Giuseppe Arcovito

Globular structure of human ovulatory cervical mucus.

Human cervical mucus is a heterogeneous mixture of mucin glycoproteins whose relative concentration changes during the ovulatory phases, thereby producing different mucus aggregation structures that can periodically permit the transit of spermatozoa for fertilization. In preovulatory phase, mucus is arranged in compact fiber‐like structures where sperm transit is hindered. Previously, through observations made of fixed and dehydrated samples, a permissive structure in the ovulatory phase was attributed to the larger diameters of pores in the mucus network. Instead, by means of atomic force microscopy, we can show, for the first time, that unfixed ovulatory mucus is composed by floating globules of mucin aggregates. This finding sheds new light on the mechanism that governs spermatozoa transit toward the uterine cavity. In addition, we demonstrate that the switch from globular ovulatory to fibrous preovulatory mucus largely depends on a pH‐driven mechanism. Analysis of mucin 5B primary sequence, the main mucin in ovulatory mucus, highlights pH‐sensitive domains that are associated to flexible regions prone to drive aggregation. We suggest an involvement of these domains in the fiber‐to‐globule switch in cervical mucus.— Brunelli R., Papi, M., Arcovito, G., Bompiani, A., Castagnola, M., Parasassi, T., Sampaolese, B., Vincenzoni, F., De Spirito M. Globular structure of human ovulatory cervical mucus. FASEB J. 21, 3872–3876 (2007)

Mechanical properties of zona pellucida hardening

We have investigated the changes in the mechanical properties of the zona pellucida (ZP), a multilayer glycoprotein coat that surrounds mammalian eggs, that occur after the maturation and fertilization process of the bovine oocyte by using atomic force spectroscopy. The response of the ZP to mechanical stress has been recovered according to a modified Hertz model. ZP of immature oocytes shows a pure elastic behavior. However, for ZPs of matured and fertilized oocyte, a transition from a purely elastic behavior, which occurs when low stress forces are applied, towards a plastic behavior has been observed. The high critical force necessary to induce deformations, which supports the noncovalent long interaction lifetimes of polymers, increases after the cortical reaction. Atomic force microscopy (AFM) images show that oocyte ZP surface appears to be composed mainly of a dense, random meshwork of nonuniformly arranged fibril bundles. More wrinkled surface characterizes matured oocytes compared with immature and fertilized oocytes. From a mechanical point of view, the transition of the matured ZP membrane toward fertilized ZP, through the hardening process, consists of the recovery of the elasticity of the immature ZP while maintaining a plastic transition that, however, occurs with a much higher force compared with that required in matured ZP.

Low density lipoprotein misfolding and amyloidogenesis

In early atherogenesis, subendothelial retention of lipidic droplets is associated with an inflammatory response‐to‐injury, culminating in the formation of foam cells and plaque. Low density lipoprotein (LDL) is the main constituent of subendothelial lipidic droplets. The process is believed to occur following LDL modification. Searching for a modified LDL in plasma, electronegative LDL [LDL(—)] was identified and found to be associated with major risk biomarkers. The apoprotein in LDL(—) is misfolded, and we show here that this modification primes the aggregation of native LDL, conforming to the typical pattern of protein amyloidogenesis. After a lag phase, whose length depends on LDL(—) concentration, light scattering and atomic force microscopy reveal early exponential growth of intermediate globules, which evolve into fibrils. These globules are remarkably similar to subendothelial droplets in atheromatous lesions and different from those produced by oxidation or biochemical manipulation. During aggregation, ellipticity and tryptophan fluorescence measurements reveal a domino‐style spread of apoprotein misfolding from LDL(—) to all of the LDL. Computational analysis of the apoprotein primary sequence predicts an unstable, aggregation‐prone domain in the regulatory α2 region. Apoprotein misfolding well represents an LDL modification able to transform this cholesterol carrier into a trigger for a response‐to‐injury in the artery wall.—Parasassi, T., De Spirito, M., Mei, G., Brunelli, R., Greco, G., Lenzi, L., Maulucci, G., Nicolai, E., Papi, M., Arcovito, G., Tosatto, S. C. E., Ursini, F. Low density lipoprotein misfolding and amyloidogenesis. FASEB J. 22, 2350–2356 (2008)

Evidence of elastic to plastic transition in the zona pellucida of oocytes using atomic force spectroscopy

We have investigated the mechanical properties of the zona pellucida ZP, a multilayer glycoprotein coat that surrounds mammalian eggs, using atomic force spectroscopy. The response of the membrane to mechanical stress has been reconstructed using a modified Hertz model. The transition from elastic behavior, which occurs when low stress forces are applied characterized by a Young’s modulus E=225 kPa, toward plastic behavior is observed. The critical indentation necessary to induce plastic deformations occurs at yield=55050 nm. This high critical value, corresponding to two ZP layers, well supports the noncovalent long lifetimes of interactions that take place between constituent glycoproteins.

Small- and wide-angle elastic light scattering study of fibrin structure

We show how small- and wide-angle elastic light scattering (q ∼ 0.03-30 μm -1 ) can be used to quantitatively characterize the structure of polymeric gels made of semi-flexible entangled fibers. We applied the technique to the study of fibrin gels grown from the polymerization of fibrinogen (FG) macromolecular monomers following activation by the enzyme thrombin (TH), at different concentrations and under different physical-chemical conditions of the gelling solution. Our findings show that the gel can be imagined as a random network of fibers of size d and density p, entangled together to form densely packed blobs of mass fractal dimension D m and average size ξ, which may overlap by a factor η and exhibit a long-range order. Provided that d ≥50-100 nm, all of the above parameters can be recovered by the use of a global fitting function developed by us on the basis on the proposed gel model. When the fibers are thinner (d < ∼50 nm), only the fiber mass/length ratio μ ∼ ρd 2 can be retrieved instead of d and p. Our data confirm and quantify the major changes in the gel structure that can be obtained by varying either the salt concentration of the solution and/or the molar ratio TH/FG. Gels formed in Tris-HCl 50 mM/NaCl 150 mM, pH 7.4 and TH/FG = 0.01 are characterized by relatively small, fairly branched (D in ∼ 1.4-2.0) fibers with a mass/length ratio independent of concentration. On reducing the TH/FG ratio, the fibers become increasingly thicker, with d ∼ 90 nm at TH/FG = 10 -5 . When the salt concentration is reduced to NaCl 100 mM (TH/FG = 0.01) the fibers are less branched (D m ∼ 1.2-1.4), but much thicker, with μ increasing by an order of magnitude. These effects are quantitatively analyzed and discussed.

Fluid viscosity determination by means of uncalibrated atomic force microscopy cantilevers

In this letter it has been proved that the vibrating resonance frequency of an atomic force microscope cantilever is strictly characterized by its thickness (α), while its width/thickness ratio (β) appears to be a less sensitive parameter that can be approximated to a constant. We therefore propose a data analysis method that, by accounting for a constant β, allows for the determination of the value of α and consequently to calculate η. This method of monitoring viscosity has the advantage of requiring short measurement times on very small sample volumes, thereby avoiding laborious, time-consuming cantilever calibration.

High-resolution imaging of redox signaling in live cells through an oxidation-sensitive yellow fluorescent protein.

Measure and visualize intracellular changes in redox state. We present the application of a redox-sensitive mutant of the yellow fluorescent protein (rxYFP) to image, with elevated sensitivity and high temporal and spatial resolution, oxidative responses of eukaryotic cells to pathophysiological stimuli. The method presented, based on the ratiometric quantitation of the distribution of fluorescence by confocal microscopy, allows us to draw real-time “redox maps” of adherent cells and to score subtle changes in the intracellular redox state, such as those induced by overexpression of redox-active proteins. This strategy for in vivo imaging of redox signaling circumvents many of the technical limitations currently encountered in the study of complex redox-based phenomena and promises to contribute substantially to this expanding area of signal transduction.

Ristocetin-induced self-aggregation of von Willebrand factor.

Von Willebrand factor (VWF) is a large multimeric adhesive glycoprotein, with complex roles in thrombosis and hemostasis, present in circulating blood and in secretory granules of endothelial cells and platelets. High shear stress triggers conformational changes responsible for both binding to the platelet receptor glycoprotein GpIb and its self-association, thus supporting the formation of platelet plug under flow. Ristocetin also promotes the interaction of VWF with GpIb and is able to induce platelet aggregation, and thus is largely used to mimic this effect in vitro. In this research paper, we followed the time course of VWF self-association in solution induced by ristocetin binding by light scattering and at the same time we collected atomic force microscopy images to clarify the nature of the assembly that is formed. In fact, this process evolves initially through the formation of fibrils that subsequently interact to form supramolecular structures whose dimensions would be capable of trapping platelets even in the absence of any degree of shear stress or interaction with external surfaces. This intrinsic property, that is the ability to self-aggregate, may be involved in some pathological settings that have been revealed in clinical practice.

Simultaneous static and dynamic light scattering approach to the characterization of the different fibrin gel structures occurring by changing chloride concentration

The structure of fibrin fibers has been investigated by simultaneous elastic and dynamic light scattering measurements. This approach allows the recovery of reliable structural parameters of the fibrin fibers while checking for the reliability of the necessarily ab initio assumptions on some nonexperimentally accessible parameters. The number of protofibrils per fiber section, N, related to the fiber diameter, and the gel mass fractal dimension Dm are obtained. Since the fiber size is largely controlled by ions in the gelling solution, a detailed characterization of the fiber structure formed by changing Cl− concentration is given. While N values decrease from 6000 to 1.5 protofibrils per fiber section, going from 0 to 300 mM in CCl−,Dm increases from 1 to 1.8. This means that the overall gel structure is affected by Cl−, being the fiber size decreased and branch points per unit volume increased.

Self-similarity properties of alpha-crystallin supramolecular aggregates

The supramolecular aggregation of alpha-crystallin, the major protein of the eye lens, was investigated by means of static and dynamic light scattering. The aggregation was induced by generating heat-modified alpha-crystallin forms and by stabilizing the clusters with calcium ions. The kinetic pattern of the aggregation and the structural features of the clusters can be described according to the reaction limited cluster-cluster aggregation theory previously adopted for the study of colloidal particles aggregation systems. Accordingly, the average mass and the hydrodynamic radius of alpha-crystallin supramolecular aggregates grow exponentially in time. The structure factor of the clusters is typical of fractal aggregates. A fractal dimension df approximately 2.15 was determined, indicating a low probability of sticking together of the primitive aggregating particles. As a consequence, the slow-forming clusters assemble a rather compact structure. The basic units forming the fractal aggregates were found to have a radius about twice (approximately 17 nm) that of the native protein and 5.3 times its size, which is consistent with an intermediate molecular assembly corresponding to the already known high molecular weight forms of alpha-crystallin.