Constitutive formation of caveolae in a bacterium.

Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest.

Application of nanotags and nanobodies for live cell single-molecule imaging of the Z-ring in Escherichia coli.

Understanding where proteins are localized in a bacterial cell is essential for understanding their function and regulation. This is particularly important for proteins that are involved in cell division, which localize at the division septum and assemble into highly regulated complexes. Current knowledge of these complexes has been greatly facilitated by super-resolution imaging using fluorescent protein fusions. Herein, we demonstrate with FtsZ that single-molecule PALM images can be obtained in-vivo using a genetically fused nanotag (ALFA), and a corresponding nanobody fused to mEos3.2. The methodology presented is applicable to other bacterial proteins.

Antibody Complexes.

Monoclonal based therapeutics have always been looked at as a futuristic natural way we could take care of pathogens and many diseases. However, in order to develop, establish and realize monoclonal based therapy we need to understand how the immune system contains or kill pathogens. Antibody complexes serve the means to decode this black box. We have discussed examples of antibody complexes both at biochemical and structural levels to understand and appreciate how discoveries in the field of antibody complexes have started to decoded mechanism of viral invasion and create potential vaccine targets against many pathogens. Antibody complexes have made advancement in our knowledge about the molecular interaction between antibody and antigen. It has also led to identification of potent protective monoclonal antibodies. Further use of selective combination of monoclonal antibodies have provided improved protection against deadly diseases. The administration of newly designed and improved immunogen has been used as potential vaccine. Therefore, antibody complexes are important tools to develop new vaccine targets and design an improved combination of monoclonal antibodies for passive immunization or protection with very little or no side effects.

Spatial separation of FtsZ and FtsN during cell division.

The division of Escherichia coli is mediated by a collection of some 34 different proteins that are recruited to the division septum and are thought to assemble into a macromolecular complex known as 'the divisome'. Herein, we have endeavored to better understand the structure of the divisome by imaging two of its core components; FtsZ and FtsN. Super resolution microscopy (SIM and gSTED) indicated that both proteins are localized in large assemblies, which are distributed around the division septum (i.e., forming a discontinuous ring). Although the rings had similar radii prior to constriction, the individual densities were often spatially separated circumferentially. As the cell envelope constricted, the discontinuous ring formed by FtsZ moved inside the discontinuous ring formed by FtsN. The radial and circumferential separation observed in our images indicates that the majority of FtsZ and FtsN molecules are organized in different macromolecular assemblies, rather than in a large super-complex. This conclusion was supported by fluorescence recovery after photobleaching measurements, which indicated that the dynamic behavior of the two macromolecular assemblies was also fundamentally different. Taken together, the data indicates that constriction of the cell envelope is brought about by (at least) two spatially separated complexes.

Coordinated disassembly of the divisome complex in Escherichia coli.

The divisome is the macromolecular complex that carries out cell division in Escherichia coli. Every generation it must be assembled, and then disassembled so that the sequestered proteins can be recycled. Whilst the assembly process has been well studied, virtually nothing is known about the disassembly process. In this study, we have used super-resolution SIM imaging to monitor pairs of fluorescently tagged divisome proteins as they depart from the division septum. These simple binary comparisons indicated that disassembly occurs in a coordinated process that consists of at least five steps: [FtsZ, ZapA] ⇒ [ZipA, FtsA] ⇒ [FtsL, FtsQ] ⇒ [FtsI, FtsN] ⇒ [FtsN]. This sequence of events is remarkably similar to the assembly process, indicating that disassembly follows a first-in, first-out principle. A secondary observation from these binary comparisons was that FtsZ and FtsN formed division rings that were spatially separated throughout the division process. Thus the data indicate that the divisome structure can be visualized as two concentric rings; a proto-ring containing FtsZ and an FtsN-ring.

Cryo-electron microscopy of IgM-VAR2CSA complex reveals IgM inhibits binding of Plasmodium falciparum to Chondroitin Sulfate A.

Placental malaria is caused by Plasmodium falciparum-infected erythrocytes (IEs) adhering to chondroitin sulfate proteoglycans in placenta via VAR2CSA-type PfEMP1. Human pentameric immunoglobulin M (IgM) binds to several types of PfEMP1, including VAR2CSA via its Fc domain. Here, a 3.6 Å cryo-electron microscopy map of the IgM-VAR2CSA complex reveals that two molecules of VAR2CSA bind to the Cµ4 of IgM through their DBL3X and DBL5ε domains. The clockwise and anti-clockwise rotation of the two VAR2CSA molecules on opposite faces of IgM juxtaposes C-termini of both VAR2CSA near the J chain, where IgM creates a wall between both VAR2CSA molecules and hinders its interaction with its receptor. To support this, we show when VAR2CSA is bound to IgM, its staining on IEs as well as binding of IEs to chondroitin sulfate A in vitro is severely compromised.

Electron lambda-tomography.

Filtered back-projection and weighted back-projection have long been the methods of choice within the electron microscopy community for reconstructing the structure of macromolecular assemblies from electron tomography data. Here, we describe electron lambda-tomography, a reconstruction method that enjoys the benefits of the above mentioned methods, namely speed and ease of implementation, but also addresses some of their shortcomings. In particular, compared to these standard methods, electron lambda-tomography is less sensitive to artifacts that come from structures outside the region that is being reconstructed, and it can sharpen boundaries.

Allosteric activation of preformed EGF receptor dimers by a single ligand binding event.

Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after ligand binding. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilized by ligand binding. This conformational flexibility stabilization most likely accompanies rotation of the entire extracellular domain and the transmembrane domain, resulting in dissociation of the intracellular kinase dimer and, thus, rearranging it into an active form. Consistently, mutations of amino acid residues at the interface of the symmetric inactive kinase dimer spontaneously activate the receptor in vivo. Optical observation also indicated that binding of only one ligand activates the receptor dimer on the cell surface. Our results suggest how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

Structural features of the tmRNA-ribosome interaction.

Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA-ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.

Spo0J and SMC are required for normal chromosome segregation in Staphylococcus aureus.

Bacterial chromosome segregation is an essential cellular process that is particularly elusive in spherical bacteria such as the opportunistic human pathogen Staphylococcus aureus. In this study, we examined the functional significance of a ParB homologue, Spo0J, in staphylococcal chromosome segregation and investigated the role of the structural maintenance of chromosomes (SMC) bacterial condensin in this process. We show that neither spo0J nor smc is essential in S. aureus; however, their absence causes abnormal chromosome segregation. We demonstrate that formation of complexes containing Spo0J and SMC is required for efficient S. aureus chromosome segregation and that SMC localization is dependent on Spo0J. Furthermore, we found that cell division and cell cycle progression are unaffected by the absence of spo0J or smc. Our results verify the role of Spo0J and SMC in ensuring accurate staphylococcal chromosome segregation and also imply functional redundancy or the involvement of additional mechanisms that might contribute to faithful chromosome inheritance.

Detecting Gold Biomineralization by Delftia acidovorans Biofilms on a Quartz Crystal Microbalance.

The extensive use of gold in sensing, diagnostics, and electronics has led to major concerns in solid waste management since gold and other heavy metals are nonbiodegradable and can easily accumulate in the environment. Moreover, gold ions are extremely reactive and potentially harmful for humans. Thus, there is an urgent need to develop reliable methodologies to detect and possibly neutralize ionic gold in aqueous solutions and industrial wastes. In this work, by using complementary measurement techniques such as quartz crystal microbalance (QCM), atomic force microscopy, crystal violet staining, and optical microscopy, we investigate a promising biologically induced gold biomineralization process accomplished by biofilms of bacterium Delftia acidovorans. When stressed by Au3+ ions, D. acidovorans is able to neutralize toxic soluble gold by excreting a nonribosomal peptide, which forms extracellular gold nanonuggets via complexation with metal ions. Specifically, QCM, a surface-sensitive transducer, is employed to quantify the production of these gold complexes directly on the D. acidovorans biofilm in real time. Detailed kinetics obtained by QCM captures the condition for maximized biomineralization yield and offers new insights underlying the biomineralization process. To the best of our knowledge, this is the first study providing an extensive characterization of the gold biomineralization process by a model bacterial biofilm. We also demonstrate QCM as a cheap, user-friendly sensing platform and alternative to standard analytical techniques for studies requiring high-resolution quantitative details, which offers promising opportunities in heavy-metal sensing, gold recovery, and industrial waste treatment.

Cell shape-independent FtsZ dynamics in synthetically remodeled bacterial cells.

FtsZ is the main regulator of bacterial cell division. It has been implicated in acting as a scaffolding protein for other division proteins, a force generator during constriction, and more recently, as an active regulator of septal cell wall production. FtsZ assembles into a heterogeneous structure coined the Z-ring due to its resemblance to a ring confined by the midcell geometry. Here, to establish a framework for examining geometrical influences on proper Z-ring assembly and dynamics, we sculpted Escherichia coli cells into unnatural shapes using division- and cell wall-specific inhibitors in a micro-fabrication scheme. This approach allowed us to examine FtsZ behavior in engineered Z-squares and Z-hearts. We use stimulated emission depletion (STED) nanoscopy to show that FtsZ clusters in sculpted cells maintain the same dimensions as their wild-type counterparts. Based on our results, we propose that the underlying membrane geometry is not a deciding factor for FtsZ cluster maintenance and dynamics in vivo.

Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography.

Nephrin is a key functional component of the slit diaphragm, the structurally unresolved molecular filter in renal glomerular capillaries. Abnormal nephrin or its absence results in severe proteinuria and loss of the slit diaphragm. The diaphragm is a thin extracellular membrane spanning the approximately 40-nm-wide filtration slit between podocyte foot processes covering the capillary surface. Using electron tomography, we show that the slit diaphragm comprises a network of winding molecular strands with pores the same size as or smaller than albumin molecules, as demonstrated in humans, rats, and mice. In the network, which is occasionally stratified, immunogold-nephrin antibodies labeled individually detectable globular cross strands, about 35 nm in length, lining the lateral elongated pores. The cross strands, emanating from both sides of the slit, contacted at the slit center but had free distal endings. Shorter strands associated with the cross strands were observed at their base. Immunolabeling of recombinant nephrin molecules on transfected cells and in vitrified solution corroborated the findings in kidney. Nephrin-deficient proteinuric patients with Finnish-type congenital nephrosis and nephrin-knockout mice had only narrow filtration slits that lacked the slit diaphragm network and the 35-nm-long strands but contained shorter molecular structures. The results suggest the direct involvement of nephrin molecules in constituting the macromolecule-retaining slit diaphragm and its pores.

A dynamical study of antibody-antigen encounter reactions.

The effects of internal dynamics in diffusion-driven encounters between macro-molecules represent a problem of broad relevance in molecular biology. In this view, we investigate a typical antigen-antibody reaction chain, based on a coarse-grained mechanical model parameterized directly upon results from single-molecule experiments. We demonstrate that the internal dynamics is a crucial factor in the encounter process. To describe our numerical results, we formulate a simple, intuitive theoretical framework, and we develop it analytically. This enables us to show that the inner dynamics of antibody molecules results in a cooperative behavior of their individual sub-units. Along the same lines, we also investigate the case of double binding to multi-valent antigens. Our results quantify the enhancement of avidity afforded by the double binding in excellent agreement with the available experimental data.

Structure and flexibility of individual immunoglobulin G molecules in solution.

In contrast to averaging methods of determining structure, such as X-ray diffraction, NMR, and single-particle tomography, cryo-electron tomography allows three-dimensional imaging of an individual object in solution. The method has previously been used to study cells and very large macromolecules. We have used cryo-electron tomography to analyze a monoclonal IgG, with a molecular weight of only 150 kDa. Tomograms reveal y-shaped IgG molecules with three protruding subunits. Docking X-ray structures enabled us to recognize the three subunits as two ellipsoidal Fab arms and a heart-shaped Fc stem. Each subunit has a similar structure in the tomograms and in the X-ray map. Notably, the positions of the Fab arms relative to the Fc stem differed greatly from one molecule to another. The large flexibility of IgG in solution is most likely of functional significance in antigen recognition. This distribution of individual structures provides a qualitative insight into the system dynamics.

FtsZ does not initiate membrane constriction at the onset of division.

The source of constriction required for division of a bacterial cell remains enigmatic. FtsZ is widely believed to be a key player, because in vitro experiments indicate that it can deform liposomes when membrane tethered. However in vivo evidence for such a role has remained elusive as it has been challenging to distinguish the contribution of FtsZ from that of peptidoglycan-ingrowth. To differentiate between these two possibilities we studied the early stages of division in Escherichia coli, when FtsZ is present at the division site but peptidoglycan synthesizing enzymes such as FtsI and FtsN are not. Our approach was to use correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) to monitor the localization of fluorescently labeled FtsZ, FtsI or FtsN correlated with the septal ultra-structural geometry in the same cell. We noted that the presence of FtsZ at the division septum is not sufficient to deform membranes. This observation suggests that, although FtsZ can provide a constrictive force, the force is not substantial at the onset of division. Conversely, the presence of FtsN always correlated with membrane invagination, indicating that allosteric activation of peptidoglycan ingrowth is the trigger for constriction of the cell envelope during cell division in E. coli.

Graph-based sinogram denoising for tomographic reconstructions.

Limited data and low-dose constraints are common problems in a variety of tomographic reconstruction paradigms, leading to noisy and incomplete data. Over the past few years, sinogram denoising has become an essential preprocessing step for low-dose Computed Tomographic (CT) reconstructions. We propose a novel sinogram denoising algorithm inspired by signal processing on graphs. Graph-based methods often perform better than standard filtering operations since they can exploit the signal structure. This makes the sinogram an ideal candidate for graph based denoising since it generally has a piecewise smooth structure. We test our method with a variety of phantoms using different reconstruction methods. Our numerical study shows that the proposed algorithm improves the performance of analytical filtered back-projection (FBP) and iterative methods such as ART (Kaczmarz), and SIRT (Cimmino). We observed that graph denoised sinograms always minimize the error measure and improve the accuracy of the solution, compared to regular reconstructions.

Architecture of Human IgM in Complex with P. falciparum Erythrocyte Membrane Protein 1.

Plasmodium falciparum virulence is associated with sequestration of infected erythrocytes. Microvascular binding mediated by PfEMP1 in complex with non-immune immunoglobulin M (IgM) is common among parasites that cause both severe childhood malaria and pregnancy-associated malaria. Here, we present cryo-molecular electron tomography structures of human IgM, PfEMP1 and their complex. Three-dimensional reconstructions of IgM reveal that it has a dome-like core, randomly oriented Fab2s units, and the overall shape of a turtle. PfEMP1 is a C- shaped molecule with a flexible N terminus followed by an arc-shaped backbone and a bulky C terminus that interacts with IgM. Our data demonstrate that the PfEMP1 binding pockets on IgM overlap with those of C1q, and the bulkiness of PfEMP1 limits the capacity of IgM to interact with PfEMP1. We suggest that P. falciparum exploits IgM to cluster PfEMP1 into an organized matrix to augment its affinity to host cell receptors.

Formation of crystal-like structures and branched networks from nonionic spherical micelles.

Crystal-like structures at nano and micron scales have promise for purification and confined reactions, and as starting points for fabricating highly ordered crystals for protein engineering and drug discovery applications. However, developing controlled crystallization techniques from batch processes remain challenging. We show that neutrally charged nanoscale spherical micelles from biocompatible nonionic surfactant solutions can evolve into nano- and micro-sized branched networks and crystal-like structures. This occurs under simple combinations of temperature and flow conditions. Our findings not only suggest new opportunities for developing controlled universal crystallization and encapsulation procedures that are sensitive to ionic environments and high temperatures, but also open up new pathways for accelerating drug discovery processes, which are of tremendous interest to pharmaceutical and biotechnological industries.

The human skin barrier is organized as stacked bilayers of fully extended ceramides with cholesterol molecules associated with the ceramide sphingoid moiety.

The skin barrier is fundamental to terrestrial life and its evolution; it upholds homeostasis and protects against the environment. Skin barrier capacity is controlled by lipids that fill the extracellular space of the skin's surface layer--the stratum corneum. Here we report on the determination of the molecular organization of the skin's lipid matrix in situ, in its near-native state, using a methodological approach combining very high magnification cryo-electron microscopy (EM) of vitreous skin section defocus series, molecular modeling, and EM simulation. The lipids are organized in an arrangement not previously described in a biological system-stacked bilayers of fully extended ceramides (CERs) with cholesterol molecules associated with the CER sphingoid moiety. This arrangement rationalizes the skin's low permeability toward water and toward hydrophilic and lipophilic substances, as well as the skin barrier's robustness toward hydration and dehydration, environmental temperature and pressure changes, stretching, compression, bending, and shearing.