Comparative Structural Analysis of 20S Proteasome Ortholog Protein Complexes by Native Mass Spectrometry(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]
(2020) Cell Host & Microbe. 27, 3, p. 418-427 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.
(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]
Guanine and 7,8-Dihydroxanthopterin Reflecting Crystals in the Zander Fish Eye: Crystal Locations, Compositions, and Structures(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]
(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]
(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.
(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.
(2019) Scientific data. 6, 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 [Cover Profile](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.
(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.
Tunable microsecond dynamics of an allosteric switch regulate the activity of a AAA+ disaggregation machine(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) Nature Communications. 9, 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.
Expression of a recombinant, 4 '-Phosphopantetheinylated, active M-tuberculosis fatty acid synthase I in E-coli(2018) PLoS One. 13, 9, e0204457. Abstract
BackgroundFatty 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).MethodsIn order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fast 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.ResultsActivation 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.ConclusionPurified 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]
Two polymorphic cholesterol monohydrate crystal structures form in macrophage culture models of atherosclerosis(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) 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]
(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.
(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 <011 > and <021 > crystal axes, respectively. By these means, the monoclinic guanine crystal mimics higher symmetry hexagonal and tetragonal structures to achieve unique morphologies.
(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.
(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-2(2) 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]
Improved Doxorubicin Encapsulation and Pharmacokinetics of Ferritin-Fusion Protein Nanocarriers Bearing Proline, Serine, and Alanine Elements(2016) Biomacromolecules. 17, 2, p. 514-522 Abstract[All authors]
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 (13), serine (5), 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 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.
(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) 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) 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) 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) 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.
(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) 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 45 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.
(2010) METHODS IN ENZYMOLOGY, VOL 483: CRYO-EM, PART C: ANALYSES, INTERPRETATION, AND CASE STUDIES. Jensen GJ.(eds.). p. 245-265 (Methods 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) 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]
(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.
Detection and separation of heterogeneity in molecular complexes by statistical analysis of their two-dimensional projections(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) 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.