F1 Domain Research Articles

Kindlin-2 directly binds actin and regulates integrin outside-in signaling.

Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2(+/-)mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK(47)/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK(47)/AA mutation were equivalent in their ability to coactivate integrin αIIbβ3 in a CHO cell system when coexpressed with talin. However, K2-LK(47)/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses.

The Membrane Lipid Composition Regulates The Activity of Talin

Integrins plays a central role in the dynamics of cell‐cell and cell to extra‐cellular matrix interactions and signaling. A key step in the activation of integrin receptors is the binding of the cytoplasmic domain of integrin subunits by talin. This interaction leads to separation of the integrin transmembrane domains and significant conformational changes in the extracellular domains, resulting in a dramatic increase in integrin's affinity for ligands. Talin is a 470 kDa adapter protein that consists of a N‐terminal head domain (THD) and a C‐terminal rod domain. THD is homologous to to the domain found in band 4.1/ezrin/radixin/moesin family of proteins (FERM domain) with uncanonical linear arrangement of F0–F3 domains. A second mechanism in the regulation of integrin activation is directly related to the fact that full‐length talin adopts an auto‐inhibited conformation. In the cytosol, the rod domain binds THD, preventing talin interactions with the membrane surface and the integrin cytoplasmic domain. The membrane bilayer also plays a critical role in the talin ‐integrin interaction, where anionic lipids are required for proper interaction, yet a detailed picture of the interplay between talin and the membrane has remained elusive. We describe a new fluorescence based assay for probing talin – membrane interactions with Nanodisc bilayers of controlled composition. We show that recruitment of THD to the membrane surface is governed by the absolute charge. Measurement of the donor‐acceptor distances reveals that anionic lipids promote a conformational change in the THD favoring a direct interaction of the F3 domain with the phospholipid bilayer. The magnitude of this conformational change is regulated by the identity of the phospholipid headgroup with phosphatidylinositides (e.g. PI4P and PIP2) promoting the largest conformational motions. In addition, the binding of THD to bilayers containing PIP2 is insensitive to the presence of the inhibitory segments of the talin rod domain, while the association of THD to other tested anionic lipid bilayers are strongly inhibited by purified talin rod domains. Furthermore, we have mapped the activation to the interaction of PIP2 with the F2–F3 domain of the THD. Thus, we show that PIP2 plays a central role in the converting talin to a conformation optimized for interactions with integrin cytoplasmic tails and regulation of the auto‐inhibited form of talin, which are essential for integrin inside‐out activation.Support or Funding InformationSupported by NIH grant GM101048.

Kindlin-2 Maintains Vascular Barrier Integrity By Stabilizing Endothelial Adherens Junctions

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, angiogenesis, thrombosis and hemostasis. Its absence is embryonically lethal in mice, emphasizing its biological importance. In the present study, we examined the role of Kindlin-2 in regulation of vascular integrity. In vivo, Kindlin-2+/- mice showed enhanced (70%) permeability of tracheal vasculature to fluorescent beads as compared to wild-type (WT) littermates under both basal conditions or when stimulated with Platelet Activating Factor (PAF). Consistent with these in vivo observations, confluent monolayers of aortic endothelial cells (ECs) isolated from Kindlin-2+/- mice had increased (2-3-fold) baseline, PAF- and thrombin-induced permeability for Alexa-488-labeled bovine serum albumin (BSA) or FITC-dextran-10000 as compared to WT cells. Also, reduction of Kindlin-2 expression by 60-70% in human umbilical vein endothelial cell (HUVEC) monolayers with Kindlin-2-specific siRNA resulted in 3-fold increase in their baseline or thrombin/(PAF)-induced permeability of these markers. Mechanistically, Kindlin-2 co-localized with VE-cadherin and actin within adherens junctions in resting, confluent HUVEC monolayers as well as co-immunoprecipitated with these proteins and other components of adherens junctions, including α-, β- and γ-catenin. In contrast, Kindlin-2 did not co-localize or co-immunoprecipitate with any components of GAP or tight junctions. VE-cadherin-based complexes had less associated Kindlin-2 and actin in Kindlin-2+/- ECs and in HUVECs treated with Kindlin-2-specifc siRNA. Also, upon stimulation of HUVECs with PAF or thrombin, Kindlin-2 along with actin dissociated from VE-cadherin-based complexes, suggesting that Kindlin-2 stabilizes adherens junctions. In direct binding studies with recombinant proteins the SPR sensograms revealed direct interaction of Kindlin-2 with β- and γ-catenin, but not with α-catenin nor the VE-cadherin cytoplasmic tail. In addition, using actin spin-down assays we demonstrated that Kindlin-2 directly interacted with actin filaments and linked them to β- or γ-catenin. Using WT and deletion mutants of Kindlin-2 we mapped the β- and γ-catenin binding site to the F1 and F3 domains in Kindlin-2, while actin binding is very dependent on the F0 domain.Taken together, these data identify a previously unappreciated function of Kindlin-2. It plays a crucial role in maintaining vascular integrity by linking adherens junctions to actin filaments. DisclosuresNo relevant conflicts of interest to declare.

Structural analysis of the KRIT1 ankyrin repeat and FERM domains reveals a conformationally stable ARD-FERM interface.

Cerebral cavernous malformations (CCM) are vascular dysplasias that usually occur in the brain and are associated with mutations in the KRIT1/CCM1, CCM2/MGC4607/OSM/Malcavernin, and PDCD10/CCM3/TFAR15 genes. Here we report the 2.9 Å crystal structure of the ankyrin repeat domain (ARD) and FERM domain of the protein product of KRIT1 (KRIT1; Krev interaction trapped 1). The crystal structure reveals that the KRIT1 ARD contains 4 ankyrin repeats. There is an unusual conformation in the ANK4 repeat that is stabilized by Trp-404, and the structure reveals a solvent exposed ankyrin groove. Domain orientations of the three copies within the asymmetric unit suggest a stable interaction between KRIT1 ARD and FERM domains, indicating a globular ARD-FERM module. This resembles the additional F0 domain found N-terminal to the FERM domain of talin. Structural analysis of KRIT1 ARD-FERM highlights surface regions of high evolutionary conservation, and suggests potential sites that could mediate interaction with binding partners. The structure therefore provides a better understanding of KRIT1 at the molecular level.

Structure of ATP synthase from Paracoccus denitrificans determined by X-ray crystallography at 4.0 Å resolution

The structure of the intact ATP synthase from the α-proteobacterium Paracoccus denitrificans, inhibited by its natural regulatory ζ-protein, has been solved by X-ray crystallography at 4.0 Å resolution. The ζ-protein is bound via its N-terminal α-helix in a catalytic interface in the F1 domain. The bacterial F1 domain is attached to the membrane domain by peripheral and central stalks. The δ-subunit component of the peripheral stalk binds to the N-terminal regions of two α-subunits. The stalk extends via two parallel long α-helices, one in each of the related b and b' subunits, down a noncatalytic interface of the F1 domain and interacts in an unspecified way with the a-subunit in the membrane domain. The a-subunit lies close to a ring of 12 c-subunits attached to the central stalk in the F1 domain, and, together, the central stalk and c-ring form the enzyme's rotor. Rotation is driven by the transmembrane proton-motive force, by a mechanism where protons pass through the interface between the a-subunit and c-ring via two half-channels in the a-subunit. These half-channels are probably located in a bundle of four α-helices in the a-subunit that are tilted at ∼30° to the plane of the membrane. Conserved polar residues in the two α-helices closest to the c-ring probably line the proton inlet path to an essential carboxyl group in the c-subunit in the proton uptake site and a proton exit path from the proton release site. The structure has provided deep insights into the workings of this extraordinary molecular machine.

Studies on Immunogenicity and Antigenicity of Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Merozoite Ligand.

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum erythrocyte binding antigens (EBA) family, which are considered as prospective candidates for malaria vaccine development. EBA proteins were identified as important targets for naturally acquired inhibitory antibodies. Natural antibody response against EBA-140 ligand was found in individuals living in malaria-endemic areas. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They both share homology of domain structure, including the binding region (Region II), which consists of two homologous F1 and F2 domains and is responsible for ligand–erythrocyte receptor interaction during merozoite invasion. It was shown that the erythrocyte receptor for EBA-140 ligand is glycophorin C-a minor human erythrocyte sialoglycoprotein. In studies on the immunogenicity of P. falciparum EBA ligands, the recombinant proteins are of great importance. In this report, we have demonstrated that the recombinant baculovirus-obtained EBA-140 Region II is immunogenic and antigenic. It can raise specific antibodies in rabbits, and it is recognized by natural antibodies present in sera of patients with malaria, and thus, it may be considered for inclusion in multicomponent blood-stage vaccines.

Structural transitions during prothrombin activation: On the importance of fragment 2

Prothrombin is activated to thrombin by the prothrombinase complex through sequential cleavage at two distinct sites. This occurs at sites of vascular injury in a highly regulated cascade of serine protease and cofactor activation, where activated platelets provide a suitable surface for protease/cofactor/substrate assembly. The precise structural and conformational changes undergone during the transition from prothrombin to thrombin have been studied for decades, and several structures of prothrombin fragments along the activation pathway have been solved. Here we present a new structure analyzed in context of other recent structures and biochemical studies. What emerges is an unexpected mechanism that involves a change in the mode of binding of the F2 domain (fragment 2) on the catalytic domain after cleavage at Arg320, and a subsequent reorientation of the linker between the F2 and catalytic domain to present the Arg271 site for cleavage.

GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM

We developed a method, named GraDeR, which substantially improves the preparation of membrane protein complexes for structure determination by single-particle cryo-electron microscopy (cryo-EM). In GraDeR, glycerol gradient centrifugation is used for the mild removal of free detergent monomers and micelles from lauryl maltose-neopentyl glycol detergent stabilized membrane complexes, resulting in monodisperse and stable complexes to which standard processes for water-soluble complexes can be applied. We demonstrate the applicability of the method on three different membrane complexes, including the mammalian FoF1 ATP synthase. For this highly dynamic and fragile rotary motor, we show that GraDeR allows visualizing the asymmetry of the F1 domain, which matches the ground state structure of the isolated domain. Therefore, the present cryo-EM structure of FoF1 ATP synthase provides direct structural evidence for Boyer's binding change mechanism in the context of the intact enzyme.

The conformational states of talin autoinhibition complex and its activation under forces

Talin is an integrin-binding protein located at focal adhesion site and serves as both an adapter and a force transmitter. Its integrin binding activity is regulated by the intramolecular autoinhibition interaction between its F3 and RS domains. Here, we used atomic force microscopy to measure the strength of talin autoinhibition complex. Our results suggest that the lifetime of talin autoinhibition complex shows weak catch bond behavior and does not change significantly at smaller forces, while it drops rapidly at larger forces (>10 pN). Moreover, besides the complex conformation revealed by crystal structure, our molecular dynamics (MD) simulations indicate the possible existence of another stable conformation. Further analysis indicates that forces may regulate the equilibrium of the two stable binding states and result in the non-exponential force dependence of the binding lifetime. Our findings reveal a negative regulation mechanism on talin activation and provide a new point of view on the function of talin in focal adhesion.

The Molecular Motor F-ATP Synthase Is Targeted by the Tumoricidal Protein HAMLET

HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET–α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s−1, no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes.

The baculovirus-expressed binding region of Plasmodium falciparum EBA-140 ligand and its glycophorin C binding specificity.

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during invasion. In this report we describe, for the first time, the glycophorin C specificity of the recombinant, baculovirus-expressed binding region (Region II) of P. falciparum EBA-140 ligand. It was found that the recombinant EBA-140 Region II binds to the endogenous and recombinant glycophorin C, but does not bind to Gerbich-type glycophorin C, neither normal nor recombinant, which lacks amino acid residues 36–63 of its polypeptide chain. Our results emphasize the crucial role of this glycophorin C region in EBA-140 ligand binding. Moreover, the EBA-140 Region II did not bind either to glycophorin D, the truncated form of glycophorin C lacking the N-glycan or to desialylated GPC. These results draw attention to the role of glycophorin C glycans in EBA-140 binding. The full identification of the EBA-140 binding site on glycophorin C molecule, consisting most likely of its glycans and peptide backbone, may help to design therapeutics or vaccines that target the erythrocyte binding merozoite ligands.

Effects of horseback riding exercise therapy on background electroencephalograms of elderly people.

[Purpose] This study investigated the effect of horseback riding exercise on the background electroencephalograms of elderly people who performed horseback riding for 8 weeks. [Subjects] Twenty elderly people were divided into the horseback riding exercise and control group (n = 10 each). [Methods] The exercise was performed for 15 minutes, 3 times per week for 8 weeks. Electroencephalograms were analyzed. Post-exercise evaluation was performed after 8 weeks. [Results] After the horseback riding exercise, the relative slower alpha power index was active in the T3 and P4 domains but suppressed in the Fp1, Fp2, F3, F4, T4, and P3 domains. Moreover, the relative faster alpha power index was active in all domains of the horseback riding exercise group but was suppressed in all domains of the control group. There was a significant difference between groups in the F3 domain. [Conclusion] The alpha power index increased significantly after horseback riding exercise, suggesting the exercise improved background electroencephalogram.

Domain analyses of Usher syndrome causing Clarin-1 and GPR98 protein models.

Usher syndrome is an autosomal recessive disorder that causes hearing loss, Retinitis Pigmentosa (RP) and vestibular dysfunction. It is clinically and genetically heterogeneous disorder which is clinically divided into three types i.e. type I, type II and type III. To date, there are about twelve loci and ten identified genes which are associated with Usher syndrome. A mutation in any of these genes e.g. CDH23, CLRN1, GPR98, MYO7A, PCDH15, USH1C, USH1G, USH2A and DFNB31 can result in Usher syndrome or non-syndromic deafness. These genes provide instructions for making proteins that play important roles in normal hearing, balance and vision. Studies have shown that protein structures of only seven genes have been determined experimentally and there are still three genes whose structures are unavailable. These genes are Clarin-1, GPR98 and Usherin. In the absence of an experimentally determined structure, homology modeling and threading often provide a useful 3D model of a protein. Therefore in the current study Clarin-1 and GPR98 proteins have been analyzed for signal peptide, domains and motifs. Clarin-1 protein was found to be without any signal peptide and consists of prokar lipoprotein domain. Clarin-1 is classified within claudin 2 super family and consists of twelve motifs. Whereas, GPR98 has a 29 amino acids long signal peptide and classified within GPCR family 2 having Concanavalin A-like lectin/glucanase superfamily. It was found to be consists of GPS and G protein receptor F2 domains and twenty nine motifs. Their 3D structures have been predicted using I-TASSER server. The model of Clarin-1 showed only α-helix but no beta sheets while model of GPR98 showed both α-helix and β sheets. The predicted structures were then evaluated and validated by MolProbity and Ramachandran plot. The evaluation of the predicted structures showed 78.9% residues of Clarin-1 and 78.9% residues of GPR98 within favored regions. The findings of present study has resulted in the three dimensional structure prediction and conserved domain analysis which will be quite beneficial in better understanding of molecular components, protein-protein interaction, clinical heterogeneity and pathophysiology of Usher syndrome.

The C Ring of the F1Fo ATP Synthase Forms the Mitochondrial Permeability Transition Pore: A Critical Appraisal.

The C Ring of the F1Fo ATP Synthase Forms the Mitochondrial Permeability Transition Pore: A Critical Appraisal.

Cross-Protective Immunity to Leishmania amazonensis is Mediated by CD4+ and CD8+ Epitopes of Leishmania donovani Nucleoside Hydrolase Terminal Domains.

The nucleoside hydrolase (NH) of Leishmania donovani (NH36) is a phylogenetic marker of high homology among Leishmania parasites. In mice and dog vaccination, NH36 induces a CD4+ T cell-driven protective response against Leishmania chagasi infection directed against its C-terminal domain (F3). The C-terminal and N-terminal domain vaccines also decreased the footpad lesion caused by Leishmania amazonensis. We studied the basis of the crossed immune response using recombinant generated peptides covering the whole NH36 sequence and saponin for mice prophylaxis against L. amazonensis. The F1 (amino acids 1–103) and F3 peptide (amino acids 199–314) vaccines enhanced the IgG and IgG2a anti-NH36 antibodies to similar levels. The F3 vaccine induced the strongest DTH response, the highest proportions of NH36-specific CD4+ and CD8+ T cells after challenge and the highest expression of IFN-γ and TNF-α. The F1 vaccine, on the other hand, induced a weaker but significant DTH response and a mild enhancement of IFN-γ and TNF-α levels. The in vivo depletion with anti-CD4 or CD8 monoclonal antibodies disclosed that cross-protection against L. amazonensis infection was mediated by a CD4+ T cell response directed against the C-terminal domain (75% of reduction of the size of footpad lesion) followed by a CD8+ T cell response against the N-terminal domain of NH36 (57% of reduction of footpad lesions). Both vaccines were capable of inducing long-term cross-immunity. The amino acid sequence of NH36 showed 93% identity to the sequence of the NH A34480 of L. amazonensis, which also showed the presence of completely conserved predicted epitopes for CD4+ and CD8+ T cells in F1 domain, and of CD4+ epitopes differing by a single amino acid, in F1 and F3 domains. The identification of the C-terminal and N-terminal domains as the targets of the immune response to NH36 in the model of L. amazonensis infection represents a basis for the rationale development of a bivalent vaccine against leishmaniasis.

Fibronectin matrix assembly is essential for cell condensation during chondrogenesis.

Mesenchymal cell condensation is the initiating event in endochondral bone formation. Cell condensation is followed by differentiation into chondrocytes, which is accompanied by induction of chondrogenic gene expression. Gene mutations involved in chondrogenesis cause chondrodysplasias and other skeletal defects. Using mesenchymal stem cells (MSCs) in an in vitro chondrogenesis assay, we found that knockdown of the diastrophic dysplasia (DTD) sulfate transporter (DTDST, also known as SLC26A2), which is required for normal cartilage development, blocked cell condensation and caused a significant reduction in fibronectin matrix. Knockdown of fibronectin with small interfering RNAs (siRNAs) also blocked condensation. Fibrillar fibronectin matrix was detected prior to cell condensation, and its levels increased during and after condensation. Inhibition of fibronectin matrix assembly by use of the functional upstream domain (FUD) of adhesin F1 from Streptococcus pyogenes prevented cell condensation by MSCs and also by the chondrogenic cell line ATDC5. Our data show that cell condensation and induction of chondrogenesis depend on fibronectin matrix assembly and DTDST, and indicate that this transporter is required earlier in chondrogenesis than previously appreciated. They also raise the possibility that certain of the skeletal defects in DTD patients might derive from the link between DTDST, fibronectin matrix and condensation.

Human erythrocyte glycophorin C as the receptor for EBA-140 Plasmodium falciparum merozoite ligand

Erythrocyte invasion by the blood-stage Plasmodium falciparum parasites is a multistep process involving specific interactions between parasites and red blood cells. Several proteins are involved in this process, including EBL ligands. The structure of the EBA-140 ligand, a member of the EBL protein family, provides a full description of its molecular interactions with the erythrocyte receptor. The crystal structure of the EBA-140 Region II in a complex with sialolactose revealed that the binding region is monomeric. Two glycan binding pockets, one in each F1 or F2 domain, were identified. Stark differences in the receptor binding for the F1 and F2 domains suggests that each domain performs a distinct function. Although both domains are required for effective glycan binding, it seems that the interaction may be mediated solely by the F1 domain. The structure of the binding region and the interaction with glycan are unique to the EBA-140 ligand and not shared by other EBL ligands. The EBA-140 ligand binds specifically to human erythrocytes through the membrane sialoglycoprotein glycophorin C. The receptor site for the EBA-140 ligand was suggested to be a cluster of N-and O-linked sialylated glycans on the GPC molecule, whose conformation is dependent on the polypeptide chain region composed of amino acid residues 36-63. Precise definition of the binding site for the EBA-140 ligand on glycophorin C may be important with respect to human erythrocyte invasion inhibition strategies based on a receptor.

Conformational Changes in Talin on Binding to Anionic Phospholipid Membranes Facilitate Signaling by Integrin Transmembrane Helices

Integrins are heterodimeric (αβ) cell surface receptors that are activated to a high affinity state by the formation of a complex involving the α/β integrin transmembrane helix dimer, the head domain of talin (a cytoplasmic protein that links integrins to actin), and the membrane. The talin head domain contains four sub-domains (F0, F1, F2 and F3) with a long cationic loop inserted in the F1 domain. Here, we model the binding and interactions of the complete talin head domain with a phospholipid bilayer, using multiscale molecular dynamics simulations. The role of the inserted F1 loop, which is missing from the crystal structure of the talin head, PDB:3IVF, is explored. The results show that the talin head domain binds to the membrane predominantly via cationic regions on the F2 and F3 subdomains and the F1 loop. Upon binding, the intact talin head adopts a novel V-shaped conformation which optimizes its interactions with the membrane. Simulations of the complex of talin with the integrin α/β TM helix dimer in a membrane, show how this complex promotes a rearrangement, and eventual dissociation of, the integrin α and β transmembrane helices. A model for the talin-mediated integrin activation is proposed which describes how the mutual interplay of interactions between transmembrane helices, the cytoplasmic talin protein, and the lipid bilayer promotes integrin inside-out activation.

Kindlin-1 Mutant Zebrafish as an In Vivo Model System to Study Adhesion Mechanisms in the Epidermis

From a forward genetic screen for epidermal defects in zebrafish, we identified a loss-of-function mutation in Kindlin-1, an essential regulator of integrin function. The mutation generates a premature stop codon, deleting the integrin-binding site. The mutant zebrafish develops cell-matrix and cell-cell adhesion defects in the basal epidermis leading to progressive fin rupturing, and was therefore designated rupturing-of-fins (rof). Similar defects were observed in the epidermis of Kindler syndrome patients, carrying a loss-of-function mutation in kindlin-1. Mutational analysis and rescue experiments in zebrafish revealed that residues K610, W612, and I647 in the F3 domain are essential for Kindlin-1 function in vivo, and that Kindlin-2 can functionally compensate for the loss of Kindlin-1. The fin phenotype of rof/kindlin-1 mutants resembles that of badfin mutants, carrying a mutation in integrin α3. We show here that this mutation impairs the biosynthesis of integrin α3β1 and causes cell-matrix and cell-cell defects in vivo. Whereas both Integrin-linked kinase (Ilk) and Kindlin-1 cooperate with Integrin α3β1 to resist trauma-induced epidermal defects, Kindlin-1 and Ilk, surprisingly, do not act synergistically but in parallel. Thus, the rof/kindlin-1 mutant zebrafish provides a unique model system to study epidermal adhesion mechanisms in vivo.

Subthreshold traits of the broad autistic spectrum are distributed across different subgroups in parents, but not siblings, of probands with autism

Autism is a categorical developmental disorder characterized by impairment in socialization, communication, and by restricted and circumscribed interests. Several authors have described the presence of subthreshold autistic traits in the general population, pervasive developmental disorders representing the extreme end of their distribution. In this study, we explored the presence of autistic traits in siblings and parents of a proband with autism, and in siblings and parents of a normally developing child, using the previously validated self-report French Autism Quotient, an adaptation of the AQ developed by S. Baron-Cohen. Scores were distributed between two main factors, F1 corresponding to socialization and communication, F2 to imagination and rigidity. Here, we show that both parents and siblings of a child with autism have more symptomatic scores in the domains of communication and socialization. In addition, we show that in these families the parents, but not the siblings, are distributed across different subcategories, according to their scores for the F1 and F2 domains. We hypothesize that these different subgroups may correspond to different underlying genetic mechanisms.