Microfluidics-Based Force Spectroscopy Enables High-Throughput Force Experiments with Sub-Nanometer Resolution and Sub-Piconewton Sensitivity.

Several techniques have been established to quantify the mechanicals of single molecules. However, most of them show only limited capabilities of parallelizing the measurement by performing many individual measurements simultaneously. Herein, a microfluidics-based single-molecule force spectroscopy method, which achieves sub-nanometer spatial resolution and sub-piconewton sensitivity and is capable of simultaneously quantifying hundreds of single-molecule targets in parallel, is presented. It relies on a combination of total internal reflection microscopy and microfluidics, in which monodisperse fluorescent beads are immobilized on the bottom of a microfluidic channel by macromolecular linkers. Application of a flow generates a well-defined shear force acting on the beads, whereas the nanomechanical linker response is quantified based on the force-induced displacement of individual beads. To handle the high amount of data generated, a cluster analysis which is capable of a semi-automatic identification of measurement artifacts and molecular populations is implemented. The method is validated by probing the mechanical response polyethylene glycol linkers and binding strength of biotin-NeutrAvidin complexes. Two energy barriers (at 3 and 5.7 Å, respectively) in the biotin-NeutrAvidin interaction are resolved and the unfolding behavior of talin's rod domain R3 in the force range between 1 to ≈10 pN is probed.

Biochemical and Biophysical Characterization of Carbonic Anhydrase VI from Human Milk and Saliva.

Carbonic anhydrases (CA, EC 4.2.1.1) catalyze the hydration of carbon dioxide and take part in many essential physiological processes. In humans, 15 CAs are characterized, including the only secreted isoenzyme CA VI. CA VI has been linked to specific processes in the mouth, namely bitter taste perception, dental caries, and maintenance of enamel pellicle, and implicated in several immunity-related phenomena. However, little is known of the mechanisms of the above. In this study, we characterized human CA VI purified from saliva and milk with biophysical methods and measured their enzyme activities and acetazolamide inhibition. Size-exclusion chromatography showed peaks of salivary and milk CA VI corresponding to hexameric state or larger at pH 7.5. At pH 5.0 the hexamer peaks dominated. SDS- PAGE of milk CA VI protein treated with a bifunctional crosslinker further confirmed that a majority of CA VI is oligomers of similar sizes in solution. Mass spectrometry experiments confirmed that both of the two putative N-glycosylation sites, Asn67 and Asn256, are heterogeneously glycosylated. The attached glycans in milk CA VI were di- and triantennary complex-type glycans, carrying both a core fucose and 1 to 2 additional fucose units, whereas the glycans in salivary CA VI were smaller, seemingly degraded forms of core fucosylated complex- or hybrid-type glycans. Mass spectrometry also verified the predicted signal peptide cleavage site and the terminal residue, Gln 18, being in pyroglutamate form. Thorough characterization of CA VI paves way to better understanding of the biological function of the protein.

Talin variant P229S compromises integrin activation and associates with multifaceted clinical symptoms.

Adhesion of cells to the extracellular matrix (ECM) must be exquisitely coordinated to enable development and tissue homeostasis. Cell-ECM interactions are regulated by multiple signalling pathways that coordinate the activation state of the integrin family of ECM receptors. The protein talin is pivotal in this process, and talin's simultaneous interactions with the cytoplasmic tails of the integrins and the plasma membrane are essential to enable robust, dynamic control of integrin activation and cell-ECM adhesion. Here, we report the identification of a de novo heterozygous c.685C>T (p.Pro229Ser) variant in the TLN1 gene from a patient with a complex phenotype. The mutation is located in the talin head region at the interface between the F2 and F3 domains. The characterization of this novel p.P229S talin variant reveals the disruption of adhesion dynamics that result from disturbance of the F2-F3 domain interface in the talin head. Using biophysical, computational and cell biological techniques, we find that the variant perturbs the synergy between the integrin-binding F3 and the membrane-binding F2 domains, compromising integrin activation, adhesion and cell migration. Whilst this remains a variant of uncertain significance, it is probable that the dysregulation of adhesion dynamics we observe in cells contributes to the multifaceted clinical symptoms of the patient and may provide insight into the multitude of cellular processes dependent on talin-mediated adhesion dynamics.

Biochemical and structural characterization of beta-carbonic anhydrase from the parasite Trichomonas vaginalis.

Trichomonas vaginalis is a unicellular parasite and responsible for one of the most common sexually transmittable infections worldwide, trichomoniasis. Carbonic anhydrases (CAs) are enzymes found in all lifeforms and are known to play a vital role in many biochemical processes in organisms including the maintenance of acid-base homeostasis. To date, eight evolutionarily divergent but functionally convergent forms of CAs (α, ß, γ, δ, ζ, η, θ, and ι) have been discovered. The human genome contains only α-CAs, whereas many clinically significant pathogens express only ß-CAs and/or γ-CAs. The characterization of pathogenic ß- and γ-CAs provides important knowledge for targeting these biomolecules to develop novel anti-invectives against trichomoniasis. Here, we report the recombinant production and characterization of the second ß-CA of T. vaginalis (TvaCA2). Light scattering analysis revealed that TvaCA2 is a dimeric protein, which was further supported with in silico modeling, suggesting similar structures between TvaCA2 and the first ß-CA of T. vaginalis (TvaCA1). TvaCA2 exhibited moderate catalytic activity with the following kinetic parameters: kcat of 3.8 × 105 s-1 and kcat/KM of 4.4 × 107 M-1 s-1. Enzyme activity inhibition was studied with a set of clinically used sulfonamides and sulfonamide derivates. Twenty-seven out of the 39 compounds resulted in inhibition with a nanomolar range. These initial results encourage for future work entailing the design of more potent inhibitors against TvaCA2, which may provide new assets to fight trichomoniasis. KEY MESSAGES: • Protozoan parasite Trichomonas vaginalis has two ß-carbonic anhydrases (TvaCA1/2). • TvaCA1/TvaCA2 represents promising targets for antitrichomonal drug development. • TvaCA2 is a dimer of 20.3 kDa and possesses moderate catalytic activity. • The most efficient inhibitor was clinical drug acetazolamide with KI of 222.9 nM. • The 39 tested sulfonamides form the basis for the design of more potent inhibitors.

Surface Modification of Bioactive Glass Promotes Cell Attachment and Spreading.

Phosphate glasses have several advantages over traditional silicate-based bioglasses but are inferior in the crucial step of cell attachment to their surface. Here, as a proof of concept, we analyze fibroblast attachment to the phosphate glass surface subjected to basic treatment and silanization. Silicate (S53P4)- and phosphate (Sr50)-based bioactive glasses were either untreated or surface-treated with basic buffer and functionalized with silane. The surface-treated samples were studied as such and after fibronectin was adsorbed on to their surface. With both glass types, surface treatment enhanced fibroblast adhesion and spreading in comparison to the untreated glass. The surface-treated Sr50 glass allowed for cell adhesion, proliferation, and spreading to a similar extent as seen with S53P4 and borosilicate control glasses. Here, we show that surface treatment of bioactive glass can be used to attract cell adhesion factors found in the serum and promote cell-material adhesion, both important for efficient tissue integration.

Surface Modification of Bioresorbable Phosphate Glasses for Controlled Protein Adsorption.

The traditional silicate bioactive glasses exhibit poor thermal processability, which inhibits fiber drawing or sintering into scaffolds. The composition of the silicate glasses has been modified to enable hot processing. However, the hot forming ability is generally at the expense of bioactivity. Metaphosphate glasses, on the other hand, possess excellent thermal processability, congruent dissolution, and a tailorable degradation rate. However, due to the layer-by-layer dissolution mechanism, cells do not attach to the material surface. Furthermore, the congruent dissolution leads to a low density of OH groups forming on the glass surface, limiting the adsorption of proteins. It is well regarded that the initial step of protein adsorption is critical as the cells interact with this protein layer, rather than the biomaterial itself. In this paper, we explore the possibility of improving protein adsorption on the surface of phosphate glasses through a variety of surface treatments, such as washing the glass surface in acidic (pH 5), neutral, and basic (pH 9) buffer solutions followed or not by a treatment with (3-aminopropyl)triethoxysilane (APTS). The impact of these surface treatments on the surface chemistry (contact angle, ζ-potential) and glass structure (FTIR) was assessed. In this manuscript, we demonstrate that understanding of the material surface chemistry enables to selectively improve the adsorption of albumin and fibronectin (used as model proteins). Furthermore, in this study, well-known silicate bioactive glasses (i.e., S53P4 and 13-93) were used as controls. While surface treatments clearly improved proteins adsorption on the surface of both silicate and phosphate glasses, it is of interest to note that protein adsorption on phosphate glasses was drastically improved to reach similar protein grafting ability to the silicate bioactive glasses. Overall, this study demonstrates that the limited cell/phosphate glass biological response can easily be overcome through deep understanding and control of the glass surface chemistry.

Structural and functional analysis of LIM domain-dependent recruitment of paxillin to αvß3 integrin-positive focal adhesions.

The LIM domain-dependent localization of the adapter protein paxillin to ß3 integrin-positive focal adhesions (FAs) is not mechanistically understood. Here, by combining molecular biology, photoactivation and FA-isolation experiments, we demonstrate specific contributions of each LIM domain of paxillin and reveal multiple paxillin interactions in adhesion-complexes. Mutation of ß3 integrin at a putative paxillin binding site (ß3VE/YA) leads to rapidly inward-sliding FAs, correlating with actin retrograde flow and enhanced paxillin dissociation kinetics. Induced mechanical coupling of paxillin to ß3VE/YA integrin arrests the FA-sliding, thereby disclosing an essential structural function of paxillin for the maturation of ß3 integrin/talin clusters. Moreover, bimolecular fluorescence complementation unveils the spatial orientation of the paxillin LIM-array, juxtaposing the positive LIM4 to the plasma membrane and the ß3 integrin-tail, while in vitro binding assays point to LIM1 and/or LIM2 interaction with talin-head domain. These data provide structural insights into the molecular organization of ß3 integrin-FAs.

Extrusion-Based Bioprinting of Multilayered Nanocellulose Constructs for Cell Cultivation Using In Situ Freezing and Preprint CaCl2 Cross-Linking.

Extrusion-based bioprinting with a preprint cross-linking agent and an in situ cooling stage provides a versatile method for the fabrication of 3D structures for cell culture. We added varying amounts of calcium chloride as a precross-linker into native nanofibrillated cellulose (NFC) hydrogel prior to 3D bioprinting to fabricate structurally stable multilayered constructs without the need for a separate cross-linking bath. To further enhance their stability, we bioprinted the multilayered structures onto an in situ temperature-controlled printing stage at 25, 0, and -10 °C. The extruded and subsequently freeze-dried volumetric constructs maintained their structures after being immersed into a cell culture medium. The ability to maintain the shape after immersion in cell media is an essential feature for the fabrication of stem cell-based artificial organs. We studied the viability and distribution of mouse embryonic fibroblast cells into the hydrogels using luminescence technique and confocal microscopy. Adding CaCl2 increased the stability of the multilayered nanocellulose structures, making them suitable for culturing cells inside the 3D hydrogel environment. Lower stage temperature considerably improved the structural stability of the 3D printed structures, however, had no effect on cell viability.

Cancer associated talin point mutations disorganise cell adhesion and migration.

Talin-1 is a key component of the multiprotein adhesion complexes which mediate cell migration, adhesion and integrin signalling and has been linked to cancer in several studies. We analysed talin-1 mutations reported in the Catalogue of Somatic Mutations in Cancer database and developed a bioinformatics pipeline to predict the severity of each mutation. These predictions were then assessed using biochemistry and cell biology experiments. With this approach we were able to identify several talin-1 mutations affecting integrin activity, actin recruitment and Deleted in Liver Cancer 1 localization. We explored potential changes in talin-1 signalling responses by assessing impact on migration, invasion and proliferation. Altogether, this study describes a pipeline approach of experiments for crude characterization of talin-1 mutants in order to evaluate their functional effects and potential pathogenicity. Our findings suggest that cancer related point mutations in talin-1 can affect cell behaviour and so may contribute to cancer progression.

Crystal structure of the FERM-folded talin head reveals the determinants for integrin binding.

Binding of the intracellular adapter proteins talin and its cofactor, kindlin, to the integrin receptors induces integrin activation and clustering. These processes are essential for cell adhesion, migration, and organ development. Although the talin head, the integrin-binding segment in talin, possesses a typical FERM-domain sequence, a truncated form has been crystallized in an unexpected, elongated form. This form, however, lacks a C-terminal fragment and possesses reduced ß3-integrin binding. Here, we present a crystal structure of a full-length talin head in complex with the ß3-integrin tail. The structure reveals a compact FERM-like conformation and a tightly associated N-P-L-Y motif of ß3-integrin. A critical C-terminal poly-lysine motif mediates FERM interdomain contacts and assures the tight association with the ß3-integrin cytoplasmic segment. Removal of the poly-lysine motif or disrupting the FERM-folded configuration of the talin head significantly impairs integrin activation and clustering. Therefore, structural characterization of the FERM-folded active talin head provides fundamental understanding of the regulatory mechanism of integrin function.

The F1 loop of the talin head domain acts as a gatekeeper in integrin activation and clustering.

Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the ß integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn2+ Here we show that kindlin-1 can replace Mn2+ to mediate ß3 integrin clustering induced by the talin head, but not that induced by the F2-F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for ß3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of α- and ß3-integrins, in order to activate the ß3 integrin heterodimer, further detailing the mechanism by which the talin-kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the ß3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer.This article has an associated First Person interview with the first author of the paper.

Biochemical and structural characterisation of a protozoan beta-carbonic anhydrase from Trichomonas vaginalis.

We report the biochemical and structural characterisation of a beta-carbonic anhydrase (ß-CA) from Trichomonas vaginalis, a unicellular parasite responsible for one of the world's leading sexually transmitted infections, trichomoniasis. CAs are ubiquitous metalloenzymes belonging to eight evolutionarily divergent groups (α, ß, γ, δ, ζ, η, θ, and ι); humans express only α-CAs, whereas many clinically significant pathogens express only ß- and/or γ-CAs. For this reason, the latter two groups of CAs are promising biomedical targets for novel antiinfective agents. The ß-CA from T. vaginalis (TvaCA1) was recombinantly produced and biochemically characterised. The crystal structure was determined, revealing the canonical dimeric fold of ß-CAs and the main features of the enzyme active site. The comparison with the active site of human CA enzymes revealed significant differences that can be exploited for the design of inhibitors selective for the protozoan enzyme with respect to the human ones.

ß1D integrin splice variant stabilizes integrin dynamics and reduces integrin signaling by limiting paxillin recruitment.

Heterodimeric integrin receptors control cell adhesion, migration and extracellular matrix assembly. While the α integrin subunit determines extracellular ligand specificity, the ß integrin chain binds to an acidic residue of the ligand, and cytoplasmic adapter protein families such as talins, kindlins and paxillin, to form mechanosensing cell matrix adhesions. Alternative splicing of the ß1 integrin cytoplasmic tail creates ubiquitously expressed ß1A, and the heart and skeletal muscle-specific ß1D form. To study the physiological difference between these forms, we developed fluorescent ß1 integrins and analyzed their dynamics, localization, and cytoplasmic adapter recruitment and effects on cell proliferation. On fibronectin, GFP-tagged ß1A integrin showed dynamic exchange in peripheral focal adhesions, and long, central fibrillar adhesions. In contrast, GFP-ß1D integrins exchanged slowly, forming immobile and short central adhesions. While adhesion recruitment of GFP-ß1A integrin was sensitive to C-terminal tail mutagenesis, GFP-ß1D integrin was recruited independently of the distal NPXY motif. In addition, a P786A mutation in the proximal, talin-binding NPXY783 motif switched ß1D to a highly dynamic integrin. In contrast, the inverse A786P mutation in ß1A integrin interfered with paxillin recruitment and proliferation. Thus, differential ß1 integrin splicing controls integrin-dependent adhesion signaling, to adapt to the specific physiological needs of differentiated muscle cells.

Molecular features of steroid-binding antidins and their use for assaying serum progesterone.

Chicken avidin (Avd) and streptavidin from Streptomyces avidinii are extensively used in bionanotechnology due to their extremely tight binding to biotin (Kd ~ 10-15 M for chicken Avd). We previously reported engineered Avds known as antidins, which have micro- to nanomolar affinities for steroids, non-natural ligands of Avd. Here, we report the 2.8 Å X-ray structure of the sbAvd-2 (I117Y) antidin co-crystallized with progesterone. We describe the creation of new synthetic phage display libraries and report the experimental as well as computational binding analysis of progesterone-binding antidins. We introduce a next-generation antidin with 5 nM binding affinity for progesterone, and demonstrate the use of antidins for measuring progesterone in serum samples. Our data give insights on how to engineer and alter the binding preferences of Avds and to develop better molecular tools for modern bionanotechnological applications.

Identification and characterization of a novel zebrafish (Danio rerio) pentraxin-carbonic anhydrase.

BACKGROUND: Carbonic anhydrases (CAs) are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H+ ions. Vertebrate genomes generally contain gene loci for 15-21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only secretory protein of the enzymatically active isoforms. We discovered that non-mammalian CA VI contains a C-terminal pentraxin (PTX) domain, a novel combination for both CAs and PTXs. METHODS: We isolated and sequenced zebrafish (Danio rerio) CA VI cDNA, complete with the sequence coding for the PTX domain, and produced the recombinant CA VI-PTX protein. Enzymatic activity and kinetic parameters were measured with a stopped-flow instrument. Mass spectrometry, analytical gel filtration and dynamic light scattering were used for biophysical characterization. Sequence analyses and Bayesian phylogenetics were used in generating hypotheses of protein structure and CA VI gene evolution. A CA VI-PTX antiserum was produced, and the expression of CA VI protein was studied by immunohistochemistry. A knock-down zebrafish model was constructed, and larvae were observed up to five days post-fertilization (dpf). The expression of ca6 mRNA was quantitated by qRT-PCR in different developmental times in morphant and wild-type larvae and in different adult fish tissues. Finally, the swimming behavior of the morphant fish was compared to that of wild-type fish. RESULTS: The recombinant enzyme has a very high carbonate dehydratase activity. Sequencing confirms a 530-residue protein identical to one of the predicted proteins in the Ensembl database (ensembl.org). The protein is pentameric in solution, as studied by gel filtration and light scattering, presumably joined by the PTX domains. Mass spectrometry confirms the predicted signal peptide cleavage and disulfides, and N-glycosylation in two of the four observed glycosylation motifs. Molecular modeling of the pentamer is consistent with the modifications observed in mass spectrometry. Phylogenetics and sequence analyses provide a consistent hypothesis of the evolutionary history of domains associated with CA VI in mammals and non-mammals. Briefly, the evidence suggests that ancestral CA VI was a transmembrane protein, the exon coding for the cytoplasmic domain was replaced by one coding for PTX domain, and finally, in the therian lineage, the PTX-coding exon was lost. We knocked down CA VI expression in zebrafish embryos with antisense morpholino oligonucleotides, resulting in phenotype features of decreased buoyancy and swim bladder deflation in 4 dpf larvae. DISCUSSION: These findings provide novel insights into the evolution, structure, and function of this unique CA form.

Mechanical stability of talin rod controls cell migration and substrate sensing.

Cells adhere to the surrounding tissue and probe its mechanical properties by forming cell-matrix adhesions. Talin is a critical adhesion protein and participates in the transmission of mechanical signals between extracellular matrix and cell cytoskeleton. Force induced unfolding of talin rod subdomains has been proposed to act as a cellular mechanosensor, but so far evidence linking their mechanical stability and cellular response has been lacking. Here, by utilizing computationally designed mutations, we demonstrate that stepwise destabilization of the talin rod R3 subdomain decreases cellular traction force generation, which affects talin and vinculin dynamics in cell-matrix adhesions and results in the formation of talin-rich but unstable adhesions. We observed a connection between talin stability and the rate of cell migration and also found that talin destabilization affects the usage of different integrin subtypes and sensing of extracellular matrix proteins. Experiments with truncated forms of talin confirm the mechanosensory role of the talin R3 subdomain and exclude the possibility that the observed effects are caused by the release of talin head-rod autoinhibition. In conclusion, this study provides evidence into how the controlled talin rod domain unfolding acts as a key regulator of adhesion structure and function and consequently controls central cellular processes such as cell migration and substrate sensing.

The molecular basis of talin2's high affinity toward ß1-integrin.

Talin interacts with ß-integrin tails and actin to control integrin activation, thus regulating focal adhesion dynamics and cell migration. There are two talin genes, Tln1 and Tln2, which encode talin1 and talin2, and it is generally believed that talin2 functions redundantly with talin1. However, we show here that talin2 has a higher affinity to ß1-integrin tails than talin1. Mutation of talin2 S339 to leucine, which can cause Fifth Finger Camptodactyly, a human genetic disease, completely disrupted its binding to ß-integrin tails. Also, substitution of talin1 C336 with Ser enhanced the affinity of talin1, whereas substitution of talin2 S339 with Cys diminished that of talin2. Further computational modeling analysis shows that talin2 S339 formed a hydrogen bond with E353, which is critical for inducing key hydrogen bonds between talin2 N326 and ß1-integrin R760, and between talin2 K327 and ß1-integrin D759. Mutation at any of these residues significantly diminished the interaction of talin2 with ß1- integrin tails. These hydrogen bonds were not observed in talin1/ß1-integrin, but did exist in talin1C336S/ß1-integrin complex. These results suggest that talin2 S339 forms a hydrogen bond with E353 to mediate its high affinity to ß1-integrin.

Talin-bound NPLY motif recruits integrin-signaling adapters to regulate cell spreading and mechanosensing.

Integrin-dependent cell adhesion and spreading are critical for morphogenesis, tissue regeneration, and immune defense but also tumor growth. However, the mechanisms that induce integrin-mediated cell spreading and provide mechanosensing on different extracellular matrix conditions are not fully understood. By expressing ß3-GFP-integrins with enhanced talin-binding affinity, we experimentally uncoupled integrin activation, clustering, and substrate binding from its function in cell spreading. Mutational analysis revealed Tyr747, located in the first cytoplasmic NPLY(747) motif, to induce spreading and paxillin adapter recruitment to substrate- and talin-bound integrins. In addition, integrin-mediated spreading, but not focal adhesion localization, was affected by mutating adjacent sequence motifs known to be involved in kindlin binding. On soft, spreading-repellent fibronectin substrates, high-affinity talin-binding integrins formed adhesions, but normal spreading was only possible with integrins competent to recruit the signaling adapter protein paxillin. This proposes that integrin-dependent cell-matrix adhesion and cell spreading are independently controlled, offering new therapeutic strategies to modify cell behavior in normal and pathological conditions.