Changes in seam number and location induce holes within microtubules assembled from porcine brain tubulin and in Xenopus egg cytoplasmic extracts.

Microtubules are tubes of about 25 nm in diameter that are critically involved in a variety of cellular functions, including motility, compartmentalization, and division. They are considered as pseudo-helical polymers whose constituent αß-tubulin heterodimers share lateral homotypic interactions, except at one unique region called the seam. Here, we used a segmented sub-tomogram averaging strategy to reassess this paradigm and analyze the organization of the αß-tubulin heterodimers in microtubules assembled from purified porcine brain tubulin in the presence of GTP and GMPCPP, and in Xenopus egg cytoplasmic extracts. We find that in almost all conditions, microtubules incorporate variable protofilament and/or tubulin subunit helical-start numbers, as well as variable numbers of seams. Strikingly, the seam number and location vary along individual microtubules, generating holes of one to a few subunits in size within their lattices. Together, our results reveal that the formation of mixed and discontinuous microtubule lattices is an intrinsic property of tubulin that requires the formation of unique lateral interactions without longitudinal ones. They further suggest that microtubule assembly is tightly regulated in a cytoplasmic environment.

Structural model for differential cap maturation at growing microtubule ends.

Microtubules (MTs) are hollow cylinders made of tubulin, a GTPase responsible for essential functions during cell growth and division, and thus, key target for anti-tumor drugs. In MTs, GTP hydrolysis triggers structural changes in the lattice, which are responsible for interaction with regulatory factors. The stabilizing GTP-cap is a hallmark of MTs and the mechanism of the chemical-structural link between the GTP hydrolysis site and the MT lattice is a matter of debate. We have analyzed the structure of tubulin and MTs assembled in the presence of fluoride salts that mimic the GTP-bound and GDP•Pi transition states. Our results challenge current models because tubulin does not change axial length upon GTP hydrolysis. Moreover, analysis of the structure of MTs assembled in the presence of several nucleotide analogues and of taxol allows us to propose that previously described lattice expansion could be a post-hydrolysis stage involved in Pi release.

Microtubule Seeded-assembly in the Presence of Poorly Nucleating Nucleotide Analogues.

Microtubule dynamic instability is driven by the hydrolysis of the GTP bound to the ß-subunit of the α-ß tubulin heterodimer. Nucleotide analogues are commonly used to mimic the different steps of the tubulin GTPase cycle, but most of them are poor microtubule nucleators. Usually, microtubule assembly is seeded by guanylyl-(α, ß)-methylene-diphosphonate (GMPCPP) or glycerol that can be limiting factors in monitoring the effect of other nucleotide analogs on their polymerization. Here, we describe a protocol that allows the assembly of microtubules in the presence of nucleotide analogues without the need of heterogeneous seeds and at a low final glycerol concentration. Microtubules are first assembled in the presence of the analogue of interest and glycerol to promote assembly. These microtubules are then sonicated to produce seeds that will be used to assemble microtubules in the absence of glycerol. This strategy produces homogeneous nucleotide-bound microtubules that can be further analyzed by biochemical or structural methods such as cryo-electron microscopy.

Should the GCM2 gene be tested when screening for familial primary hyperparathyroidism?

OBJECTIVE: Primary hyperparathyroism (PHPT) is a disease with either sporadic or inherited presentation. Germline mutations responsible for this disease can be found in different genes, the most frequently involved being MEN1, CDC73 = HRPT2 and CASR. During the last few years, new genes have been described as responsible for the development of PHPT such as GCM2. These genes are not systematically included in PHPT genetic screening yet. The aim of this work was to assess the importance of GCM2 genetic analysis in PHPT to determine if this gene should be included in gene panel investigated for this disease. DESIGN AND METHODS: The TENGEN network (French Oncogenetic Network of Neuroendocrine Tumors) collected and interpreted allelic variants according to the clinical characteristics of the GCM2-positive patients identified through genetic testing performed in French laboratories (713 patients with PHPT). RESULTS: From 713 patients with PHPT included in this study, 85 (6.6%) carried at least one GCM2 variant. A total of 12 variants classified as uncertain significance or likely pathogenic were reported in 47 patients. Their mean age at PHPT diagnosis was 49 years. Additionally, the investigation of a large family showed that GCM2 variants could be associated with low penetrance. CONCLUSION: We provide a description and interpretation for GCM2 variants identified in a French population. We suggest that this gene should be included in genetic screening of patients with PHPT and propose the follow-up of asymptomatic patients carrying such variants for calcemia.

Enhanced cell-cell contact stability and decreased N-cadherin-mediated migration upon fibroblast growth factor receptor-N-cadherin cross talk.

N-cadherin adhesion has been reported to enhance cancer and neuronal cell migration either by mediating actomyosin-based force transduction or initiating fibroblast growth factor receptor (FGFR)-dependent biochemical signalling. Here we show that FGFR1 reduces N-cadherin-mediated cell migration. Both proteins are co-stabilised at cell-cell contacts through direct interaction. As a consequence, cell adhesion is strengthened, limiting the migration of cells on N-cadherin. Both the inhibition of migration and the stabilisation of cell adhesions require the FGFR activity stimulated by N-cadherin engagement. FGFR1 stabilises N-cadherin at the cell membrane through a pathway involving Src and p120. Moreover, FGFR1 stimulates the anchoring of N-cadherin to actin. We found that the migratory behaviour of cells depends on an optimum balance between FGFR-regulated N-cadherin adhesion and actin dynamics. Based on these findings we propose a positive feed-back loop between N-cadherin and FGFR at adhesion sites limiting N-cadherin-based single-cell migration.

EB1 interacts with outwardly curved and straight regions of the microtubule lattice.

EB1 is a microtubule plus-end tracking protein that recognizes GTP-tubulin dimers in microtubules and thus represents a unique probe to investigate the architecture of the GTP cap of growing microtubule ends. Here, we conjugated EB1 to gold nanoparticles (EB1-gold) and imaged by cryo-electron tomography its interaction with dynamic microtubules assembled in vitro from purified tubulin. EB1-gold forms comets at the ends of microtubules assembled in the presence of GTP, and interacts with the outer surface of curved and straight tubulin sheets as well as closed regions of the microtubule lattice. Microtubules assembled in the presence of GTP, different GTP analogues or cell extracts display similarly curved sheets at their growing ends, which gradually straighten as their protofilament number increases until they close into a tube. Together, our data provide unique structural information on the interaction of EB1 with growing microtubule ends. They further offer insights into the conformational changes that tubulin dimers undergo during microtubule assembly and the architecture of the GTP-cap region.

Hsp90 directly interacts, in vitro, with amyloid structures and modulates their assembly and disassembly.

BACKGROUND: The 90kDa heat shock protein (Hsp90) participates in regulating the homeostasis of cellular proteins and was considered one of the key chaperones involved in the control and regulation of amyloid deposits. Hsp90 interacts with the amyloid protein tau through tau aggregation-prone regions, including the VQIVYK hexapeptide motif. This hexapeptide, which self-aggregates, forming amyloid fibrils, is widely used to model amyloid formation mechanisms. Despite evidence showing that Hsp90 interacts directly with Ac-VQIVYK-NH2, its role in the hexapeptide fibrillation process and its binding to peptide structures have not yet been determined. METHODS: Various biochemical and biophysical techniques, including ultracentrifugation, spectrophotometry, spectrofluorimetry, and electron microscopy, were employed to assess the effects of Hsp90 on Ac-VQIVYK-NH2 assembly and disassembly processes. RESULTS: At sub-stoichiometric concentrations, Hsp90 bound directly to Ac-VQIVYK-NH2 amyloid structures in vitro, with each Hsp90 dimer interacting with an amyloid structure made of around 50 hexapeptide subunits. Hsp90 inhibited Ac-VQIVYK-NH2 assembly by increasing the critical concentrations of Ac-VQIVYK-NH2 required for assembly. Hsp90 also inhibited the disassembly of Ac-VQIVYK-NH2 amyloid fibrils and promoted their rescue. CONCLUSIONS: A model explaining the dual effect of Hsp90 on the Ac-VQIVYK-NH2 amyloid fibrillation process has been proposed. GENERAL SIGNIFICANCE: These in vitro results provide new insights into the possible roles of molecular chaperones in modulating amyloid structures by limiting the spread of toxic species.

The formation of ordered nanoclusters controls cadherin anchoring to actin and cell-cell contact fluidity.

Oligomerization of cadherins could provide the stability to ensure tissue cohesion. Cadherins mediate cell-cell adhesion by forming trans-interactions. They form cis-interactions whose role could be essential to stabilize intercellular junctions by shifting cadherin clusters from a fluid to an ordered phase. However, no evidence has been provided so far for cadherin oligomerization in cellulo and for its impact on cell-cell contact stability. Visualizing single cadherins within cell membrane at a nanometric resolution, we show that E-cadherins arrange in ordered clusters, providing the first demonstration of the existence of oligomeric cadherins at cell-cell contacts. Studying the consequences of the disruption of the cis-interface, we show that it is not essential for adherens junction formation. Its disruption, however, increased the mobility of junctional E-cadherin. This destabilization strongly affected E-cadherin anchoring to actin and cell-cell rearrangement during collective cell migration, indicating that the formation of oligomeric clusters controls the anchoring of cadherin to actin and cell-cell contact fluidity.

Transport of fibroblast growth factor 2 in the pericellular matrix is controlled by the spatial distribution of its binding sites in heparan sulfate.

The heparan sulfate (HS) chains of proteoglycans are a key regulatory component of the extracellular matrices of animal cells, including the pericellular matrix around the plasma membrane. In these matrices they regulate transport, gradient formation, and effector functions of over 400 proteins central to cell communication. HS from different matrices differs in its selectivity for its protein partners. However, there has been no direct test of how HS in the matrix regulates the transport of its partner proteins. We address this issue by single molecule imaging and tracking in fibroblast pericellular matrix of fibroblast growth factor 2 (FGF2), stoichiometrically labelled with small gold nanoparticles. Transmission electron microscopy and photothermal heterodyne imaging (PHI) show that the spatial distribution of the HS-binding sites for FGF2 in the pericellular matrix is heterogeneous over length scales ranging from 22 nm to several µm. Tracking of individual FGF2 by PHI in the pericellular matrix of living cells demonstrates that they undergo five distinct types of motion. They spend much of their time in confined motion (∼110 nm diameter), but they are not trapped and can escape by simple diffusion, which may be slow, fast, or directed. These substantial translocations (µm) cover distances far greater than the length of a single HS chain. Similar molecular motion persists in fixed cells, where the movement of membrane PGs is impeded. We conclude that FGF2 moves within the pericellular matrix by translocating from one HS-binding site to another. The binding sites on HS chains form non-random, heterogeneous networks. These promote FGF2 confinement or substantial translocation depending on their spatial organisation. We propose that this spatial organisation, coupled to the relative selectivity and the availability of HS-binding sites, determines the transport of FGF2 in matrices. Similar mechanisms are likely to underpin the movement of many other HS-binding effectors.

Bipartite design of a self-fibrillating protein copolymer with nanopatterned peptide display capabilities.

The development of biomatrices for technological and biomedical applications employs self-assembled scaffolds built from short peptidic motifs. However, biopolymers composed of protein domains would offer more varied molecular frames to introduce finer and more complex functionalities in bioreactive scaffolds using bottom-up approaches. Yet, the rules governing the three-dimensional organization of protein architectures in nature are complex and poorly understood. As a result, the synthetic fabrication of ordered protein association into polymers poses major challenges to bioengineering. We have now fabricated a self-assembling protein nanofiber with predictable morphologies and amenable to bottom-up customization, where features supporting function and assembly are spatially segregated. The design was inspired by the cross-linking of titin filaments by telethonin in the muscle sarcomere. The resulting fiber is a two-protein system that has nanopatterned peptide display capabilities as shown by the recruitment of functionalized gold nanoparticles at regular intervals of ∼ 5 nm, yielding a semiregular linear array over micrometers. This polymer promises the uncomplicated display of biologically active motifs to selectively bind and organize matter in the fine nanoscale. Further, its conceptual design has high potential for controlled plurifunctionalization.

The heparan sulfate co-receptor and the concentration of fibroblast growth factor-2 independently elicit different signalling patterns from the fibroblast growth factor receptor.

BACKGROUND: The fibroblast growth factor receptor (FGFR) interprets concentration gradients of FGF ligands and structural changes in the heparan sulfate (HS) co-receptor to generate different cellular responses. However, whether the FGFR generates different signals is not known. RESULTS: We have previously shown in rat mammary fibroblasts that in cells deficient in sulfation, and so in HS co-receptor, FGF-2 can only stimulate a transient phosphorylation of p42/44 MAPK and so cannot stimulate DNA synthesis. Here we demonstrate that this is because in the absence of HS, FGF-2 fails to stimulate the phosphorylation of the adaptor FGFR substrate 2 (FRS2). In cells possessing the HS co-receptor, FGF-2 elicits a bell-shaped dose response: optimal concentrations stimulate DNA synthesis, but supramaximal concentrations (>/= 100 ng/mL) have little effect. At optimal concentrations (300 pg/mL) FGF-2 stimulates a sustained dual phosphorylation of p42/44 MAPK and tyrosine phosphorylation of FRS2. In contrast, 100 ng/mL FGF-2 only stimulates a transient early peak of p42/44 MAPK phosphorylation and fails to stimulate appreciably the phosphorylation of FRS2 on tyrosine. CONCLUSIONS: These results suggest that the nature of the FGFR signal produced is determined by a combination of the HS co-receptor and the concentration of FGF ligand. Both the phosphorylation of the adaptor FRS2, the kinetics (sustained or transient) of phosphorylation of p42/44(MAPK) are varied, and so differing cellular responses are produced.

N-Glycosylation regulates fibroblast growth factor receptor/EGL-15 activity in Caenorhabditis elegans in vivo.

The regulation of cell function by fibroblast growth factors (FGFs) classically occurs through a dual receptor system of a tyrosine kinase receptor (FGFR) and a heparan sulfate proteoglycan co-receptor. Mutations in some consensus N-glycosylation sites in human FGFR result in skeletal disorders and craniosynostosis syndromes, and biophysical studies in vitro suggest that N-glycosylation of FGFR alters ligand and heparan sulfate binding properties. The evolutionarily conserved FGFR signaling system of Caenorhabditis elegans has been used to assess the role of N-glycosylation in the regulation of FGFR signaling in vivo. The C. elegans FGF receptor, EGL-15, is N-glycosylated in vivo, and genetic substitution of specific consensus N-glycosylation sites leads to defects in the maintenance of fluid homeostasis and differentiation of sex muscles, both of which are phenotypes previously associated with hyperactive EGL-15 signaling. These phenotypes are suppressed by hypoactive mutations in EGL-15 downstream signaling components or activating mutations in the phosphatidylinositol 3-kinase pathway, respectively. The results show that N-glycans negatively regulate FGFR activity in vivo supporting the notion that mutation of N-glycosylation sites in human FGFR may lead to inappropriate activation of the receptor.

Photothermal absorption correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a popular technique, complementary to cell imaging for the investigation of dynamic processes in living cells. Based on fluorescence, this single molecule method suffers from artifacts originating from the poor fluorophore photophysics: photobleaching, blinking, and saturation. To circumvent these limitations we present here a new correlation method called photothermal absorption correlation spectroscopy (PhACS) which relies on the absorption properties of tiny nano-objects. PhACS is based on the photothermal heterodyne detection technique and measures akin FCS, the time correlation function of the detected signals. Application of this technique to the precise determination of the hydrodynamic sizes of different functionalized gold nanoparticles are presented, highlighting the potential of this method.

Robust ligand shells for biological applications of gold nanoparticles.

An important point regarding the development of stable biofunctional nanoparticles for biomedical applications is their potential for aspecific interactions with the molecules of the biological environment. Here we report a new self-assembled ligand monolayer system for gold nanoparticles called Mix-matrices, formed by a mixture of HS-PEG and alcohol peptides (peptidols) molecules. Stability of the Mix-capped nanoparticles prepared in various conditions was assessed using tests of increasing stringency. The results highlight the importance of identifying a concentration of ligands sufficiently high to obtain a compact matrix when preparing nanoparticles and that the stability of capped nanoparticles in biological environments cannot be predicted solely on their resistance to electrolyte-induced aggregation. The Mix-capped nanoparticles are resistant to aggregation induced by electrolytes and to aspecific interactions with proteins and ligand exchange. In addition, Mix-matrices allow the easy introduction of a single recognition function per nanoparticle, allowing the specific and stoichiometric labeling of proteins with gold nanoparticles. Therefore, the Mix-matrices provide a useful tool for the development of nanoparticle-based quantitative bioanalytical and imaging techniques, as well as for therapeutic purposes, such as the specific targeting of cancerous cells for photothermal destruction.

Supramolecular domains in mixed peptide self-assembled monolayers on gold nanoparticles.

Self-organization in mixed self-assembled monolayers of small molecules provides a route towards nanoparticles with complex molecular structures. Inspired by structural biology, a strategy based on chemical cross-linking is introduced to probe proximity between functional peptides embedded in a mixed self-assembled monolayer at the surface of a nanoparticle. The physical basis of the proximity measurement is a transition from intramolecular to intermolecular cross-linking as the functional peptides get closer. Experimental investigations of a binary peptide self-assembled monolayer show that this transition happens at an extremely low molar ratio of the functional versus matrix peptide. Molecular dynamics simulations of the peptide self-assembled monolayer are used to calculate the volume explored by the reactive groups. Comparison of the experimental results with a probabilistic model demonstrates that the peptides are not randomly distributed at the surface of the nanoparticle, but rather self-organize into supramolecular domains.

Influence of substitution pattern and cation binding on conformation and activity in heparin derivatives.

As model compounds for the biologically important heparan sulfate, eight systematically modified heparin derivatives were studied by synchrotron radiation circular dichroism (SRCD), which is sensitive to uronic acid conformation. Substitution pattern altered uronic acid conformation, even when structural changes were made in adjacent glucosamine residues (e.g. 6-O-desulfation) and did not involve a chromophore. SRCD spectra of these derivatives following conversion to the Na+, K+, Mg2+, Ca2+, Mn2+, Cu2+ and Fe3+ cation forms revealed that almost all substitution/cation combinations resulted in unique spectra, showing that each was structurally distinct. The detailed effects that binding Na+, K+, Mg2+ and Ca2+ ions had on a 2-de-O-sulfated derivative was also studied by NMR, revealing that subtle changes in conformation (by NOE) and flexibility (by T2 measurements) resulted. Conversion to the K+ and Cu2+ ion forms also drastically modified biological activity, from inactive to active, in a cell-based assay of fibroblast growth factor-receptor (FGF2/FGFR1c) signalling and this effect was not reproduced by free cations. These observations could explain the often-contradictory data concerning structure-activity relationships for these derivatives in the literature and, furthermore, argue strongly against the established trend of considering sequence as a complete structural definition. It also provides additional means of modifying the activity of these polysaccharides and suggests a possible additional level of control in biological systems. There are also obvious potential applications for these findings in the biotechnology sphere.

N-glycosylation of fibroblast growth factor receptor 1 regulates ligand and heparan sulfate co-receptor binding.

The regulation of cell function by fibroblast growth factors (FGF) occurs through a dual receptor system consisting of a receptor-tyrosine kinase, FGFR and the glycosaminoglycan heparan sulfate (HS). Mutations of some potential N-glycosylation sites in human fgfr lead to phenotypes characteristic of receptor overactivation. To establish how N-glycosylation may affect FGFR function, soluble- and membrane-bound recombinant receptors corresponding to the extracellular ligand binding domain of FGFR1-IIIc were produced in Chinese Hamster Ovary cells. Both forms of FGFR1-IIIc were observed to be heavily N-glycosylated and migrated on SDS-PAGE as a series of multiple bands between 50 and 75 kDa, whereas the deglycosylated receptors migrated at 32 kDa, corresponding to the expected molecular weight of the polypeptides. Optical biosensor and quartz crystal microbalance-dissipation binding assays show that the removal of the N-glycans from FGFR1-IIIc caused an increase in the binding of the receptor to FGF-2 and to heparin-derived oligosaccharides, a proxy for cellular HS. This effect is mediated by N-glycosylation reducing the association rate constant of the receptor for FGF-2 and heparin oligosaccharides. N-Glycans were analyzed by mass spectrometry, which demonstrates a predominance of bi- and tri-antennary core-fucosylated complex type structures carrying one, two, and/or three sialic acids. Modeling of such glycan structures on the receptor protein suggests that at least some may be strategically positioned to interfere with interactions of the receptor with FGF ligand and/or the HS co-receptor. Thus, the N-glycans of the receptor represent an additional pathway for the regulation of the activity of FGFs.