Structural analysis of receptors and actin polarity in platelet protrusions.

During activation the platelet cytoskeleton is reorganized, inducing adhesion to the extracellular matrix and cell spreading. These processes are critical for wound healing and clot formation. Initially, this task relies on the formation of strong cellular-extracellular matrix interactions, exposed in subendothelial lesions. Despite the medical relevance of these processes, there is a lack of high-resolution structural information on the platelet cytoskeleton controlling cell spreading and adhesion. Here, we present in situ structural analysis of membrane receptors and the underlying cytoskeleton in platelet protrusions by applying cryoelectron tomography to intact platelets. We utilized three-dimensional averaging procedures to study receptors at the plasma membrane. Analysis of substrate interaction-free receptors yielded one main structural class resolved to 26 Å, resembling the αIIbß3 integrin folded conformation. Furthermore, structural analysis of the actin network in pseudopodia indicates a nonuniform polarity of filaments. This organization would allow generation of the contractile forces required for integrin-mediated cell adhesion.

Unveiling the polarity of actin filaments by cryo-electron tomography.

The actin cytoskeleton plays a fundamental role in numerous cellular processes, such as cell motility, cytokinesis, and adhesion to the extracellular matrix. Revealing the polarity of individual actin filaments in intact cells would foster an unprecedented understanding of cytoskeletal processes and their associated mechanical forces. Cryo-electron tomography provides the means for high-resolution structural imaging of cells. However, the low signal-to-noise ratio of cryo-tomograms obscures the high frequencies, and therefore the polarity of actin filaments cannot be directly measured. Here, we developed a method that enables us to determine the polarity of actin filaments in cellular cryo-tomograms. We applied it to reveal the actin polarity distribution in focal adhesions, and show a linear relation between actin polarity and distance from the apical boundary of the adhesion site.

Differential dynamics of early stages of platelet adhesion and spreading on collagen IV- and fibrinogen-coated surfaces.

Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet's periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbß3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.

Glycoprotein Ib clustering in platelets can be inhibited by α-linolenic acid as revealed by cryo-electron tomography.

Platelet adhesion to the sub-endothelial matrix and damaged endothelium occurs through a multi-step process mediated in the initial phase by glycoprotein Ib binding to von Willebrand factor (vWF), which leads to the subsequent formation of a platelet plug. The plant-derived ω-3 fatty acid α-linolenic acid is an abundant alternative to fish-derived n-3 fatty acids and has anti-inflammatory and antithrombotic properties. In this study, we investigated the impact of α-linolenic acid on human platelet binding to vWF under high-shear flow conditions (mimicking blood flow in stenosed arteries). Pre-incubation of fresh human blood from healthy donors with α-linolenic acid at dietary relevant concentrations reduced platelet binding and rolling on vWF-coated microchannels at a shear rate of 100 dyn/cm2 Depletion of membrane cholesterol by incubation of platelet-rich plasma with methyl-ß cyclodextrin abrogated platelet rolling on vWF. Analysis of glycoprotein Ib by applying cryo-electron tomography to intact platelets revealed local clusters of glycoprotein Ib complexes upon exposure to shear force: the formation of these complexes could be prevented by treatment with α-linolenic acid. This study provides novel findings on the rapid local rearrangement of glycoprotein Ib complexes in response to high-shear flow and highlights the mechanism of in vitro inhibition of platelet binding to and rolling on vWF by α-linolenic acid.

The structure of a membrane adenylyl cyclase bound to an activated stimulatory G protein.

Membrane-integral adenylyl cyclases (ACs) are key enzymes in mammalian heterotrimeric GTP-binding protein (G protein)-dependent signal transduction, which is important in many cellular processes. Signals received by the G protein-coupled receptors are conveyed to ACs through G proteins to modulate the levels of cellular cyclic adenosine monophosphate (cAMP). Here, we describe the cryo-electron microscopy structure of the bovine membrane AC9 bound to an activated G protein αs subunit at 3.4-angstrom resolution. The structure reveals the organization of the membrane domain and helical domain that spans between the membrane and catalytic domains of AC9. The carboxyl-terminal extension of the catalytic domain occludes both the catalytic and the allosteric sites of AC9, inducing a conformation distinct from the substrate- and activator-bound state, suggesting a regulatory role in cAMP production.

Tiopronin-Protected Gold Nanoparticles as a Potential Marker for Cryo-EM and Tomography.

Gold nanoparticles (AuNPs) and their conjugation to biological samples have numerous potential applications. When combined with cryo-electron microscopy and tomography analysis, AuNPs may provide a versatile and powerful tool to identify and precisely localize proteins even when attached to cellular components. Here, we describe a general and facile approach for the synthesis of homogeneous and stable AuNPs, which can readily be conjugated to a molecule of interest and imaged by cryo-electron tomography (cryo-ET). We demonstrate the synthesis of 2.2 ± 0.45-nm tiopronin-protected AuNPs, followed by their conjugation with recombinant proteins and peptides. Visualization of the ∼2.2-nm gold-tagged peptides by cryo-ET reveals the potential use of this strategy to label and localize accessible proteins in a cellular environment with nanometric resolution.

Morphometric analysis of spread platelets identifies integrin αIIbß3-specific contractile phenotype.

Haemostatic platelet function is intimately linked to cellular mechanics and cytoskeletal morphology. How cytoskeletal reorganizations give rise to a highly contractile phenotype that is necessary for clot contraction remains poorly understood. To elucidate this process in vitro, we developed a morphometric screen to quantify the spatial organization of actin fibres and vinculin adhesion sites in single spread platelets. Platelets from healthy donors predominantly adopted a bipolar morphology on fibrinogen and fibronectin, whereas distinguishable, more isotropic phenotypes on collagen type I or laminin. Specific integrin αIIbß3 inhibitors induced an isotropic cytoskeletal organization in a dose-dependent manner. The same trend was observed with decreasing matrix stiffness. Circular F-actin arrangements in platelets from a patient with type II Glanzmann thrombasthenia (GT) were consistent with the residual activity of a small number of αIIbß3 integrins. Cytoskeletal morphologies in vitro thus inform about platelet adhesion receptor identity and functionality, and integrin αIIbß3 mechanotransduction fundamentally determines the adoption of a bipolar phenotype associated with contraction. Super-resolution microscopy and electron microscopies further confirmed the stress fibre-like contractile actin architecture. For the first time, our assay allows the unbiased and quantitative assessment of platelet morphologies and could help to identify defective platelet behaviour contributing to elusive bleeding phenotypes.

Toward correlating structure and mechanics of platelets.

The primary physiological function of blood platelets is to seal vascular lesions after injury and form hemostatic thrombi in order to prevent blood loss. This task relies on the formation of strong cellular-extracellular matrix interactions in the subendothelial lesions. The cytoskeleton of a platelet is key to all of its functions: its ability to spread, adhere and contract. Despite the medical significance of platelets, there is still no high-resolution structural information of their cytoskeleton. Here, we discuss and present 3-dimensional (3D) structural analysis of intact platelets by using cryo-electron tomography (cryo-ET) and atomic force microscopy (AFM). Cryo-ET provides in situ structural analysis and AFM gives stiffness maps of the platelets. In the future, combining high-resolution structural and mechanical techniques will bring new understanding of how structural changes modulate platelet stiffness during activation and adhesion.

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.

Surgical treatment of open globe trauma complicated with the presence of an intraocular foreign body.

BACKGROUND: Open globe injuries complicated with the presence of an intraocular foreign body constitute a vision threatening condition. PURPOSE: To present the results of pars plana vitrectomy in patients with intraocular foreign body. MATERIAL AND METHODS: Medical records of 22 patients were analyzed. Retrospective analysis of data included visual acuity, age, gender and type of injury. RESULTS: All patients were men and the mean age was 37 years. All injuries occurred while working with a hammer. All patients were treated with pars plana vitrectomy combined with intraocular foreign body removal and internal limiting membrane peeling. The visual acuities improved in 9 cases (41%), in 13 cases (59%) the deterioration of visual acuity was observed, no eye was enucleated. In 14 eyes pars plana vitrectomy was combined with lens removal, in 14 eyes silicone oil was used as a tamponade. CONCLUSIONS: Surgical intervention with pars plana vitrectomy combined with intraocular foreign body removal and cataract extraction may preserve severely traumatized eyes and maintain or even improve vision. ocular trauma, vitrectomy, intraocular foreign body.

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.

Roll, adhere, spread and contract: structural mechanics of platelet function.

Platelets are involved in life-sustaining processes such as hemostasis, wound healing, atherothrombosis and angiogenesis. Mechanical trauma to blood vessels causes platelet activation resulting in their adherence and clot formation at the damaged site, culminating in clot retraction and tissue repair. Two of the major players underlying this process are the cytoskeleton, i.e., actin and microtubules, and the membrane integrin receptors. Rare congenital bleeding disorders such as Glanzmann thrombasthenia and Bernard-Soulier syndrome are associated with genetic alterations of platelet surface receptors, also affecting the platelet cytoskeletal structure. In this review, we summarize the current knowledge about platelet structure and adhesion, and delve into the mechanical aspects of platelet function. Platelets lack a nucleus, and can thus provide a minimal model of a biological cell. New biophysical tools may help to scrutinize platelets anew and to extend the existing knowledge on cell biology.

αTAT1 is the major α-tubulin acetyltransferase in mice.

Post-translational modification of tubulin serves as a powerful means for rapidly adjusting the functional diversity of microtubules. Acetylation of the ε-amino group of K40 in α-tubulin is one such modification that is highly conserved in ciliated organisms. Recently, αTAT1, a Gcn5-related N-acetyltransferase, was identified as an α-tubulin acetyltransferase in Tetrahymena and C. elegans. Here we generate mice with a targeted deletion of Atat1 to determine its function in mammals. Remarkably, we observe a loss of detectable K40 α-tubulin acetylation in these mice across multiple tissues and in cellular structures such as cilia and axons where acetylation is normally enriched. Mice are viable and develop normally, however, the absence of Atat1 impacts upon sperm motility and male mouse fertility, and increases microtubule stability. Thus, αTAT1 has a conserved function as the major α-tubulin acetyltransferase in ciliated organisms and has an important role in regulating subcellular specialization of subsets of microtubules.

Effects of gonadotropin-releasing hormone agonist/recombinant follicle-stimulating hormone versus gonadotropin-releasing hormone antagonist/recombinant follicle-stimulating hormone on follicular fluid levels of adhesion molecules during in vitro fertilization.

OBJECTIVE: To compare the effects of GnRH-agonist/recombinant rFSH versus GnRH-antagonist/recombinant FSH stimulation on follicular fluid levels of soluble intercellular adhesion molecule (sICAM)-1 and vascular cell adhesion molecule-1 (sVCAM-1) during in vitro fertilization (IVF). DESIGN: Prospective, randomized study. SETTING: University hospital. PATIENT(S): Seventy-three women underwent IVF. INTERVENTION(S): GnRH-agonist/rFSH or GnRH-antagonist/rFSH administration and collection of follicular fluid from 3 small (11-14 mm in diameter) and 3 large (18-21 mm in diameter) follicles on the day of oocyte retrieval. MAIN OUTCOME MEASURE(S): Follicular fluid levels of sICAM-1 and sVCAM-1 and intrafollicular estradiol and progesterone were also measured. RESULT(S): Women who underwent GnRH-agonist/rFSH showed higher concentrations of sICAM-1 in both small and large follicles were compared with patients who received GnRH-antagonist/rFSH treatment; follicular fluid levels of sVCAM-1 were similar between the 2 stimulation protocols. Content of sICAM-1 in small and large follicles positively correlated with the number of follicles of > or =15 mm and the number of oocytes that were retrieved in both study groups. Concentrations of follicular fluid sVCAM-1 and progesterone were higher in large than in small follicles and were correlated positively to each other in both follicular classes. CONCLUSION(S): In IVF, GnRH-agonist/rFSH is associated with higher follicular fluid levels of sICAM-1 compared with GnRH-antagonist/rFSH regimen. Intrafollicular sICAM-1 content may predict ovarian response, and sVCAM-1 appears as an indicator of the degree of follicular luteinization.