How instructive cues present on the cell surface have their precise effects on the actin cytoskeleton is poorly understood. Semaphorins are one of the largest families of these instructive cues and are widely studied for their effects on cell movement, navigation, angiogenesis, immunology and cancer1. Semaphorins/collapsins were characterized in part on the basis of their ability to drastically alter actin cytoskeletal dynamics in neuronal processes2, but despite considerable progress in the identification of semaphorin receptors and their signalling pathways3, the molecules linking them to the precise control of cytoskeletal elements remain unknown. Recently, highly unusual proteins of the Mical family of enzymes have been found to associate with the cytoplasmic portion of plexins, which are large cell-surface semaphorin receptors, and to mediate axon guidance, synaptogenesis, dendritic pruning and other cell morphological changes4–7. Mical enzymes perform reduction–oxidation (redox) enzymatic reactions4,5,8–10 and also contain domains found in proteins that regulate cell morphology4,11. However, nothing is known of the role of Mical or its redox activity in mediating morphological changes. Here we report that Mical directly links semaphorins and their plexin receptors to the precise control of actin filament (F-actin) dynamics. We found that Mical is both necessary and sufficient for semaphorin–plexin-mediated F-actin reorganization in vivo. Likewise, we purified Mical protein and found that it directly binds F-actin and disassembles both individual and bundled actin filaments. We also found that Mical utilizes its redox activity to alter F-actin dynamics in vivo and in vitro, indicating a previously unknown role for specific redox signalling events in actin cytoskeletal regulation. Mical therefore is a novel F-actin-disassembly factor that provides a molecular conduit through which actin reorganization—a hallmark of cell morphological changes including axon navigation—can be precisely achieved spatiotemporally in response to semaphorins.

Cardiac hypertrophy occurs in up to 95% of patients with CKD and increases their risk for cardiovascular death. In the kidney, full-length membranous Klotho forms the coreceptor for fibroblast growth factor 23 (FGF23) to regulate phosphate metabolism. The prevailing view is that the decreased level of Klotho in CKD causes cardiomyopathy through increases in serum FGF23 and/or phosphate levels. However, we reported recently that soluble Klotho protects against cardiac hypertrophy by inhibiting abnormal calcium signaling in the heart. Here, we tested whether this protective effect requires changes in FGF23 and/or phosphate levels. Heterozygous Klotho-deficient CKD mice exhibited aggravated cardiac hypertrophy compared with wild-type CKD mice. Cardiac magnetic resonance imaging studies revealed that Klotho-deficient CKD hearts had worse functional impairment than wild-type CKD hearts. Normalization of serum phosphate and FGF23 levels by dietary phosphate restriction did not abrogate the aggravated cardiac hypertrophy observed in Klotho-deficient CKD mice. Circulating levels of the cleaved soluble ectodomain of Klotho were lower in wild-type CKD mice than in control mice and even lower in Klotho-deficient CKD mice. Intravenous delivery of a transgene encoding soluble Klotho ameliorated cardiac hypertrophy in Klotho-deficient CKD mice. These results suggest that the decreased level of circulating soluble Klotho in CKD is an important cause of uremic cardiomyopathy independent of FGF23 and phosphate, opening new avenues for treatment of this disease.

We describe methods for generating artificial transcription factors capable of up- or downregulating the expression of genes whose promoter regions contain the target DNA sequences. To accomplish this, we screened zinc fingers derived from sequences in the human genome and isolated 56 zinc fingers with diverse DNA-binding specificities. We used these zinc fingers as modular building blocks in the construction of novel, sequence-specific DNA-binding proteins. Fusion of these zinc-finger proteins with either a transcriptional activation or repression domain yielded potent transcriptional activators or repressors, respectively. These results show that the human genome encodes zinc fingers with diverse DNA-binding specificities and that these domains can be used to design sequence-specific DNA-binding proteins and artificial transcription factors.

Soluble klotho, the shed ectodomain of the antiaging membrane protein α-klotho, is a pleiotropic endocrine/paracrine factor with no known receptors and poorly understood mechanism of action. Soluble klotho down-regulates growth factor-driven PI3K signaling, contributing to extension of lifespan, cardioprotection, and tumor inhibition. Here we show that soluble klotho binds membrane lipid rafts. Klotho binding to rafts alters lipid organization, decreases membrane's propensity to form large ordered domains for endocytosis, and down-regulates raft-dependent PI3K/Akt signaling. We identify α2-3-sialyllactose present in the glycan of monosialogangliosides as targets of soluble klotho. α2-3-Sialyllactose is a common motif of glycans. To explain why klotho preferentially targets lipid rafts we show that clustering of gangliosides in lipid rafts is important. In vivo, raft-dependent PI3K signaling is up-regulated in klotho-deficient mouse hearts vs. wild-type hearts. Our results identify ganglioside-enriched lipid rafts to be receptors that mediate soluble klotho regulation of PI3K signaling. Targeting sialic acids may be a general mechanism for pleiotropic actions of soluble klotho.soluble klotho | lipid rafts | gangliosides | sialic acids | TRPC6 M ice homozygous for a severe hypomorphic α-klotho allele manifest multiple aging-related phenotypes and die prematurely at 2-3 mo after birth (1). α-Klotho is predominantly expressed in renal tubules, parathyroid glands, and epithelial cells of the choroids plexus, but not in myocardium. Overexpression of α-klotho extends life span in mice, indicating that it is an agingsuppression molecule (2). The full-length α-klotho protein is a single-pass membrane protein with a large extracellular domain, a membrane-spanning segment, and a short intracellular carboxyl terminus (1). Membranous α-klotho associates with FGF receptors to form coreceptors for the ligand FGF23, a bone-derived circulating hormone that plays an important role in phosphate homeostasis (3).The ectodomain of α-klotho, ∼950 aa in length, is composed of two internal repeats, KL1 and KL2, each sharing amino acid sequence homology to family-1 glycosidases (4). The ectodomain (soluble klotho, sKL) is released into the systemic circulation, urine, and cerebrospinal fluid and functions as a humoral factor (2, 5). To date, more than 10 different functions of sKL have been described (2, 6-9). sKL regulates ion transporters, antagonizes Wnt and TGF-β1signaling to suppress cellular senescence, tissue fibrosis, and cancer metastasis, and inhibits insulin and insulin-like growth factor-1 (IGF1)-driven PI3K/Akt signaling contributing to extension of lifespan in mice, cardioprotection, and inhibition of tumor cell proliferation. A fundamental gap in the understanding of sKL's mechanism of action is the lack of knowledge of potential membrane receptor(s) that mediate cellular responses to sKL.Lipid rafts are highly dynamic, cholesterol-and sphingolipidrich membrane microdomains that compartmentalize cellular processes such as ...

Cellular form and function – and thus normal development and physiology – are specified via proteins that control the organization and dynamic properties of the actin cytoskeleton. Using the Drosophila model, we have recently identified an unusual actin regulatory enzyme, Mical, which is directly activated by F-actin to selectively post-translationally oxidize and destabilize filaments – regulating numerous cellular behaviors. Mical proteins are also present in mammals, but their actin regulatory properties, including comparisons among different family members, remain poorly defined. We now find that each human MICAL family member, MICAL-1, MICAL-2, and MICAL-3, directly induces F-actin dismantling and controls F-actin-mediated cellular remodeling. Specifically, each human MICAL selectively associates with F-actin, which directly induces MICALs catalytic activity. We also find that each human MICAL uses an NADPH-dependent Redox activity to post-translationally oxidize actin’s methionine (M) M44/M47 residues, directly dismantling filaments and limiting new polymerization. Genetic experiments also demonstrate that each human MICAL drives F-actin disassembly in vivo, reshaping cells and their membranous extensions. Our results go on to reveal that MsrB/SelR reductase enzymes counteract each MICAL’s effect on F-actin in vitro and in vivo. Collectively, our results therefore define the MICALs as an important phylogenetically-conserved family of catalytically-acting F-actin disassembly factors.

SUMMARY Extracellular cues that regulate cellular shape, motility, and navigation are generally classified as growth-promoting (i.e., growth factors/chemoattractants and attractive guidance cues) or growth-preventing (i.e., repellents and inhibitors). Yet, these designations are often based on complex assays and undefined signaling pathways, and thus may misrepresent direct roles of specific cues. Herein, we find that a recognized growth-promoting signaling pathway amplifies the F-actin disassembly and repulsive effects of a growth-preventing pathway. Focusing on Semaphorin/Plexin repulsion, we identified an interaction between the F-actin-disassembly enzyme Mical and the Abl tyrosine kinase. Biochemical assays revealed Abl phosphorylates Mical to directly amplify Mical Redox-mediated F-actin disassembly. Genetic assays revealed Abl allows growth factors and Semaphorin/Plexin repellents to combinatorially increase Mical-mediated F-actin disassembly, cellular remodeling, and repulsive axon guidance. Similar roles for Mical in growth factor/Abl-related cancer cell behaviors further revealed contexts in which characterized positive effectors of growth/guidance stimulate such negative cellular effects as F-actin disassembly/repulsion.

Background To determine the effect of missing teeth on the risk of dementia onset among individuals who received tooth extractions and those who did not, based on the number of missing teeth. Methods We selected individuals who had not been diagnosed or treated for dementia between 2002 to 2011 from the National Health Insurance Service-Elderly Cohort Database (NHIS-ECD). We divided participants into two cohorts, a tooth extraction and non-extraction cohort, based on tooth loss from 2002 to 2011. After propensity score matching, there were 104,903 individuals in each cohort, and we included a total of 209,806 individuals in this study. Each cohort was grouped by sex, age, residential area, health insurance eligibility, income level, history of dental caries, history of periodontal treatment, and number of extracted teeth. We analyzed the relationship between dementia onset and these variables using logistic regression analysis. Results Individuals with tooth loss had a higher risk for dementia than those without tooth loss (odds ratio [OR] = 1.18; 95% confidence interval [CI]: 1.146–1.215). Regarding the incidence of dementia, the OR increased as the number of missing teeth and age increased, and the OR was higher for women (OR = 1.33; 95% CI: 1.286–1.367) than for men, and this difference was statistically significant ( P < 0.01). The incidence of dementia decreased with periodontal treatment (OR = 0.96; 95% CI: 0.932–0.992) and increased with dental caries (OR = 1.07; 95% CI: 1.035–1.101). Conclusions These results suggest that it is important to delay tooth loss and preserve the stable remaining teeth to help prevent dementia. Electronic supplementary material The online version of this article (10.1186/s12903-019-0750-4) contains supplementary material, which is available to authorized users.

We estimated the energy barrier of proton transfer on ice film surfaces through the measurement of the H/D exchange kinetics of H(2)O and D(2)O molecules. The isotopomeric populations of water molecules and hydronium ions on the surface were monitored by using the techniques of reactive ion scattering and low energy sputtering, respectively, along the progress of the H/D reaction. When hydronium ions were externally added onto an ice film at a temperature of 70 K, a proton was transferred from the hydronium ion mostly to an adjacent water molecule. The proton transfer distance and the H/D exchange rate increased as the temperature increased for 90-110 K. The activation energy of the proton transfer was estimated to be 10+/-3 kJ mol(-1) on a polycrystalline ice film grown at 135 K. The existence of a substantial energy barrier for proton transfer on the ice surface agreed with proton stabilization at the surface. We also examined the H/D exchange reaction on a pure ice film surface at temperatures of 110-130 K. The activation energy of the reaction was estimated to be 17+/-4 kJ mol(-1), which was contributed from the ion pair formation and proton transfer processes on the surface.