pigments.

[All authors]

2019

Guanine and 7,8-Dihydroxanthopterin Reflecting Crystals in the Zander Fish Eye: Crystal Locations, Compositions, and Structures
Zhang G., Hirsch A., Shmul G., Avram L., Elad N., Brumfeld V., Pinkas I., Feldman Y., Ben Asher R., Palmer B. A., Kronik L., Leiserowitz L., Weiner S., Addadi L. et al. (2019) Journal of the American Chemical Society. 141, 50, p. 19736-19745 Abstract
The eyes of many fish contain a reflecting layer of organic crystals partially surrounding the photoreceptors of the retina, which are commonly believed to be composed of guanine. Here we study an unusual fish eye from Stizostedion lucioperca that contains two layers of organic crystals. The crystals in the outer layer are thin plates, whereas the crystals in the inner tapetum layer are block-shaped. We show that the outer layer indeed contains guanine crystals. Analyses of solutions of crystals from the inner layer indicated that the block-shaped crystals are composed of xanthopterin. A model of the structure of the block-shaped crystals was produced using symmetry arguments based on electron diffraction data followed by dispersion-augmented DFT calculations. The resulting crystal structure of xanthopterin included, however, a problematic repulsive interaction between C=O and N of two adjacent molecules. Knowing that dissolved 7,8-dihydroxanthopterin can oxidize to xanthopterin, we replaced xanthopterin with 7,8-dihydroxanthopterin in the model. An excellent fit was obtained with the powder X-ray diffraction pattern of the biogenic crystals. We then analyzed the biogenic block-shaped crystals in their solid state, using MALDI-TOF and Raman spectroscopy. All three methods unequivocally prove that the block-shaped crystals in the eye of S. lucioperca are crystals of 7,8-dihydroxanthopterin. On the basis of the eye anatomy, we deduce that the guanine crystals form a reflective layer producing the silvery color present on part of the eye surface, whereas the block-shaped crystals backscatter light into the retina in order to increase the light sensitivity of the eye.

[All authors]
Polyclonal and convergent antibody response to Ebola virus vaccine rVSV-ZEBOV
Ehrhardt S. A., Zehner M., Kraehling V., Cohen-Dvashi H., Kreer C., Elad N., Gruell H., Ercanoglu M. S., Schommers P., Gieselmann L., Eggeling R., Dahlke C., Wolf T., Pfeifer N., Addo M. M., Diskin R., Becker S., Klein F. et al. (2019) Nature Medicine. 25, 10, p. 1589-1600 Abstract
Recombinant vesicular stomatitis virus-Zaire Ebola virus (rVSV-ZEBOV) is the most advanced Ebola virus vaccine candidate and is currently being used to combat the outbreak of Ebola virus disease (EVD) in the Democratic Republic of the Congo (DRC). Here we examine the humoral immune response in a subset of human volunteers enrolled in a phase 1 rVSV-ZEBOV vaccination trial by performing comprehensive single B cell and electron microscopy structure analyses. Four studied vaccinees show polyclonal, yet reproducible and convergent B cell responses with shared sequence characteristics. EBOV-targeting antibodies cross-react with other Ebolavirus species, and detailed epitope mapping revealed overlapping target epitopes with antibodies isolated from EVD survivors. Moreover, in all vaccinees, we detected highly potent EBOV-neutralizing antibodies with activities comparable or superior to the monoclonal antibodies currently used in clinical trials. These include antibodies combining the IGHV3-15/IGLV1-40 immunoglobulin gene segments that were identified in all investigated individuals. Our findings will help to evaluate and direct current and future vaccination strategies and offer opportunities for novel EVD therapies.

[All authors]
Anhydrous beta-guanine crystals in a marine dinoflagellate: Structure and suggested function
Jantschke A., Pinkas I., Hirsch A., Elad N., Schertel A., Addadi L. & Weiner S. (2019) Journal of Structural Biology. 207, 1, p. 12-20 Abstract
Guanine crystals are used by certain animals, including vertebrates, to produce structural colors or to enhance vision, because of their distinctive reflective properties. Here we use cryo-SEM, cryo-FIB SEM and Raman spectroscopic imaging to characterize crystalline inclusions in a single celled photosynthesizing marine dinoflagellate species. We demonstrate spectroscopically that these inclusions are blocky crystals of anhydrous guanine in the beta-polymorph. Two-dimensional cryo-SEM and three-dimensional cryo-FIB-SEM serial block face imaging show that the deposits of anhydrous guanine crystals are closely associated with the chloroplasts. We suggest that the crystalline deposits scatter light either to enhance light exploitation by the chloroplasts, or possibly for protection from UV radiation. This is consistent with the crystal locations within the cell, their shapes and their sizes. As the dinoflagellates are extremely abundant in the oceans and are a major group of photosynthesizing marine organisms, the presence of guanine crystals in this marine organism may have broad significance.
Controlled crystallization of anhydrous guanine beta nano-platelets via an amorphous precursor: Via an amorphous precursor
Chen F., Wu B., Elad N., Gal A., Liu Y., Ma Y. & Qi L. (2019) CrystEngComm. 21, 23, p. 3586-3591 Abstract
As important optical devices, including diffuse scatterers, broadband reflectors, and image-forming mirrors with superior properties, biological guanine crystals have been thoroughly investigated in recent years. However, controlling the polymorph and morphology of synthetic guanine crystals is still highly challenging. Herein, a pure phase of the anhydrous guanine (AG) ss form was obtained via transformation of a hydrated amorphous guanine phase (HAmG) in solvents such as formamide, DMSO and DMF. The AG ss crystals can be stable for up to three months in the above solvents. The AG ss nano-platelets obtained in DMSO exposed the (100) plane when polyvinylpyrrolidone (PVP) was applied as an additive. The AG ss nano-platelets were about 100 nm in length, 40 nm in width, and 10-20 nm in thickness. Solid-state NMR (ssNMR) characterization indicated that the HAmG precursor had a similar short-range order as AG ss, which might be the reason for the formation of AG ss instead of the thermodynamically more stable AG a. The delicate control of the polymorph and morphology of the guanine crystals via an amorphous phase strategy may inspire the formation of highly ordered hierarchical structures of guanine crystals with unique optical properties.
Geometric description of self-interaction potential in symmetric protein complexes
Empereur-Mot C., Garcia-Seisdedos H., Elad N., Dey S. & Levy E. D. (2019) Scientific data. 6, 1, 64. Abstract
Proteins can self-associate with copies of themselves to form symmetric complexes called homomers. Homomers are widespread in all kingdoms of life and allow for unique geometric and functional properties, as reflected in viral capsids or allostery. Once a protein forms a homomer, however, its internal symmetry can compound the effect of point mutations and trigger uncontrolled self-assembly into high-order structures. We identified mutation hot spots for supramolecular assembly, which are predictable by geometry. Here, we present a dataset of descriptors that characterize these hot spot positions both geometrically and chemically, as well as computer scripts allowing the calculation and visualization of these properties for homomers of choice. Since the biological relevance of homomers is not readily available from their X-ray crystallographic structure, we also provide reliability estimates obtained by methods we recently developed. These data have implications in the study of disease-causing mutations, protein evolution and can be exploited in the design of biomaterials.
The Effect of the Phospholipid Bilayer Environment on Cholesterol Crystal Polymorphism
Varsano N., Beghi F., Dadosh T., Elad N., Pereiro E., Haran G., Leiserowitz L. & Addadi L. (2019) ChemPlusChem. 84, 4, p. 338-344 Abstract
Cholesterol crystallization from mixtures of unesterified cholesterol with phospholipids and cholesterol esters is believed to be a key event in atherosclerosis progression. Not much is understood, however, about the influence of the lipid environment on cholesterol crystallization. Here we study cholesterol monohydrate crystal formation from mixed bilayers with palmitoyl-oleoyl-phosphatidylcholine (POPC), dipalmitoyl-phosphatidylcholine (DPPC) and sphingomyelin. We show that disordered phospholipids and sphingomyelin stabilize the formation of crystal plates of the triclinic cholesterol monohydrate polymorph, whereas saturated glycerolipids stabilize helical and tubular crystals of the metastable monoclinic polymorph. We followed the subsequent transformation of these helical crystals into the stable triclinic plates. Discovering the relations between membrane lipid composition and cholesterol crystal polymorphism may provide important clues to the understanding of cholesterol crystal formation in atherosclerosis.
The Effect of the Phospholipid Bilayer Environment on Cholesterol Crystal Polymorphism [Cover Profile]
Varsano N., Beghi F., Dadosh T., Elad N., Pereiro E., Haran G., Leiserowitz L. & Addadi L. (2019) ChemPlusChem. 84, 4, p. 317-317 Abstract
Invited for this month's cover are the group of Prof. Lia Addadi at the Weizmann Institute of Science, Israel and collaborators at the Università Degli Studi di Milano, Italy, and the ALBA Synchrotron Light Source, Spain. The front cover shows how cholesterol crystals form in macrophage cells and in lipid bilayers of different compositions. Cholesterol monohydrate stable triclinic crystals form in vitro as rhomb-shaped plates, whereas the monoclinic crystals fold into tubular or helical shapes. Read the full text of the article at 10.1002/cplu.201800632.
Tunable microsecond dynamics of an allosteric switch regulate the activity of a AAA+ disaggregation machine
Mazal H., Iljina M., Barak Y., Elad N., Rosenzweig R., Goloubinoff P., Riven I. & Haran G. (2019) Nature Communications. 10, 1, 1438. Abstract
Large protein machines are tightly regulated through allosteric communication channels. Here we demonstrate the involvement of ultrafast conformational dynamics in allosteric regulation of CIpB, a hexameric AAA+ machine that rescues aggregated proteins. Each subunit of CIpB contains a unique coiled-coil structure, the middle domain (M domain), proposed as a control element that binds the co-chaperone DnaK. Using single-molecule FRET spectroscopy, we probe the M domain during the chaperone cycle and find it to jump on the microsecond time scale between two states, whose structures are determined. The M-domain jumps are much faster than the overall activity of CIpB, making it an effectively continuous, tunable switch. Indeed, a series of allosteric interactions are found to modulate the dynamics, including binding of nucleotides, DnaK and protein substrates. This mode of dynamic control enables fast cellular adaptation and may be a general mechanism for the regulation of cellular machineries.

2018

Expression of a recombinant, 4'-Phosphopantetheinylated, active M. tuberculosis fatty acid synthase I in E. coli
Baron S., Peleg Y., Grunwald J., Morgenstern D., Elad N., Peretz M., Albeck S., Levin Y., Welch J. T., DeWeerd K. A., Schwarz A., Burstein Y., Diskin R., Shakked Z., Zimhony O. et al. (2018) PLoS ONE. 9, e0204457. Abstract
Fatty acid synthase 1 (FAS I) from Mycobacterium tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4'-Phosphopantetheine (4'-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4'-PP transferase, termed acyl carrier protein synthase (AcpS). In order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid. We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography. Activation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM. Purified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structure-function analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.

[All authors]
Structure of Type-I Mycobacterium tuberculosis fatty acid synthase at 3.3 angstrom resolution
Elad N., Baron S., Peleg Y., Albeck S., Grunwald J., Raviv G., Shakked Z., Zimhony O. & Diskin R. (2018) Nature Communications. 9, 1, 3886. Abstract
Tuberculosis (TB) is a devastating and rapidly spreading disease caused by Mycobacterium tuberculosis (Mtb). Therapy requires prolonged treatment with a combination of multiple agents and interruptions in the treatment regimen result in emergence and spread of multidrug resistant (MDR) Mtb strains. MDR Mtb poses a significant global health problem, calling for urgent development of novel drugs to combat TB. Here, we report the 3.3 angstrom resolution structure of the similar to 2 MDa type-I fatty acid synthase (FAS-I) from Mtb, determined by single particle cryo-EM. Mtb FAS-I is an essential enzymatic complex that contributes to the virulence of Mtb, and thus a prime target for anti-TB drugs. The structural information for Mtb FAS-I we have obtained enables computer-based drug discovery approaches, and the resolution achieved by cryo-EM is sufficient for elucidating inhibition mechanisms by putative small molecular weight inhibitors.
Two polymorphic cholesterol monohydrate crystal structures form in macrophage culture models of atherosclerosis
Varsano N., Beghi F., Elad N., Pereiro E., Dadosh T., Pinkas I., Perez-Berna A. J., Jin X., Kruth H. S., Leiserowitz L., Addadi L. et al. (2018) Proceedings Of The National Academy Of Sciences Of The United States Of America-Biological Sciences. 115, 30, p. 7662-7669 Abstract
The formation of atherosclerotic plaques in the blood vessel walls is the result of LDL particle uptake, and consequently of cholesterol accumulation in macrophage cells. Excess cholesterol accumulation eventually results in cholesterol crystal deposition, the hallmark of mature atheromas. We followed the formation of cholesterol crystals in J774A.1 macrophage cells with time, during accumulation of LDL particles, using a previously developed correlative cryosoft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM) technique. We show, in the initial accumulation stages, formation of small quadrilateral crystal plates associated with the cell plasma membrane, which may subsequently assemble into large aggregates. These plates match crystals of the commonly observed cholesterol monohydrate triclinic structure. Large rod-like cholesterol crystals form at a later stage in intracellular locations. Using cryotransmission electron microscopy (cryo-TEM) and cryoelectron diffraction (cryo-ED), we show that the structure of the large elongated rods corresponds to that of monoclinic cholesterol monohydrate, a recently determined polymorph of the triclinic crystal structure. These monoclinic crystals form with an unusual hollow cylinder or helical architecture, which is preserved in the mature rodlike crystals. The rod-like morphology is akin to that observed in crystals isolated from atheromas. We suggest that the crystals in the atherosclerotic plaques preserve in their morphology the memory of the structure in which they were formed. The identification of the polymorph structure, besides explaining the different crystal morphologies, may serve to elucidate mechanisms of cholesterol segregation and precipitation in atherosclerotic plaques.

[All authors]

2017

The image-forming mirror in the eye of the scallop
Palmer B. A., Taylor G. J., Brumfeld V., Gur D., Shemesh M., Elad N., Osherov A., Oron D., Weiner S., Addadi L. et al. (2017) Science. 358, 6367, p. 1172-1175 Abstract
Scallops possess a visual system comprising up to 200 eyes, each containing a concave mirror rather than a lens to focus light. The hierarchical organization of the multilayered mirror is controlled for image formation, from the component guanine crystals at the nanoscale to the complex three-dimensional morphology at the millimeter level. The layered structure of the mirror is tuned to reflect the wavelengths of light penetrating the scallop's habitat and is tiled with a mosaic of square guanine crystals, which reduces optical aberrations. The mirror forms images on a double-layered retina used for separately imaging the peripheral and central fields of view. The tiled, off-axis mirror of the scallop eye bears a striking resemblance to the segmented mirrors of reflecting telescopes.

[All authors]
Detection of isolated protein-bound metal ions by single-particle cryo-STEM
Elad N., Bellapadrona G., Houben L., Sagi I. & Elbaum M. (2017) Proceedings Of The National Academy Of Sciences Of The United States Of America-Physical Sciences. 114, 42, p. 11139-11144 Abstract
Metal ions play essential roles in many aspects of biological chemistry. Detecting their presence and location in proteins and cells is important for understanding biological function. Conventional structural methods such as X-ray crystallography and cryo-transmission electron microscopy can identify metal atoms on protein only if the protein structure is solved to atomic resolution. We demonstrate here the detection of isolated atoms of Zn and Fe on ferritin, using cryogenic annular dark-field scanning transmission electron microscopy (cryo-STEM) coupled with single-particle 3D reconstructions. Zn atoms are found in a pattern that matches precisely their location at the ferroxidase sites determined earlier by X-ray crystallography. By contrast, the Fe distribution is smeared along an arc corresponding to the proposed path from the ferroxidase sites to the mineral nucleation sites along the twofold axes. In this case the single-particle reconstruction is interpreted as a probability distribution function based on the average of individual locations. These results establish conditions for detection of isolated metal atoms in the broader context of electron cryo-microscopy and tomography.
Biologically Controlled Morphology and Twinning in Guanine Crystals
Hirsch A., Palmer B. A., Elad N., Gur D., Weiner S., Addadi L., Kronik L. & Leiserowitz L. (2017) ANGEWANDTE CHEMIE-INTERNATIONAL EDITION. 56, 32, p. 9420-9424 Abstract
Guanine crystals are widely used in nature as components of multilayer reflectors. Guanine-based reflective systems found in the copepod cuticle and in the mirror of the scallop eye are unique in that the multilayered reflectors are tiled to form a contiguous packed array. In the copepod cuticle, hexagonal crystals are closely packed to produce brilliant colors. In the scallop eye, square crystals are tiled to obtain an image-forming reflecting mirror. The tiles are about 1 mm in size and 70 nm thick. According to analysis of their electron diffraction patterns, the hexagon and square tiles are not single crystals. Rather, each tile type is a composite of what appears to be three crystalline domains differently oriented and stacked onto one another, achieved through a twice-repeated twinning about their and crystal axes, respectively. By these means, the monoclinic guanine crystal mimics higher symmetry hexagonal and tetragonal structures to achieve unique morphologies.
Proteins evolve on the edge of supramolecular self-assembly
Garcia-Seisdedos H., Empereur-Mot C., Elad N. & Levy E. D. (2017) Nature. 548, 7666, p. 244-247 Abstract
The self-association of proteins into symmetric complexes is ubiquitous in all kingdoms of life(1-6). Symmetric complexes possess unique geometric and functional properties, but their internal symmetry can pose a risk. In sickle-cell disease, the symmetry of haemoglobin exacerbates the effect of a mutation, triggering assembly into harmful fibrils(7). Here we examine the universality of this mechanism and its relation to protein structure geometry. We introduced point mutations solely designed to increase surface hydrophobicity among 12 distinct symmetric complexes from Escherichia coli. Notably, all responded by forming supramolecular assemblies in vitro, as well as in vivo upon heterologous expression in Saccharomyces cerevisiae. Remarkably, in four cases, micrometre-long fibrils formed in vivo in response to a single point mutation. Biophysical measurements and electron microscopy revealed that mutants self-assembled in their folded states and so were not amyloid-like. Structural examination of 73 mutants identified supramolecular assembly hot spots predictable by geometry. A subsequent structural analysis of 7,471 symmetric complexes showed that geometric hot spots were buffered chemically by hydrophilic residues, suggesting a mechanism preventing mis-assembly of these regions. Thus, point mutations can frequently trigger folded proteins to self-assemble into higher-order structures. This potential is counterbalanced by negative selection and can be exploited to design nanomaterials in living cells.
A new israeli Tobamovirus isolate infects tomato plants harboring Tm-2² resistance genes
Luria N., Smith E., Reingold V., Bekelman I., Lapidot M., Levin I., Elad N., Tam Y., Sela N., Abu-Ras A., Ezra N., Haberman A., Yitzhak L., Lachman O., Dombrovsky A. et al. (2017) PLoS ONE. 12, 1, e0170429. Abstract
An outbreak of a new disease infecting tomatoes occurred in October-November 2014 at the Ohad village in Southern Israel. Symptomatic plants showed a mosaic pattern on leaves accompanied occasionally by narrowing of leaves and yellow spotted fruit. The disease spread mechanically and rapidly reminiscent of tobamovirus infection. Epidemiological studies showed the spread of the disease in various growing areas, in the South and towards the Southeast and Northern parts of the country within a year. Transmission electron microscope (TEM) analysis showed a single rod-like form characteristic to the Tobamovirus genus. We confirmed Koch's postulates for the disease followed by partial host range determination and revealed that tomato cultivars certified to harbor the Tm-2² resistance gene are susceptible to the new viral disease. We further characterized the viral source of the disease using a range of antisera for serological detection and analyzed various virus genera and families for cross-reactivity with the virus. In addition, next generation sequencing of total small RNA was performed on two cultivars grown in two different locations. In samples collected from commercial cultivars across Israel, we found a single virus that caused the disease. The complete genome sequence of the new Israeli tobamovirus showed high sequence identity to the Jordanian isolate of tomato brown rugose fruit virus.

[All authors]

2016

Improved Doxorubicin Encapsulation and Pharmacokinetics of Ferritin-Fusion Protein Nanocarriers Bearing Proline, Serine, and Alanine Elements
Falvo E., Tremante E., Arcovito A., Papi M., Elad N., Boffi A., Morea V., Conti G., Toffoli G., Fracasso G., Giacomini P., Ceci P. et al. (2016) Biomacromolecules. 17, 2, p. 514-522 Abstract
A novel human ferritin-based nanocarrier, composed of 24 modified monomers able to auto-assemble into a modified protein cage, was produced and used as selective carrier of anti-tumor payloads. Each modified monomer derives from the genetic fusion of two distinct modules, namely the heavy chain of human ferritin (HFt) and a stabilizing/protective PAS polypeptide sequence rich in proline (P), serine (S), and alanine (A) residues. Two genetically fused protein constructs containing PAS polymers with 40- and 75-residue lengths, respectively, were compared. They were produced and purified as recombinant proteins in Escherichia coli at high yields. Both preparations were highly soluble and stable in vitro as well as in mouse plasma. Size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy results indicated that PASylated ferritins are fully assembled and highly monodispersed. In addition, yields and stability of encapsulated doxorubicin were significantly better for both HFt-PAS proteins than for wild-type HFt. Importantly, PAS sequences considerably prolonged the half-life of HFt in the mouse bloodstream. Finally, our doxorubicin-loaded nanocages preserved the pharmacological activity of the drug. Taken together, these results indicate that both of the developed HFt-PAS fusion proteins are promising nanocarriers for future applications in cancer therapy.

[All authors]

2015

The dynamic conformational landscape of gamma-secretase
Elad N., De Strooper B., Lismont S., Hagen W., Veugelen S., Arimon M., Horre K., Berezovska O., Sachse C., Chavez-Gutierrez L. et al. (2015) Journal of Cell Science. 128, 3, p. 589-598 Abstract
The structure and function of the gamma-secretase proteases are of great interest because of their crucial roles in cellular and disease processes. We established a novel purification protocol for the gamma-secretase complex that involves a conformation-and complexspecific nanobody, yielding highly pure and active enzyme. Using single particle electron microscopy, we analyzed the gamma-secretase structure and its conformational variability. Under steady-state conditions, the complex adopts three major conformations, which differ in overall compactness and relative position of the nicastrin ectodomain. Occupancy of the active or substrate-binding sites by inhibitors differentially stabilizes subpopulations of particles with compact conformations, whereas a mutation linked to familial Alzheimer disease results in enrichment of extended-conformation complexes with increased flexibility. Our study presents the gamma-secretase complex as a dynamic population of interconverting conformations, involving rearrangements at the nanometer scale and a high level of structural interdependence between subunits. The fact that protease inhibition or clinical mutations, which affect amyloid beta (A beta) generation, enrich for particular subpopulations of conformers indicates the functional relevance of the observed dynamic changes, which are likely to be instrumental for highly allosteric behavior of the enzyme.

[All authors]

2014

Signature Amyloid beta Profiles Are Produced by Different gamma-Secretase Complexes
Acx H., Chavez-Gutierrez L., Serneels L., Lismont S., Benurwar M., Elad N. & De Strooper B. (2014) The journal of Biological chemistry. 289, 7, p. 4346-4355 Abstract
Background: -Secretase complexes generate amyloid- (A) in Alzheimer disease. Results: A profiles of the four -secretase complexes expressed in humans show that PSEN regulates total peptide levels and the A(38) pathway, whereas APH1 affects mainly the efficiency of the carboxypeptidase-like activity. Conclusion: -Secretase subunit composition regulates A generation. Significance: These intrinsic differences could be used to advance AD therapeutic development.-Secretase complexes are involved in the generation of amyloid- (A) in the brain. Therefore, -secretase has been proposed as a potential therapeutic target in Alzheimer disease (AD). Targeting -secretase activity in AD requires the pharmacological dissociation of the processing of physiological relevant substrates and the generation of toxic A. Previous reports suggest the differential targeting of -secretase complexes, based on their subunit composition, as a valid strategy. However, little is known about the biochemical properties of the different complexes, and key questions regarding their A product profiles should be first addressed. Here, we expressed, purified, and analyzed, under the same conditions, the endopeptidase and carboxypeptidase-like activities of the four -secretase complexes present in humans. We find that the nature of the catalytic subunit in the complex affects both activities. Interestingly, PSEN2 complexes discriminate between the A(40) and A(38) production lines, indicating that A generation in one or the other pathway can be dissociated. In contrast, the APH1 subunit mainly affects the carboxypeptidase-like activity, with APH1B complexes favoring the generation of longer A peptides. In addition, we determined that expression of a single human -secretase complex in cell lines retains the intrinsic attributes of the protease while present in the membrane, providing validation for the in vitro studies. In conclusion, our data show that each -secretase complex produces a characteristic A signature. The qualitative and quantitative differences between different -secretase complexes could be used to advance drug development in AD and other disorders.

2013

The role of integrin-linked kinase in the molecular architecture of focal adhesions
Elad N., Volberg T., Patla I., Hirschfeld-Warneken V., Grashoff C., Spatz J. P., Faessler R., Geiger B. & Medalia O. (2013) Journal of Cell Science. 126, 18, p. 4099-4107 Abstract
Integrin-mediated focal adhesions (FAs) are large, multi-protein complexes that link the actin cytoskeleton to the extracellular matrix and take part in adhesion-mediated signaling. These adhesions are highly complex and diverse at the molecular level; thus, assigning particular structural or signaling functions to specific components is highly challenging. Here, we combined functional, structural and biophysical approaches to assess the role of a major FA component, namely, integrin-linked kinase (ILK), in adhesion formation. We show here that ILK plays a key role in the formation of focal complexes, early forms of integrin adhesions, and confirm its involvement in the assembly of fibronectin-bound fibrillar adhesions. Examination of ILK-null fibroblasts by cryo-electron tomography pointed to major structural changes in their FAs, manifested as disarray of the associated actin filaments and an increase in the packing density of FA-related particles. Interestingly, adhesion of the mutant cells to the substrate required a higher ligand density than in control cells. These data indicate that ILK has a key role in integrin adhesion assembly and sub-structure, and in the regulation of the FA-associated cytoskeleton.

2012

The Human Nuclear Pore Complex as Revealed by Cryo-Electron Tomography
Maimon T., Elad N., Dahan I. & Medalia O. (2012) Structure (London, England : 1993). 20, 6, p. 998-1006 Abstract
Nuclear pore complexes (NPCs) are the sole passage through the nuclear envelope, connecting the cytoplasm to the nucleoplasm. These gigantic molecular machines, over 100 MDa in molecular weight, allow free diffusion of small molecules and ions while mediating selective energy-dependent nucleocytoplasmic transport of large macromolecules. Here, we applied cryo-electron tomography to human fibroblast cells, reconstructing their nuclear envelopes without applying any purification steps. From these reconstructions, we extracted subtomograms containing individual NPCs and utilized in silico subtomogram averaging procedures to determine the structure of the mammalian pore complex at a resolution of similar to 6.6 nm. Beyond revealing the canonical features of the human NPC, our analysis identified inner lateral channels and fusing bridge-like structures, suggesting alternative routes of peripheral nuclear passage. Finally, we concluded from our structural analysis that the human NPC is structurally distinct from that of lower eukaryotes in terms of dimension and organization but resembles its amphibian (frog) counterpart.

2011

Assembly and breakdown of microtubules within the midbody
Tamir A., Elad N. & Medalia O. (2011) Communicative and Integrative Biology. 4, 5, p. 552-3 Abstract
In animal cells, cell division concludes with the separation of two daughter cells during a process called cytokinesis. Abscission, the termination of cytokinesis, is performed through formation of the midbody, a vis-á-vis microtubule (MT)-rich structure bridging the daughter cells. Disassembly of the midbody is the final stage of daughter cell separation and occurs in parallel to membrane fusion in this area. To shed light on this process and to better understand MT organization within the dense area of the midbody structure, an integrative fluorescence microscopy and cryo-electron tomography (cryo-ET) approach was taken.1 These efforts led to a resolving of MT architecture at single-fiber resolution, resulting in a refined model of abscission.
Microtubule organization in the final stages of cytokinesis as revealed by cryo-electron tomography
Elad N., Abramovitch S., Sabanay H. & Medalia O. (2011) Journal of Cell Science. 124, 2, p. 207-215 Abstract
The completion of cytokinesis is dominated by the midbody, a tightly-packed microtubule (MT)-based bridge that transiently connects the two daughter cells. Assembled from condensed, spindle-MTs and numerous associated proteins, the midbody gradually narrows down until daughter cell partitioning occurs at this site. Although described many years ago, detailed understanding of the abscission process remains lacking. Applying cryo-electron tomography to purified midbodies, in combination with fluorescence microscopy, we present here new insight into MT organization within the midbody. We find that the midbody is spatially divided into a core bundle of MTs that traverses the electron-dense overlap region (continuous MTs), surrounded by MTs that terminate within the overlap region (polar MTs). Residual continuous MTs remained intact up to the verge of abscission, whereas the residual polar MTs lost their organization and retreated from the overlap region at late cytokinesis stages. A detailed localization of the microtubule-bundling protein PRC1 supports the above notion. Our study thus provides a detailed account of the abscission process and suggests that the midbody, having acquired a distinct MT architecture as compared to the preceding central spindle, actively facilitates the final stage of cytokinesis.

2010

Dissecting the molecular architecture of integrin adhesion sites by cryo-electron tomography
Patla I., Volberg T., Elad N., Hirschfeld-Warneken V., Grashoff C., Faessler R., Spatz J. P., Geiger B. & Medalia O. (2010) Nature Cell Biology. 12, 9, p. 909-915 Abstract
Focal adhesions are integrin-based multiprotein complexes, several micrometres in diameter, that mechanically link the extracellular matrix with the termini of actin bundles. The molecular diversity of focal adhesions and their role in cell migration and matrix sensing has been extensively studied, but their ultrastructural architecture is still unknown. We present the first three-dimensional structural reconstruction of focal adhesions using cryo-electron tomography. Our analyses reveal that the membrane-cytoskeleton interaction at focal adhesions is mediated through particles located at the cell membrane and attached to actin fibres. The particles have diameters of 25 ± 5 nm, and an average interspacing of approximately 45Å nm. Treatment with the Rho-kinase inhibitor Y-27632 induces a rapid decrease in particle diameter, suggesting that they are highly mechanosensitive. Our findings clarify the internal architecture of focal adhesions at molecular resolution, and provide insights into their scaffolding and mechanosensory functions.
CRYOELECTRON TOMOGRAPHY OF EUKARYOTIC CELLS
Mader A., Elad N. & Medalia O. (2010) METHODS IN ENZYMOLOGY, VOL 483: CRYO-EM, PART C: ANALYSES, INTERPRETATION, AND CASE STUDIES. Jensen GJ.(eds.). C ed. p. 245-265 (trueMethods in Enzymology). Abstract
Biological processes involve a high degree of protein dynamics resulting in a constant remodeling of the cellular landscape at the molecular level. Orchestrated changes lead to significant rearrangement of the eukaryotic cytoskeleton and nuclear structures. Visualization of the cellular landscape in the unperturbed state is essential for understanding these processes. The development of cryoelectron tomography (cryo-ET) and its application to eukaryotic cells has provided a major step forward toward better realizing these processes. In conjunction with rapid freezing techniques, that is, vitrification by plunge-freezing and high-pressure freezing, cryo-ET is most suitable for investigating cellular ultrastructures in a close-to-life state. Here, we review the application of cryo-ET to the study of eukaryotic cells, with special emphasis on sample preparation, cytoskeleton organization, and macromolecular structures observed at a resolution of 4-6 nm.

2009

Profilin 1 is required for abscission during late cytokinesis of chondrocytes
Boettcher R. T., Wiesner S., Braun A., Wimmer R., Berna A., Elad N., Medalia O., Pfeifer A., Aszodi A., Costell M., Faessler R. et al. (2009) EMBO Journal. 28, 8, p. 1157-1169 Abstract
Profilins are key factors for dynamic rearrangements of the actin cytoskeleton. However, the functions of profilins in differentiated mammalian cells are uncertain because profilin deficiency is early embryonic lethal for higher eukaryotes. To examine profilin function in chondrocytes, we disrupted the profilin 1 gene in cartilage (Col2pfn1). Homozygous Col2pfn1 mice develop progressive chondrodysplasia caused by disorganization of the growth plate and defective chondrocyte cytokinesis, indicated by the appearance of binucleated cells. Surprisingly, Col2pfn1 chondrocytes assemble and contract actomyosin rings normally during cell division; however, they display defects during late cytokinesis as they frequently fail to complete abscission due to their inability to develop strong traction forces. This reduced force generation results from an impaired formation of lamellipodia, focal adhesions and stress fibres, which in part could be linked to an impaired mDia1-mediated actin filament elongation. Neither an actin nor a poly-proline binding-deficient profilin 1 is able to rescue the defects. Taken together, our results demonstrate that profilin 1 is not required for actomyosin ring formation in dividing chondrocytes but necessary to generate sufficient force for abscission during late cytokinesis.

[All authors]
Structural analysis of the nuclear pore complex by integrated approaches
Elad N., Maimon T., Frenkiel-Krispin D., Lim R. Y. H. & Medalia O. (2009) Current Opinion in Structural Biology. 19, 2, p. 226-232 Abstract
In eukaryotic cells, the nucleus is surrounded by a double membrane system, the nuclear envelope (NE), in which the outer membrane is continuous with the endoplasmic reticulum (ER). Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes to form aqueous translocation channels that allow the free diffusion of small molecules and ions, as well as receptor-mediated transport of large macromolecules. Being the sole gateways for import and export to and from the nucleus, NPCs regulate the nucleocytoplasmic transport of macromolecules in a highly selective manner to maintain cellular functions. The large size and complexity of these multimolecular assemblies, which are composed of similar to 30 different proteins (termed nucleoporins), present a major challenge for structural biologists. Here, we discuss the latest structural findings related to the functional organization of the NPC.

2008

Detection and separation of heterogeneity in molecular complexes by statistical analysis of their two-dimensional projections
Elad N., Clare D. K., Salbil H. R. & Orlova E. V. (2008) Journal of Structural Biology. 162, 1, p. 108-120 Abstract
Progress in molecular structure determination by cryo electron microscopy and single particle analysis has led to improvements in the resolution achievable. However, in many cases the limiting factor is structural heterogeneity of the sample. To address this problem, we have developed a method based on statistical analysis of the two-dimensional images to detect and sort localised structural variations caused, for example, by variable occupancy of a ligand. Images are sorted by two consecutive stages of multivariate statistical analysis (MSA) to dissect out the two main sources of variation, namely out of plane orientation and local structural changes. Heterogeneity caused by local changes is detected by MSA that reveals significant peaks in the higher order eigenimages. The eigenimages revealing local peaks are used for automated classification. Evaluation of differences between classes allows discrimination of molecular images with and without ligand. This method is very rapid, independent of any initial three-dimensional model, and can detect even minor subpopulations in an image ensemble. A strategy for using this technique was developed on model data sets. Here, we demonstrate the successful application of this method to both model and real EM data on chaperonin-substrate and ribosome-ligand complexes. (C) 2007 Elsevier Inc. All rights reserved.

2007

Topologies of a substrate protein bound to the chaperonin GroEL
Elad N., Farr G. W., Clare D. K., Orlova E. V., Horwich A. L. & Saibil H. R. (2007) Molecular Cell. 26, 3, p. 415-426 Abstract
The chaperonin GroEL assists polypeptide folding through sequential steps of binding nonnative protein in the central cavity of an open ring, via hydrophobic surfaces of its apical domains, followed by encapsulation in a hydrophilic cavity. To examine the binding state, we have classified a large data set of GroEL binary complexes with nonnative malate dehydrogenase (MDH), imaged by cryo-electron microscopy, to sort them into homogeneous subsets. The resulting electron density maps show MDH associated in several characteristic binding topologies either deep inside the cavity or at its inlet, contacting three to four consecutive GroEL apical domains. Consistent with visualization of bound polypeptide distributed over many parts of the central cavity, disulfide crosslinking could be carried out between a cysteine in a bound substrate protein and cysteines substituted anywhere inside GroEL. Finally, substrate binding induced adjustments in GroEL itself, observed mainly as clustering together of apical domains around sites of substrate binding.