Caspase-2 is required for skeletal muscle differentiation and myogenesis

Caspase activation plays a crucial role in skeletal muscle differentiation. We previously found that caspase-2 activity increases during skeletal muscle cell differentiation; however, its direct effect on differentiation has not been fully investigated. Here, we found that caspase-2 activity transiently increased more than two-fold within 24 h following induction of differentiation. Both pharmacological inhibition and shRNA-mediated knockdown of caspase-2 suppressed myogenic differentiation and dramatically impaired myotube formation. Furthermore, shRNA-mediated knockdown prevented induction of p21 and altered cell cycle profiles. Interestingly, caspase-3 activity was also dramatically reduced following caspase-2 inhibition or ablation. Moreover, caspase-2 and p21 were localized to the nucleus during early differentiation. Given the nuclear localization of caspase-2 and p21, as well as the impairment in p21 induction in caspase-2 knockdown cells, we propose that the role of caspase-2 is to regulate p21 induction at the onset of differentiation, which may regulate the myogenic program. Collectively, these results highlight a novel function for caspase-2 in myocyte differentiation and myogenesis.     

Kindlin-3 is essential for integrin activation and platelet aggregation

Integrin-mediated platelet adhesion and aggregation are essential for sealing injured blood vessels and preventing blood loss, and excessive platelet aggregation can initiate arterial thrombosis, causing heart attacks and stroke. To ensure that platelets aggregate only at injury sites, integrins on circulating platelets exist in a low-affinity state and shift to a high-affinity state (in a process known as integrin activation or priming) after contacting a wounded vessel. The shift is mediated through binding of the cytoskeletal protein Talin to the beta subunit cytoplasmic tail. Here we show that platelets lacking the adhesion plaque protein Kindlin-3 cannot activate integrins despite normal Talin expression. As a direct consequence, Kindlin-3 deficiency results in severe bleeding and resistance to arterial thrombosis. Mechanistically, Kindlin-3 can directly bind to regions of beta-integrin tails distinct from those of Talin and trigger integrin activation. We have therefore identified Kindlin-3 as a novel and essential element for platelet integrin activation in hemostasis and thrombosis.     

Kindlin-2 in Sertoli cells is essential for testis development and male fertility in mice

Kindlin-2 is known to play important roles in the development of mesoderm-derived tissues including myocardium, smooth muscle, cartilage and blood vessels. However, nothing is known for the role of Kindlin-2 in mesoderm-derived reproductive organs. Here, we report that loss of Kindlin-2 in Sertoli cells caused severe testis hypoplasia, abnormal germ cell development and complete infertility in male mice. Functionally, loss of Kindlin-2 inhibits proliferation, increases apoptosis, impairs phagocytosis in Sertoli cells and destroyed the integration of blood-testis barrier structure in testes. Mechanistically, Kindlin-2 interacts with LATS1 and YAP, the key components of Hippo pathway. Kindlin-2 impedes LATS1 interaction with YAP, and depletion of Kindlin-2 enhances LATS1 interaction with YAP, increases YAP phosphorylation and decreases its nuclear translocation. For clinical relevance, lower Kindlin-2 expression and decreased nucleus localization of YAP was found in SCOS patients. Collectively, we demonstrated that Kindlin-2 in Sertoli cells is essential for sperm development and male reproduction.Subject terms: Apoptosis, Male factor infertility     

R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis

We have carried out a small pool expression screen for modulators of the Wnt/beta-catenin pathway and identified Xenopus R-spondin2 (Rspo2) as a secreted activator of this cascade. Rspo2 is coexpressed with and positively regulated by Wnt signals and synergizes with Wnts to activate beta-catenin. Analyses of functional interaction with components of the Wnt/beta-catenin pathway suggest that Rspo2 functions extracellularly at the level of receptor ligand interaction. In addition to activating the Wnt/beta-catenin pathway, Rspo2 overexpression blocks Activin, Nodal, and BMP4 signaling in Xenopus, raising the possibility that it may negatively regulate the TGF-beta pathway. Antisense Morpholino experiments in Xenopus embryos and RNAi experiments in HeLa cells reveal that Rspo2 is required for Wnt/beta-catenin signaling. In Xenopus embryos depleted of Rspo2, the muscle markers myoD and myf5 fail to be activated and later muscle development is impaired. Thus, Rspo2 functions in a positive feedback loop to stimulate the Wnt/beta-catenin cascade.     

Centrobin-tubulin interaction is required for centriole elongation and stability

Centrobin is a daughter centriole protein that is essential for centrosome duplication. However, the molecular mechanism by which centrobin functions during centriole duplication remains undefined. In this study, we show that centrobin interacts with tubulin directly, and centrobin-tubulin interaction is pivotal for the function of centrobin during centriole duplication. We found that centrobin is recruited to the centriole biogenesis site via its interaction with tubulins during the early stage of centriole biogenesis, and its recruitment is dependent on hSAS-6 but not centrosomal P4.1-associated protein (CPAP) and CP110. The function of centrobin is also required for the elongation of centrioles, which is likely mediated by its interaction with tubulin. Furthermore, disruption of centrobin-tubulin interaction led to destabilization of existing centrioles and the preformed procentriole-like structures induced by CPAP expression, indicating that centrobin-tubulin interaction is critical for the stability of centrioles. Together, our study demonstrates that centrobin facilitates the elongation and stability of centrioles via its interaction with tubulins.     

Tomato BRASSINOSTEROID INSENSITIVE1 is required for systemin-induced root elongation in Solanum pimpinellifolium but is not essential for wound signaling

The tomato Leu-rich repeat receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) has been implicated in both peptide (systemin) and steroid (brassinosteroid [BR]) hormone perception. In an attempt to dissect these signaling pathways, we show that transgenic expression of BRI1 can restore the dwarf phenotype of the tomato curl3 (cu3) mutation. Confirmation that BRI1 is involved in BR signaling is highlighted by the lack of BR binding to microsomal fractions made from cu3 mutants and the restoration of BR responsiveness following transformation with BRI1. In addition, wound and systemin responses in the cu3 mutants are functional, as assayed by proteinase inhibitor gene induction and rapid alkalinization of culture medium. However, we observed BRI1-dependent root elongation in response to systemin in Solanum pimpinellifolium. In addition, ethylene perception is required for normal systemin responses in roots. These data taken together suggest that cu3 is not defective in systemin-induced wound signaling and that systemin perception can occur via a non-BRI1 mechanism.     

Kindlin-3-mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption

The blood cell-specific kindlin-3 protein is required to activate leukocyte and platelet integrins. In line with this function, mutations in the KINDLIN-3 gene in man cause immunodeficiency and severe bleeding. Some patients also suffer from osteopetrosis, but the underlying mechanism leading to abnormal bone turnover is unknown. Here we show that kindlin-3-deficient mice develop severe osteopetrosis because of profound adhesion and spreading defects in bone-resorbing osteoclasts. Mechanistically, loss of kindlin-3 impairs the activation of β1, β2, and β3 integrin classes expressed on osteoclasts, which in turn abrogates the formation of podosomes and sealing zones required for bone resorption. In agreement with these findings, genetic ablation of all integrin classes abolishes the development of podosomes, mimicking kindlin-3 deficiency. Although loss of single integrin classes gives rise to podosomes, their resorptive activity is impaired. These findings show that osteoclasts require their entire integrin repertoire to be regulated by kindlin-3 to orchestrate bone homeostasis.     

Identification of a new hybrid serum response factor and myocyte enhancer factor 2-binding element in MyoD enhancer required for MyoD expression during myogenesis

MyoD is a critical myogenic factor induced rapidly upon activation of quiescent satellite cells, and required for their differentiation during muscle regeneration. One of the two enhancers of MyoD, the distal regulatory region, is essential for MyoD expression in postnatal muscle. This enhancer contains a functional divergent serum response factor (SRF)-binding CArG element required for MyoD expression during myoblast growth and muscle regeneration in vivo. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and microinjection analyses show this element is a hybrid SRF- and MEF2 Binding (SMB) sequence where myocyte enhancer factor 2 (MEF2) complexes can compete out binding of SRF at the onset of differentiation. As cells differentiate into postmitotic myotubes, MyoD expression no longer requires SRF but instead MEF2 binding to this dual-specificity element. As such, the MyoD enhancer SMB element is the site for a molecular relay where MyoD expression is first initiated in activated satellite cells in an SRF-dependent manner and then increased and maintained by MEF2 binding in differentiated myotubes. Therefore, SMB is a DNA element with dual and stage-specific binding activity, which modulates the effects of regulatory proteins critical in controlling the balance between proliferation and differentiation.     

Brachyury is required for elongation and vasculogenesis in the murine allantois

Mouse conceptuses homozygous for mutations in brachyury (T) exhibit a short, misshapen allantois that fails to fuse with the chorion. Ultimately, mutant embryos die during mid-gestation. In the 60 years since this discovery, the role of T in allantoic development has remained obscure. T protein was recently identified in several new sites during mouse gastrulation, including the core of the allantois, where its function is not known. Here, using molecular, genetic and classical techniques of embryology, we have investigated the role of T in allantoic development. Conceptuses homozygous for the T(Curtailed) (T(C)) mutation (T(C)/T(C)) exhibited allantoic dysmorphogenesis shortly after the allantoic bud formed. Diminution in allantoic cell number and proliferation was followed by cell death within the core. Fetal liver kinase (Flk1)-positive angioblasts were significantly decreased in T(C)/T(C) allantoises and did not coalesce into endothelial tubules, possibly as a result of the absence of platelet endothelial cell adhesion molecule 1 (Pecam1), whose spatiotemporal relationship to Flk1 suggested a role in patterning the umbilical vasculature. Remarkably, microsurgical perturbation of the wild-type allantoic core phenocopied the T(C)/T(C) vascularization defect, providing further support that an intact core is essential for vascularization. Last, abnormalities were observed in the T(C)/T(C) heart and yolk sac, recently reported sites of T localization. Our findings reveal that T is required to maintain the allantoic core, which is essential for allantoic elongation and vascular patterning. In addition, morphological defects in other extraembryonic and embryonic vascular organs suggest a global role for T in vascularization of the conceptus.