The molecular architecture of lamins in somatic cells.

The nuclear lamina is a fundamental constituent of metazoan nuclei. It is composed mainly of lamins, which are intermediate filament proteins that assemble into a filamentous meshwork, bridging the nuclear envelope and chromatin. Besides providing structural stability to the nucleus, the lamina is involved in many nuclear activities, including chromatin organization, transcription and replication. However, the structural organization of the nuclear lamina is poorly understood. Here we use cryo-electron tomography to obtain a detailed view of the organization of the lamin meshwork within the lamina. Data analysis of individual lamin filaments resolves a globular-decorated fibre appearance and shows that A- and B-type lamins assemble into tetrameric filaments of 3.5 nm thickness. Thus, lamins exhibit a structure that is remarkably different from the other canonical cytoskeletal elements. Our findings define the architecture of the nuclear lamin meshworks at molecular resolution, providing insights into their role in scaffolding the nuclear lamina.

Structural analysis of multicellular organisms with cryo-electron tomography.

We developed a method for visualizing tissues from multicellular organisms using cryo-electron tomography. Our protocol involves vitrifying samples with high-pressure freezing, thinning them with cryo-FIB-SEM (focused-ion-beam scanning electron microscopy) and applying fiducial gold markers under cryogenic conditions to the lamellae post-milling. We applied this protocol to acquire tomograms of vitrified Caenorhabditis elegans embryos and worms, which showed the intracellular organization of selected tissues at particular developmental stages in otherwise intact specimens.

The macromolecular architecture of platelet-derived microparticles.

Platelets are essential for hemostasis and wound healing. They are involved in fundamental processes of vascular biology such as angiogenesis, tissue regeneration, and tumor metastasis. Upon activation, platelets shed small plasma membrane vesicles termed platelet-derived microparticles (PMPs). PMPs include functional cell adhesion machinery that comprises transmembrane receptors (most abundant are the αIIbß3 integrins), cytoskeletal systems and a large variety of adapter and signaling molecules. Glanzmann thrombasthenia (GT) is a condition characterized by platelets that are deficient of the integrin αIIbß3 heterodimer. Here, we use cryo-electron tomography (cryo-ET) to study the structural organization of PMPs (in both healthy and GT patients), especially the cytoskeleton organization and receptor architecture. PMPs purified from GT patients show a significantly altered cytoskeletal organization, characterized by a reduced number of filaments present, compared to the healthy control. Furthermore, our results show that incubating healthy PMPs with manganese ions (Mn(2+)), in the presence of fibrinogen, induces a major conformational change of integrin receptors, whereas thrombin activation yields a moderate response. These results provide the first insights into the native molecular organization of PMPs.

Developments in cryo-electron tomography for in situ structural analysis.

Structural analysis of macromolecular assemblies and their remodeling during physiological processes is instrumental to defining the fundament of cellular and molecular biology. Recent advances in computational and analytical tools for cryo-electron tomography have enabled the study of macromolecular structures in their native environment, providing unprecedented insights into cell function. Moreover, the recent implementation of direct electron detectors has progressed cryo-electron tomography to a stage where it can now be applied to the reconstruction of macromolecular structures at high resolutions. Here, we discuss some of the recent technical developments in cryo-electron tomography to reveal structures of macromolecular complexes in their physiological medium, focusing mainly on eukaryotic cells.

Evolving protocells to prototissues: rational design of a missing link.

Realization of a functional artificial cell, the so-called protocell, is a major challenge posed by synthetic biology. A subsequent goal is to use the protocellular units for the bottom-up assembly of prototissues. There is, however, a looming chasm in our knowledge between protocells and prototissues. In the present paper, we give a brief overview of the work on protocells to date, followed by a discussion on the rational design of key structural elements specific to linking two protocellular bilayers. We propose that designing synthetic parts capable of simultaneous insertion into two bilayers may be crucial in the hierarchical assembly of protocells into a functional prototissue.

Bend, Push, Stretch: Remarkable Structure and Mechanics of Single Intermediate Filaments and Meshworks.

The cytoskeleton of the eukaryotic cell provides a structural and functional scaffold enabling biochemical and cellular functions. While actin and microtubules form the main framework of the cell, intermediate filament networks provide unique mechanical properties that increase the resilience of both the cytoplasm and the nucleus, thereby maintaining cellular function while under mechanical pressure. Intermediate filaments (IFs) are imperative to a plethora of regulatory and signaling functions in mechanotransduction. Mutations in all types of IF proteins are known to affect the architectural integrity and function of cellular processes, leading to debilitating diseases. The basic building block of all IFs are elongated α-helical coiled-coils that assemble hierarchically into complex meshworks. A remarkable mechanical feature of IFs is the capability of coiled-coils to metamorphize into ß-sheets under stress, making them one of the strongest and most resilient mechanical entities in nature. Here, we discuss structural and mechanical aspects of IFs with a focus on nuclear lamins and vimentin.

Dual energy landscape: the functional state of the ß-barrel outer membrane protein G molds its unfolding energy landscape.

We applied dynamic single-molecule force spectroscopy to quantify the parameters (free energy of activation and distance of the transition state from the folded state) characterizing the energy barriers in the unfolding energy landscape of the outer membrane protein G (OmpG) from Escherichia coli. The pH-dependent functional switching of OmpG directs the protein along different regions on the unfolding energy landscape. The two functional states of OmpG take the same unfolding pathway during the sequential unfolding of ß-hairpins I-IV. After the initial unfolding events, the unfolding pathways diverge. In the open state, the unfolding of ß-hairpin V in one step precedes the unfolding of ß-hairpin VI. In the closed state, ß-hairpin V and ß-strand S11 with a part of extracellular loop L6 unfold cooperatively, and subsequently ß-strand S12 unfolds with the remaining loop L6. These two unfolding pathways in the open and closed states join again in the last unfolding step of ß-hairpin VII. Also, the conformational change from the open to the closed state witnesses a rigidified extracellular gating loop L6. Thus, a change in the conformational state of OmpG not only bifurcates its unfolding pathways but also tunes its mechanical properties for optimum function.

Nonlinear mechanics of lamin filaments and the meshwork topology build an emergent nuclear lamina.

The nuclear lamina-a meshwork of intermediate filaments termed lamins-is primarily responsible for the mechanical stability of the nucleus in multicellular organisms. However, structural-mechanical characterization of lamin filaments assembled in situ remains elusive. Here, we apply an integrative approach combining atomic force microscopy, cryo-electron tomography, network analysis, and molecular dynamics simulations to directly measure the mechanical response of single lamin filaments in three-dimensional meshwork. Endogenous lamin filaments portray non-Hookean behavior - they deform reversibly at a few hundred picoNewtons and stiffen at nanoNewton forces. The filaments are extensible, strong and tough similar to natural silk and superior to the synthetic polymer Kevlar®. Graph theory analysis shows that the lamin meshwork is not a random arrangement of filaments but exhibits small-world properties. Our results suggest that lamin filaments arrange to form an emergent meshwork whose topology dictates the mechanical properties of individual filaments. The quantitative insights imply a role of meshwork topology in laminopathies.

Modulation of molecular interactions and function by rhodopsin palmitylation.

Rhodopsin is palmitylated at two cysteine residues in its carboxyl terminal region. We have looked at the effects of palmitylation on the molecular interactions formed by rhodopsin using single-molecule force spectroscopy and the function of rhodopsin using both in vitro and in vivo approaches. A knockin mouse model expressing palmitate-deficient rhodopsin was used for live animal in vivo studies and to obtain native tissue samples for in vitro assays. We specifically looked at the effects of palmitylation on the chromophore-binding pocket, interactions of rhodopsin with transducin, and molecular interactions stabilizing the receptor structure. The structure of rhodopsin is largely unperturbed by the absence of palmitate linkage. The binding pocket for the chromophore 11-cis-retinal is minimally altered as palmitate-deficient rhodopsin exhibited the same absorbance spectrum as wild-type rhodopsin. Similarly, the rate of release of all-trans-retinal after light activation was the same both in the presence and absence of palmitylation. Significant differences were observed in the rate of transducin activation by rhodopsin and in the force required to unfold the last stable structural segment in rhodopsin at its carboxyl terminal end. A 1.3-fold reduction in the rate of transducin activation by rhodopsin was observed in the absence of palmitylation. Single-molecule force spectroscopy revealed a 2.1-fold reduction in the normalized force required to unfold the carboxyl terminal end of rhodopsin. The absence of palmitylation in rhodopsin therefore destabilizes the molecular interactions formed in the carboxyl terminal end of the receptor, which appears to hinder the activation of transducin by light-activated rhodopsin.

Single-molecule studies of membrane proteins.

Characterizing membrane proteins with single-molecule techniques provides structural and functional insights that cannot be obtained with conventional approaches. Recent studies show that atomic force microscopy (AFM) in the context of a 'lab on a tip' enables the measurement of multiple parameters of membrane proteins. This multifunctional tool can be applied to probe the oligomeric states and conformational changes of membrane protein assemblies in their native environment. The ability to determine diverse properties at high spatial resolution facilitates the mapping of structural flexibilities, electrostatic potentials and electric currents. By using the AFM tip as tweezer, it is possible to characterize unfolding and refolding pathways of single proteins and the location of their molecular interactions. These interactions dictate the stability of the protein and might be modulated by ligands that alter the protein's functional state.

Imaging and Force Spectroscopy of Single Transmembrane Proteins with the Atomic Force Microscope.

The atomic force microscope (AFM) has opened avenues and provided opportunities to investigate biological soft matter and processes ranging from nanometer (nm) to millimeter (mm). The high temporal (millisecond) and spatial (nanometer) resolutions of the AFM are suited for studying many biological processes in their native conditions. The AFM cantilever-aptly termed as a "lab on a tip"-can be used as an imaging tool as well as a handle to manipulate single bonds and proteins. Recent examples have convincingly established AFM as a tool to study the mechanical properties and monitor processes of single proteins and cells with high sensitivity, thus affording insight into important mechanistic details. This chapter specifically focuses on practical and analytical protocols of single-molecule AFM methodologies related to high-resolution imaging and single-molecule force spectroscopy of transmembrane proteins in a lipid bilayer (reconstituted or native). Both these techniques are operator oriented, and require specialized working knowledge of the instrument, theory and practical skills.

Seeing and sensing single G protein-coupled receptors by atomic force microscopy.

G protein-coupled receptors (GPCRs) relay extracellular information across cell membranes through a continuum of conformations that are not always captured in structures. Hence, complementary approaches are required to quantify the physical and chemical properties of the dynamic conformations linking to GPCR function. Atomic force microscopy (AFM)-based high-resolution imaging and force spectroscopy are unique methods to scrutinize GPCRs and to sense their interactions. Here, we exemplify recent AFM-based applications to directly observe the supramolecular assembly of GPCRs in native membranes, to measure the ligand-binding free-energy landscape, and how interactions modulate the structural properties of GPCRs. Common trends in GPCR function are beginning to emerge. We envision that technical developments in combining AFM with superresolution fluorescence imaging will provide insights into how cellular states modulate GPCRs and vice versa.

Conformational Plasticity of Human Protease-Activated Receptor 1 upon Antagonist- and Agonist-Binding.

G protein-coupled receptors (GPCRs) show complex relationships between functional states and conformational plasticity that can be qualitatively and quantitatively described by contouring their free energy landscape. However, how ligands modulate the free energy landscape to direct conformation and function of GPCRs is not entirely understood. Here, we employ single-molecule force spectroscopy to parametrize the free energy landscape of the human protease-activated receptor 1 (PAR1), and delineate the mechanical, kinetic, and energetic properties of PAR1 being set into different functional states. Whereas in the inactive unliganded state PAR1 adopts mechanically rigid and stiff conformations, upon agonist or antagonist binding the receptor mechanically softens, while increasing its conformational flexibility, and kinetic and energetic stability. By mapping the free energy landscape to the PAR1 structure, we observe key structural regions putting this conformational plasticity into effect. Our insight, complemented with previously acquired knowledge on other GPCRs, outlines a more general framework to understand how GPCRs stabilize certain functional states.

Role of extracellular glutamic acids in the stability and energy landscape of bacteriorhodopsin.

Bacteriorhodopsin (BR), a specialized nanomachine, converts light energy into a proton gradient to power Halobacterium salinarum. In this work, we analyze the mechanical stability of a BR triple mutant in which three key extracellular residues, Glu(9), Glu(194), and Glu(204), were mutated simultaneously to Gln. These three Glu residues are involved in a network of hydrogen bonds, in cation binding, and form part of the proton release pathway of BR. Changes in these features and the robust photocycle dynamics of wild-type (WT) BR are apparent when the three extracellular Glu residues are mutated to Gln. It is speculated that such functional changes of proteins go hand in hand with changes in their mechanical properties. Here, we apply single-molecule dynamic force spectroscopy to investigate how the Glu to Gln mutations change interactions, reaction pathways, and the energy barriers of the structural regions of WT BR. The altered heights and positions of individual energy barriers unravel the changes in the mechanical and the unfolding kinetic properties of the secondary structures of WT BR. These changes in the mechanical unfolding energy landscape cause the proton pump to choose unfolding pathways differently. We suggest that, in a similar manner, the changed mechanical properties of mutated BR alter the functional energy landscape favoring different reaction pathways in the light-induced proton pumping mechanism.

From valleys to ridges: exploring the dynamic energy landscape of single membrane proteins.

Membrane proteins are involved in essential biological processes such as energy conversion, signal transduction, solute transport and secretion. All biological processes, also those involving membrane proteins, are steered by molecular interactions. Molecular interactions guide the folding and stability of membrane proteins, determine their assembly, switch their functional states or mediate signal transduction. The sequential steps of molecular interactions driving these processes can be described by dynamic energy landscapes. The conceptual energy landscape allows to follow the complex reaction pathways of membrane proteins while its modifications describe why and how pathways are changed. Single-molecule force spectroscopy (SMFS) detects, quantifies and locates interactions within and between membrane proteins. SMFS helps to determine how these interactions change with temperature, point mutations, oligomerization and the functional states of membrane proteins. Applied in different modes, SMFS explores the co-existence and population of reaction pathways in the energy landscape of the protein and thus reveals detailed insights into local mechanisms, determining its structural and functional relationships. Here we review how SMFS extracts the defining parameters of an energy landscape such as the barrier position, reaction kinetics and roughness with high precision.

Single-Molecule Force Spectroscopy of Transmembrane ß-Barrel Proteins.

Single-molecule force spectroscopy (SMFS) has been widely applied to study the mechanical unfolding and folding of transmembrane proteins. Here, we review the recent progress in characterizing bacterial and human transmembrane ß-barrel proteins by SMFS. First, we describe the mechanical unfolding of transmembrane ß-barrels, which follows a general mechanism dictated by the sequential unfolding and extraction of individual ß-strands and ß-hairpins from membranes. Upon force relaxation, the unfolded polypeptide can insert stepwise into the membrane as single ß-strands or ß-hairpins to fold as the native ß-barrel. The refolding can be followed at a high spatial and temporal resolution, showing that small ß-barrels are able to fold without assistance, whereas large and complex ß-barrels require chaperone cofactors. Applied in the dynamic mode, SMFS can quantify the kinetic and mechanical properties of single ß-hairpins and reveal complementary insight into the membrane protein structure and function relationship. We further outline the challenges that SMFS experiments must overcome for a comprehensive understanding of the folding and function of transmembrane ß-barrel proteins.

Structural Properties of the Human Protease-Activated Receptor 1 Changing by a Strong Antagonist.

The protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) involved in hemostasis, thrombosis, and inflammation, is activated by thrombin or other coagulation proteases. This activation is inhibited by the irreversible antagonist vorapaxar used for anti-platelet therapy. Despite detailed structural and functional information, how vorapaxar binding alters the structural properties of PAR1 to prevent activation is hardly known. Here we apply dynamic single-molecule force spectroscopy to characterize how vorapaxar binding changes the mechanical, kinetic, and energetic properties of human PAR1 under physiologically relevant conditions. We detect structural segments stabilizing PAR1 and quantify their properties in the unliganded and the vorapaxar-bound state. In the presence of vorapaxar, most structural segments increase conformational variability, lifetime, and free energy, and reduce mechanical rigidity. These changes highlight a general trend in how GPCRs are affected by strong antagonists.

Characterizing molecular interactions in different bacteriorhodopsin assemblies by single-molecule force spectroscopy.

Using single-molecule force spectroscopy we characterized inter- and intramolecular interactions stabilizing structural segments of individual bacteriorhodopsin (BR) molecules assembled into trimers and dimers, and monomers. While the assembly of BR did not vary the location of these structural segments, their intrinsic stability could change up to 70% increasing from monomer to dimer to trimer. Since each stable structural segment established one unfolding barrier, we conclude that the locations of unfolding barriers were determined by intramolecular interactions but that their strengths were strongly influenced by intermolecular interactions. Subtracting the unfolding forces of the BR trimer from that of monomer allowed us to calculate the contribution of inter- and intramolecular interactions to the membrane protein stabilization. Statistical analyses showed that the unfolding pathways of differently assembled BR molecules did not differ in their appearance but in their population. This suggests that in our experiments the membrane protein assembly does not necessarily change the location of unfolding barriers within the protein, but certainly their strengths, and thus alters the probability of a protein to choose certain unfolding pathways.

Multi-compartment encapsulation of communicating droplets and droplet networks in hydrogel as a model for artificial cells.

Constructing a cell mimic is a major challenge posed by synthetic biologists. Efforts to this end have been primarily focused on lipid- and polymer-encapsulated containers, liposomes and polymersomes, respectively. Here, we introduce a multi-compartment, nested system comprising aqueous droplets stabilized in an oil/lipid mixture, all encapsulated in hydrogel. Functional capabilities (electrical and chemical communication) were imparted by protein nanopores spanning the lipid bilayer formed at the interface of the encapsulated aqueous droplets and the encasing hydrogel. Crucially, the compartmentalization enabled the formation of two adjoining lipid bilayers in a controlled manner, a requirement for the realization of a functional protocell or prototissue.

Imaging and detecting molecular interactions of single transmembrane proteins.

Single-molecule atomic force microscopy (AFM) provides novel ways to characterize structure-function relationships of native membrane proteins. High-resolution AFM-topographs allow observing substructures of single membrane proteins at sub-nanometer resolution as well as their conformational changes, oligomeric state, molecular dynamics and assembly. Complementary to AFM imaging, single-molecule force spectroscopy experiments allow detecting molecular interactions established within and between membrane proteins. The sensitivity of this method makes it possible to detect the interactions that stabilize secondary structures such as transmembrane alpha-helices, polypeptide loops and segments within. Changes in temperature or protein-protein assembly do not change the position of stable structural segments, but influence their stability established by collective molecular interactions. Such changes alter the probability of proteins to choose a certain unfolding pathway. Recent examples have elucidated unfolding and refolding pathways of membrane proteins as well as their energy landscapes. We review current and future potential of these approaches to reveal insights into membrane protein structure, function, and unfolding as we recognize that they could help answering key questions in the molecular basis of certain neuro-pathological dysfunctions.