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

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.publishedVersionPeer reviewe

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.publishedVersionPeer reviewe

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.</p

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 beta 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 beta 3 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 beta 3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of alpha- and beta 3-integrins, in order to activate the beta 3 integrin heterodimer, further detailing the mechanism by which the talin-kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the beta 3 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.Peer reviewe

Structure-function of Talin1 : An integrin adaptor protein involved in health and disease

Solu on organismin pienin rakenteellinen ja toiminnallinen yksikkö. Solut ovat dynaamisesti vuorovaikutuksessa ympäröivän ympäristönsä kanssa. Solujen välisen tilan täyttävää ainetta, joka on proteiinien ja polysakkaridien verkosto, kutsutaan soluväliaineeksi (engl. extracellular matrix, lyh. ECM). Solun pintareseptoreilla on tärkeä rooli solu-ECM:n ja muiden vuorovaikutusten välittämisessä. Integriinit ovat solun pinnan reseptoreita, jotka osallistuvat monenlaisiin biologisiin prosesseihin, mukaan lukien soluadheesio, homeostaasi, immuunivaste ja syöpä. Yksi integriinien tärkeimmistä tehtävistä on kaksisuuntainen signalointi solukalvon läpi. Koordinoitu vuorovaikutus ECM:n, reseptorien ja solutukirangan välillä on olennaista solujen kiinnittymisessä. Taliini on solun sisällä ilmentyvä proteiini, joka sitoutuu integriinin β-alayksikköihin, säädellen integriinien signalointia ja yhdistäen ne solutukirangan kanssa. Julkaisuissa I ja II keskityimme taliinin pää-domeenin rakenteeseen ja toimintaan molekyylidynamiikkasimulaatioiden, solubiologian ja biokemiallisten analyysien avulla. Havaitsimme, että joustava silmukka taliiinin F1 aladomeenissa on tärkeä β3-integriinien klusteroitumisessa. Lisäksi havaitsimme, että taliinin pää-domeenissa oleva C-terminaalinen poly-lysiini-motiivi välittää taliinin pään aladomeenien välisiä kontakteja varmistaen taliinin pää-domeenin laskostumisen FERM-muotoon. Julkaisuissa III ja IV tutkittiin sairauteen liittyviä taliinin pistemutaatioita. Huomasimme, että pienetkin muutokset taliinissa voivat johtaa merkittäviin muutoksiin solujen toiminnassa. Julkaisussa III kehitimme laskennallisen menetelmän ja pisteytysjärjestelmän analysoimaan talini1-pistemutaatioita, joiden on aiemmissa tutkimuksissa havaittu liittyvän syöpään. I392N:n pistemutaatio F3-domeenissa lisäsi solujen migraatiota ja invaasiota, mutta vähensi integriini-β1-aktivaatiota. L2509P pistemutaatio taliinin dimerisaatiodomeenissa johti solujen polarisoitumisen katoamiseen ja häiritsi adheesion kypsymistä. Tämä johtuu taliinin dimerisoitumisen häiriintymisestä ja aktiinin sitoutumisen heikentymisestä. Julkaisu IV käsittelee de novo heterotsygoottisen variantin P229S analysoimista. Mutaatio löydettiin potilaalta, jolla on monimutkainen fenotyyppi. Tämä mutaatio johti paikallisiin muutoksiin taliinin F2-F3-domeenirajapinnassa, aiheuttaen muutoksia integriinin aktivoitumisessa, solujen adheesioiden rakenteessa ja solujen liikkumisessa. Lisäksi tutkittiin taliinin pistemutaatioita, joita löydettiin spontaania sepelvaltimoiden dissektiota (SCAD) sairastavilta potilailta (julkaisematon data). Havaitsimme yhteyden taliinin pistemutaatioiden ja solujen toiminnan välillä. Tuloksemme korostavat integriinin ja solutukirangan välisen yhteyden tärkeyttä solujen ja kudosten toiminnassa.A cell is the smallest structural and functional unit of an organism. Cells dynamically interact with their surrounding environment. The substance filling the space between cells is a network of proteins and polysaccharides and is called extracellular matrix the (ECM). Cell surface receptors play a vital role in mediating cell-ECM and other interactions. Integrins are cell surface receptors participating in a wide range of biological processes including cell adhesion, homeostasis, immune responses, and cancer. One of the most important aspects of integrins is the bidirectional signalling across the plasma membrane. Coordinated interaction between ECM, receptors and cytoskeleton is essential in the cell adhesion process. Talin is a cytoplasmic protein that binds directly to integrin β-subunits, regulating integrin signalling and connecting them with the actin cytoskeleton. In healthy cells, when talin undergoes force-induced mechanical unfolding, new recognition sites are exposed for binding to cytoskeletal proteins that are involved in mechanotransduction. Dysregulation of talin or its activators may lead to diseased state, deviating integrin activation and mechanotransduction. Furthermore, such disorder may cause changes in cell migration, spreading and overall survival. In publications I and II, we have focused on the structure and function of the talin head domain using molecular dynamic simulations, cell biology and biochemical analyses. We found that a flexible loop in the talin head subdomain F1 is important for β3-integrin clustering. In addition, we found that the C-terminal poly-lysine motif in the talin head mediates FERM interdomain contacts, assuring the FERM-folded configuration of the talin head. In publications III and IV, disease-associated talin point mutations were investigated. We found that even small changes in talin can lead to dramatic effects on cell function. In publication III, we developed a bioinformatic pipeline and scoring system to analyse talin1 point mutations that were found to be associated with cancer in previous studies. An I392N point mutation in the F3 domain increased cell migration and invasion but decreased integrin β1 activation. An L2509P point mutation in the dimerization domain led to a non-polarised cellular phenotype and disruption of adhesion maturation due to a lack of dimerisation and actin binding of the talin´s mutant. Publication IV discusses the identification of a de novo heterozygous variant P229S found from a patient with complex phenotype. This mutation led to local alterations of the F2-F3 domain interface compromising integrin activation, adhesion composition and cell migration. Furthermore, point mutations found from spontaneous coronary artery dissection (SCAD) patients were investigated (unpublished data), adding considerable supportive data demonstrating the impact of talin point mutations on cell function. Our findings suggest that disease associated point mutations in talin1 can affect cell behaviour and may contribute to the progression of different diseases. In addition, the importance of proper connections between integrins and the actin cytoskeleton through talin1 is a critical factor for cell´s health

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.publishedVersionPeer reviewe

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.publishedVersionPeer reviewe

Structural and functional analysis of LIM domain-dependent recruitment of paxillin to αvβ3 integrin-positive focal adhesions

The LIM domain-dependent localization of the adapter protein paxillin to β3 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 β3 integrin at a putative paxillin binding site (β3VE/YA) leads to rapidly inward-sliding FAs, correlating with actin retrograde flow and enhanced paxillin dissociation kinetics. Induced mechanical coupling of paxillin to β3VE/YA integrin arrests the FA-sliding, thereby disclosing an essential structural function of paxillin for the maturation of β3 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 β3 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 β3 integrin-FAs