Marginal band microtubules are acetylated by αTAT1.

The discoid shape of resting platelets is maintained by a peripheral, circular bundle of microtubules called marginal band. Marginal band microtubules are acetylated on lysine 40 of the alpha-tubulin subunits. We have previously shown that the deacetylase HDAC6 is responsible for tubulin deacetylation in platelets and that the hyperacetylated state of the microtubules in HDAC6KO platelets correlates with faster activation/spreading kinetics, pointing to a regulatory role of this modification. So far, the question about the reverse enzyme, responsible for tubulin acetylation in platelets, has remained unanswered. Several enzymes have been described as having tubulin acetylation activity. Here we identify αTAT1 as the enzyme responsible for the acetylation of marginal band microtubules. We show that αTAT1 deficiency has only minor consequences for platelet production and function. A residual tubulin acetylation level in αTAT1 deficient platelet lysates suggests the presence of an additional tubulin-acetylating enzyme that is unable to acetylate marginal band microtubules.

ICAP-1 monoubiquitylation coordinates matrix density and rigidity sensing for cell migration through ROCK2-MRCKα balance.

Cell migration is a complex process requiring density and rigidity sensing of the microenvironment to adapt cell migratory speed through focal adhesion and actin cytoskeleton regulation. ICAP-1 (also known as ITGB1BP1), a ß1 integrin partner, is essential for ensuring integrin activation cycle and focal adhesion formation. We show that ICAP-1 is monoubiquitylated by Smurf1, preventing ICAP-1 binding to ß1 integrin. The non-ubiquitylatable form of ICAP-1 modifies ß1 integrin focal adhesion organization and interferes with fibronectin density sensing. ICAP-1 is also required for adapting cell migration in response to substrate stiffness in a ß1-integrin-independent manner. ICAP-1 monoubiquitylation regulates rigidity sensing by increasing MRCKα (also known as CDC42BPA)-dependent cell contractility through myosin phosphorylation independently of substrate rigidity. We provide evidence that ICAP-1 monoubiquitylation helps in switching from ROCK2-mediated to MRCKα-mediated cell contractility. ICAP-1 monoubiquitylation serves as a molecular switch to coordinate extracellular matrix density and rigidity sensing thus acting as a crucial modulator of cell migration and mechanosensing.

ß1 integrin-dependent Rac/group I PAK signaling mediates YAP activation of Yes-associated protein 1 (YAP1) via NF2/merlin.

Cell adhesion to the extracellular matrix or to surrounding cells plays a key role in cell proliferation and differentiation and is critical for proper tissue homeostasis. An important pathway in adhesion-dependent cell proliferation is the Hippo signaling cascade, which is coregulated by the transcription factors Yes-associated protein 1 (YAP1) and transcriptional coactivator with PDZ-binding motif (TAZ). However, how cells integrate extracellular information at the molecular level to regulate YAP1's nuclear localization is still puzzling. Herein, we investigated the role of ß1 integrins in regulating this process. We found that ß1 integrin-dependent cell adhesion is critical for supporting cell proliferation in mesenchymal cells both in vivo and in vitro ß1 integrin-dependent cell adhesion relied on the relocation of YAP1 to the nucleus after the down-regulation of its phosphorylated state mediated by large tumor suppressor gene 1 and 2 (LATS1/2). We also found that this phenotype relies on ß1 integrin-dependent local activation of the small GTPase RAC1 at the plasma membrane to control the activity of P21 (RAC1)-activated kinase (PAK) of group 1. We further report that the regulatory protein merlin (neurofibromin 2, NF2) interacts with both YAP1 and LATS1/2 via its C-terminal moiety and FERM domain, respectively. PAK1-mediated merlin phosphorylation on Ser-518 reduced merlin's interactions with both LATS1/2 and YAP1, resulting in YAP1 dephosphorylation and nuclear shuttling. Our results highlight RAC/PAK1 as major players in YAP1 regulation triggered by cell adhesion.

Calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII)-mediated intramolecular opening of integrin cytoplasmic domain-associated protein-1 (ICAP-1α) negatively regulates ß1 integrins.

Focal adhesion turnover during cell migration is an integrated cyclic process requiring tight regulation of integrin function. Interaction of integrin with its ligand depends on its activation state, which is regulated by the direct recruitment of proteins onto the ß integrin chain cytoplasmic domain. We previously reported that ICAP-1α, a specific cytoplasmic partner of ß1A integrins, limits both talin and kindlin interaction with ß1 integrin, thereby restraining focal adhesion assembly. Here we provide evidence that the calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII) is an important regulator of ICAP-1α for controlling focal adhesion dynamics. CaMKII directly phosphorylates ICAP-1α and disrupts an intramolecular interaction between the N- and the C-terminal domains of ICAP-1α, unmasking the PTB domain, thereby permitting ICAP-1α binding onto the ß1 integrin tail. ICAP-1α direct interaction with the ß1 integrin tail and the modulation of ß1 integrin affinity state are required for down-regulating focal adhesion assembly. Our results point to a molecular mechanism for the phosphorylation-dependent control of ICAP-1α function by CaMKII, allowing the dynamic control of ß1 integrin activation and cell adhesion.

The fate of mitochondria during platelet activation.

Blood platelets undergo several successive motor-driven reorganizations of the cytoskeleton when they are recruited to an injured part of a vessel. These reorganizations take place during the platelet activation phase, the spreading process on the injured vessel or between fibrin fibers of the forming clot, and during clot retraction. All these steps require a lot of energy, especially the retraction of the clot when platelets develop strong forces similar to those of muscle cells. Platelets can produce energy through glycolysis and mitochondrial respiration. However, although resting platelets have only 5 to 8 individual mitochondria, they produce adenosine triphosphate predominantly via oxidative phosphorylation. Activated, spread platelets show an increase in size compared with resting platelets, and the question arises as to where the few mitochondria are located in these larger platelets. Using expansion microscopy, we show that the number of mitochondria per platelet is increased in spread platelets. Live imaging and focused ion beam-scanning electron microscopy suggest that a mitochondrial fission event takes place during platelet activation. Fission is Drp1 dependent because Drp1-deficient platelets have fused mitochondria. In nucleated cells, mitochondrial fission is associated with a shift to a glycolytic phenotype, and using clot retraction assays, we show that platelets have a more glycolytic energy production during clot retraction and that Drp1-deficient platelets show a defect in clot retraction.

The Role of LIM Kinases during Development: A Lens to Get a Glimpse of Their Implication in Pathologies.

The organization of cell populations within animal tissues is essential for the morphogenesis of organs during development. Cells recognize three-dimensional positions with respect to the whole organism and regulate their cell shape, motility, migration, polarization, growth, differentiation, gene expression and cell death according to extracellular signals. Remodeling of the actin filaments is essential to achieve these cell morphological changes. Cofilin is an important binding protein for these filaments; it increases their elasticity in terms of flexion and torsion and also severs them. The activity of cofilin is spatiotemporally inhibited via phosphorylation by the LIM domain kinases 1 and 2 (LIMK1 and LIMK2). Phylogenetic analysis indicates that the phospho-regulation of cofilin has evolved as a mechanism controlling the reorganization of the actin cytoskeleton during complex multicellular processes, such as those that occur during embryogenesis. In this context, the main objective of this review is to provide an update of the respective role of each of the LIM kinases during embryonic development.

Asymmetrical Forces Dictate the Distribution and Morphology of Platelets in Blood Clots.

Primary hemostasis consists in the activation of platelets, which spread on the exposed extracellular matrix at the injured vessel surface. Secondary hemostasis, the coagulation cascade, generates a fibrin clot in which activated platelets and other blood cells get trapped. Active platelet-dependent clot retraction reduces the clot volume by extruding the serum. Thus, the clot architecture changes with time of contraction, which may have an important impact on the healing process and the dissolution of the clot, but the precise physiological role of clot retraction is still not completely understood. Since platelets are the only actors to develop force for the retraction of the clot, their distribution within the clot should influence the final clot architecture. We analyzed platelet distributions in intracoronary thrombi and observed that platelets and fibrin co-accumulate in the periphery of retracting clots in vivo. A computational mechanical model suggests that asymmetric forces are responsible for a different contractile behavior of platelets in the periphery versus the clot center, which in turn leads to an uneven distribution of platelets and fibrin fibers within the clot. We developed an in vitro clot retraction assay that reproduces the in vivo observations and follows the prediction of the computational model. Our findings suggest a new active role of platelet contraction in forming a tight fibrin- and platelet-rich boundary layer on the free surface of fibrin clots.

Two Antagonistic Microtubule Targeting Drugs Act Synergistically to Kill Cancer Cells.

Paclitaxel is a microtubule stabilizing agent and a successful drug for cancer chemotherapy inducing, however, adverse effects. To reduce the effective dose of paclitaxel, we searched for pharmaceutics which could potentiate its therapeutic effect. We screened a chemical library and selected Carba1, a carbazole, which exerts synergistic cytotoxic effects on tumor cells grown in vitro, when co-administrated with a low dose of paclitaxel. Carba1 targets the colchicine binding-site of tubulin and is a microtubule-destabilizing agent. Catastrophe induction by Carba1 promotes paclitaxel binding to microtubule ends, providing a mechanistic explanation of the observed synergy. The synergistic effect of Carba1 with paclitaxel on tumor cell viability was also observed in vivo in xenografted mice. Thus, a new mechanism favoring paclitaxel binding to dynamic microtubules can be transposed to in vivo mouse cancer treatments, paving the way for new therapeutic strategies combining low doses of microtubule targeting agents with opposite mechanisms of action.

[On the road to deciphering the tubulin code: focus on acetylation and detyrosination]. / Déchiffrage du code tubuline - Le voile se lève sur le rôle de l'acétylation et de la détyrosination.

Microtubules are cytoskeletal fibers formed by the assembly of α- and ß-tubulin heterodimers. They contribute to cell morphology, mobility and polarity, as well as to cellular transport processes and cell division. The microtubular network constantly adapts to cellular needs and may be composed of very dynamic or more stable microtubules. To regulate their diverse functions in a spatio-temporal manner, microtubules are subjected to numerous reversible post-translational modifications, which generate the "tubulin code". This review focuses on two modifications characteristic of stable microtubules - acetylation and detyrosination of α-tubulin - and their deregulation in certain pathologies.

ß1 integrins mediate the BMP2 dependent transcriptional control of osteoblast differentiation and osteogenesis.

Osteoblast differentiation is a highly regulated process that requires coordinated information from both soluble factors and the extracellular matrix. Among these extracellular stimuli, chemical and physical properties of the matrix are sensed through cell surface receptors such as integrins and transmitted into the nucleus to drive specific gene expression. Here, we showed that the conditional deletion of ß1 integrins in the osteo-precursor population severely impacts bone formation and homeostasis both in vivo and in vitro. Mutant mice displayed a severe bone deficit characterized by bone fragility and reduced bone mass. We showed that ß1 integrins are required for proper BMP2 dependent signaling at the pre-osteoblastic stage, by positively modulating Smad1/5-dependent transcriptional activity at the nuclear level. The lack of ß1 integrins results in a transcription modulation that relies on a cooperative defect with other transcription factors rather than a plain blunted BMP2 response. Our results point to a nuclear modulation of Smad1/5 transcriptional activity by ß1 integrins, allowing a tight control of osteoblast differentiation.

Molecular characterization of four ADAMTS13 mutations responsible for congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome).

ADAMTS13 mutations S203P, R268P, R507Q and A596V were previously identified in French patients with hereditary thrombotic thrombocytopenic purpura (TTP) (Upshaw-Schulman syndrome). Mutated recombinant (r) ADAMTS13 were transiently expressed in COS-7 cells and characterized in comparison with wild-type (WT) rADAMTS13. ADAMTS13 antigen was qualitatively and quantitatively estimated by electrophoretic analysis and ELISA. Enzymatic activity was qualitatively and quantitatively estimated using GST-VWF73, FRETS-VWF73 fragments and full-length rVWF-WT as substrates. The four mutants and rADAMTS13-WT were present within the cells. Secretion level of rADAMTS13-WT reached 1,200 ng/ml. The four mutations strongly altered the secretion and biological activity of rADAMTS13. The percentage secretion was 21, 38 and 17% for rADAMTS13-S203P, -R268P and -A596V compared with rADAMTS13-WT. rADAMTS13-R507Q concentration was under the detection limit of the assay. In the four cases, no enzymatic activity was detected. After concentration, we confirmed that mutations S203P and R268P totally abolished the proteolytic activity of ADAMTS13. Due to the very low protease concentration, activity of rADAMTS13-R507Q was below the threshold of the assays. rADAMTS13-A596V had no proteolytic activity towards the full-length rVWF-WT whereas it exhibited a decreased specific activity of about 30% of that of rADAMTS13-WT towards FRETS-VWF73 fragment. Binding study of mutated rADAMTS13-S203P, -R268P and -A596V showed that the three mutations strongly decreased the interaction of ADAMTS13 with VWF. In conclusion, the four mutations, which led to a secretion defect, a loss of enzymatic activity and a decreased binding to the substrate, are responsible for the hereditary TTP in patients.

Impaired dimerization of von Willebrand factor subunit due to mutation A2801D in the CK domain results in a recessive type 2A subtype IID von Willebrand disease.

The CK domain of von Willebrand factor (VWF) is involved in the dimerization of the protein. We identified the homozygous substitution A2801D of the CK domain in two siblings. Patients had low levels of VWF in plasma, abnormal ristocetin-induced binding to platelets and abnormal multimeric pattern with a lack of high molecular weight (HMW) forms and the presence of intervening bands between normal multimers. Accordingly, they were classified in type 2A, subtype IID, von Willebrand disease (VWD). Both asymptomatic parents carried the mutation at the heterozygous state. Their plasmaVWF exhibited the full range of multimers found in normal plasma. When analyzed by high resolution gel electrophoresis, very faint bands corresponding to the position of intervening bands of the propositus can be observed. The mutated recombinant (r)VWF-D2801, the hybrid rVWF-A/D2801 and the mutated C-terminal VWF fragment rSPII-D2801 were expressed in COS-7 cells. rVWF-D2801 showed an abnormal multimeric distribution similar to that of the propositus'VWF with intervening bands and a lack of HMW species. rVWF-A/D2801 exhibited the full range of multimers and the aberrant sized forms observed both in propositus'VWF and in rVWF-D2801. rSPII-WT assembled correctly into a dimer of 220 kDa. rSPII-D2801 appeared as a mixture of monomeric and dimeric forms which may be related to the abnormal multimeric pattern of the propositus and both mutated rVWF. We concluded that mutation A2801D disturbs the folding of the CK domain, which may result in a mixture of monomers and dimers of VWF. Multimers containing either an odd or even number of mature subunits are produced, and the presence of monomers appears to limit the degree of multimerization. In the heterozygousVWF, the presence of normal dimers improves the multimerization process. In conclusion, the mutation A2801D appears to be responsible for a recessive type 2A, subtype IID, VWD.

Thrombotic thrombocytopenic purpura associated with von Willebrand factor-cleaving protease (ADAMTS13) deficiency in children.

The physiopathology of thrombotic thrombocytopenic purpura (TTP) has been clarified since 1998, when it was shown that TTP in adults was most often associated with an acquired deficiency of von Willebrand factor-cleaving protease (ADAMTS13) due to autoantibodies, whereas TTP in children was most often associated with a hereditary autosomal recessive severe deficiency of ADAMTS13. The hereditary form of TPP (Upshaw-Schulman syndrome) is a very rare but life-threatening disease if adequate treatment (plasma therapy) is not administered. First manifestations occur before age 10 in two thirds of cases and as soon as birth in most cases. The subsequent course is characterized by recurrent hemolytic and thrombocytopenic crises, with intervals between relapses from every 3 to 4 weeks in two thirds of cases to several months or years in one third of cases. TTP crises are associated with cerebral vascular accidents in at least 30% of patients, with a risk of neurologic sequelae in approximately 20% of patients. Renal involvement includes frequent acute renal failure due to hemoglobinuria and/or thrombotic microangiopathy during hemolytic crisis and progressive renal deterioration in approximately 50% of cases, leading to chronic or end-stage renal failure in approximately 20% of patients. The clinical phenotype may vary from the typical congenital recurrent TTP. Some mild forms are limited to a fluctuating thrombocytopenia and may be misdiagnosed as idiopathic thrombocytopenic purpura. Phenotypic variability may be observed within a single family, which suggests a role of modifier genes. Fresh frozen plasma (FFP) replaces active ADAMTS13. Ten milliliters per kilogram FFP every 2 to 4 weeks suffices to maintain remission. FFP infusions are best used preventively, given that rescue infusions may not prevent central nervous system and renal involvement. It is hoped that plasmatic or recombinant purified ADAMTS13 will be available in the years to come.

Two clusters of charged residues located in the electropositive face of the von Willebrand factor A1 domain are essential for heparin binding.

The VWF A1 domain seems to possess two heparin binding regions (residues 565-587 and 633-648) displaying positively charged amino acids, but the overall polyanion-A1 domain interaction scheme remains essentially elusive. To probe this molecular reaction as well as the role of electrostatic forces in VWF-heparin interaction, we performed mutagenesis and molecular modeling experiments. Fifteen mutated rVWFs were expressed [R571A, K572A, R573A, K585A, R571A/K572A/R573A, R578A/R579A, R578A/R579A/K585A, R571A/K572A/R573A/R578A/R579A/K585A (6A), K642G, K643G, K644G, K645G, K642G/K645G, K643G/K644G, and K642G/K643G/K644G/K645G (4G)]. Experimental results indicate that the multimeric structure of the mutants was similar to that of wild-type (WT) rVWF and that all rVWFs displayed normal binding to four conformation-dependent mAbs directed against the A1 domain. Three variants displayed significant reductions in the level of heparin binding. The 6A variant showed 39.2 +/- 1.3% of the WT rVWF level (p < 0.005), while mutants K643G/K644G and 4G showed 63.6 +/- 3.2 and 53.3 +/- 5% of the WT rVWF level, respectively (p < 0.005). Computational investigations showed that one face of the A1 domain is strongly electropositive, indicating that electrostatic forces should be essential in steering heparin onto its binding site. In agreement with our experimental data, the most striking alterations of the electrostatic potential contours were seen for mutants 4G, K643G/K644G, and 6A. Our data suggest that two clusters, one at positions 571-573, 578, 579, and 585 and the other at positions 642-645, act in concert for the recognition of heparin, forming a single extended binding surface across the electropositive face of the VWF A1 domain. A structural model of the VWF A1 domain-heparin complex is proposed, taking into account both experimental and computer modeling data.

An experimental model to study the in vivo survival of von Willebrand factor. Basic aspects and application to the R1205H mutation.

To explore the molecular basis of von Willebrand factor (VWF) clearance, an experimental model employing VWF-deficient mice was developed. Biodistribution was examined by the injection of radiolabeled VWF, which was primarily directed to the liver with minor amounts in other organs. Disappearance of VWF from plasma was characterized by a rapid initial phase (t((1/2))alpha = 13 min) and a slow secondary phase (t((1/2))beta = 3 h), with a mean residence time (MRT) of 2.8 h. A similar clearance was observed for VWF consisting of only high or low molecular weight multimers, indicating that, in our experimental model, clearance is independent of multimeric distribution. This allowed us to compare the survival of full-length VWF to truncated variants. Deletion of both the amino-terminal D'-D3 and carboxyl-terminal D4-CK domains resulted in a fragment with a similar clearance to wild-type VWF. Deletion of only the D'-D3 region was associated with an almost 2-fold lower recovery and increased clearance (MRT = 1.6 h), whereas deletion of only the D4-CK region resulted in a significantly reduced clearance (MRT = 4.5 h, p < 0.02). These results point to a role of the D'-D3 region in preventing clearance of VWF. Furthermore, replacement of D3 domain residue Arg-1205 by His resulted in a markedly increased clearance (MRT = 0.3 h; p = 0.004). Therefore, this mutation seems to abrogate the protective effect of the D'-D3 region. In vitro analysis of this mutant also revealed a 2-fold reduced affinity for VWF propeptide at low pH, showing that mutation of Arg-1205 results not only in an increased clearance rate but is also associated with an impaired pH-dependent interaction with VWF propeptide.

A new mutation, S1285F, within the A1 loop of von Willebrand factor induces a conformational change in A1 loop with abnormal binding to platelet GPIb and botrocetin causing type 2M von Willebrand disease.

We report the identification of a new mutation in exon 28 of the von Willebrand factor (VWF) gene in two related patients with type 2M von Willebrand disease (VWD). The molecular abnormality changes the Ser 1285 to Phe within the A1 loop of VWF. The S1285F mutation was reproduced by site-directed mutagenesis on the full-length VWF cDNA. The mutated recombinant VWF (rVWF), F1285rVWF, and the hybrid, S/F1285rVWF, were expressed in COS-7 cells. F1285rVWF exhibited a slight decrease of high-molecular-weight multimers and markedly reduced ristocetin- or botrocetin-induced binding of VWF to platelets in association with a decreased binding to botrocetin. The hybrid S/F1285rVWF showed a normal multimeric profile and bound to platelets in a similar way to the patients' plasma VWF, in the presence of ristocetin or botrocetin. Thus, the new S1285F mutation within the A1 loop was responsible for the type 2M VWD observed in these patients, and was involved in the binding of VWF to botrocetin and to platelet glycoprotein Ib (GPIb). Three anti-VWF monoclonal antibodies, with conformational epitopes within the A1 loop but distinct GPIb binding inhibitory properties, showed a different interaction with F1285rVWF. These results indicate that the S1285F substitution alters the folding of the A1 loop and prevents the correct exposure of the VWF binding sites to botrocetin and GPIb.