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Purpose Spatiotemporal regulation of cell membrane dynamics is a major process that promotes cancer cell invasion by acting as a driving force for cell migration. Beta-Pix (βPix), a guanine nucleotide exchange factor for Rac1, has been reported to be involved in actin-mediated cellular processes, such as cell migration, by interacting with various proteins. As yet, however, the molecular mechanisms underlying βPix-mediated cancer cell invasion remain unclear. Methods The clinical significance of βPix was analyzed in patients with colorectal cancer (CRC) using public clinical databases. Pull-down and immunoprecipitation assays were employed to identify novel binding partners for βPix. Additionally, various cell biological assays including immunocytochemistry and time-lapse video microscopy were performed to assess the effects of βPix on CRC progression. A βPix-SH3 antibody delivery system was used to determine the effects of the βPix-Dyn2 complex in CRC cells. Results We found that the Src homology 3 (SH3) domain of βPix interacts with the proline-rich domain of Dynamin 2 (Dyn2), a large GTPase. The βPix-Dyn2 interaction promoted lamellipodia formation, along with plasma membrane localization of membrane-type 1 matrix metalloproteinase (MT1-MMP). Furthermore, we found that Src kinase-mediated phosphorylation of the tyrosine residue at position 442 of βPix enhanced βPix-Dyn2 complex formation. Disruption of the βPix-Dyn2 complex by βPix-SH3 antibodies targeting intracellular βPix inhibited CRC cell invasion. Conclusions Our data indicate that spatiotemporal regulation of the Src-βPix-Dyn2 axis is crucial for CRC cell invasion by promoting membrane dynamics and MT1-MMP recruitment into the leading edge. The development of inhibitors that disrupt the βPix-Dyn2 complex may be a useful therapeutic strategy for CRC.

Background Aconitine is well-known for its potential analgesic, anti-inflammatory, and circulation promoting effects and has been widely used as a folk medicine in South Korea. Owing to its extremely toxic nature and relatively low safety margin, intoxication is sometimes fatal. The toxic compound mainly affects the central nervous system, heart, and muscle, resulting in cardiovascular complications. Purpose To determine the exact relationship between blood concentration of aconitine and clinical manifestation. Basic procedures The National Forensic Service (NFS) was commissioned to assist in a quantitative analysis of highly toxic aconitine and corresponding blood concentrations by analyzing the body fluids of three patients who were suspected of aconitine poisoning. Main findings Aconitine blood values tested by the NFS showed that patients with a blood concentration below a certain level developed symptoms slowly and showed a high severity of clinical manifestation. There was no correlation between blood concentration and symptoms or ECG results. Conclusions In case of suspected aconitine poisoning, an emergency care department should be visited, even with symptomatic improvement, and the patient should be monitored for at least 24 h, depending on the level of recovery and changes in ECG results.

Background: QT prolongation is a well-known complication when tramadol or zolpidem is ingested in large amounts acutely. However, the blood drug concentration resulting in QT prolongation or tachyarrhythmia when tramadol and zolpidem are ingested simultaneously in large amounts has not yet been reported. We report a case of acute intoxication by tramadol and zolpidem resulting in QT prolongation in a patient in whomserial blood and urine tramadol and zolpidemconcentrationswere determined.Case presentation:A 38-year-old male patient presented tothe emergency medical centrebecause ofpoisoning from 3 g of tramadol and 50 mg of zolpidemat 3 h before hisemergency department(ED) visit.During supportive treatment, he developed QT prolongation without clinical manifestations. He was discharged five days after admission without any sequelae. We measured the blood and urine concentrations of tramadol and zolpidemat various time points, which revealed ablood tramadol concentration-dependent change in QTc intervals and increasing blood tramadol concentration at 8 h after the ED visit,with a sustainedlow zolpidem concentration.Tramadol and zolpidem are metabolized by the same enzyme, cytochrome P450 3A4.Therefore, competitive inhibition bya P450 3A4isoenzyme may lead to increases indrugtoxicity. The QT interval in patients acutely intoxicated bytramadol should be evaluated carefully, particularlywhen tramadol is co-ingested with other drugs.Conclusions:Considering the half-life of tramadolor zolpidem and potential for continued absorption of drugs remaining in the gastrointestinal tract, it is necessary to observe patients viacardiac monitoring for more than 36 hafteracute intoxication.The blood concentration of tramadol may increaseand result in QT prolongationeven afterappropriate initial treatment.

In Korea, an increasing number of people attempt to evade military conscription by posing as mental health patients. To verify the authenticity of mental illness, there is a need to detect wide range of psychoactive agents in biological specimens of conscription candidates. In this study, we developed and validated a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of 18 psychoactive agents and 6 metabolites in human plasma. The method was characterized by the use of a simple, fast and cheap protein precipitation as sample preparation, a rapid run time (11 min) and a low volume of plasma sample (200 μL). The analytes were monitored under the scheduled multiple reaction monitoring (sMRM) positive and negative mode using electrospray ionization (ESI). The essential validation parameters including selectivity, linearity, accuracy, precision, matrix effect and recovery were satisfactory. The limit of detection ranged from 0.0005 to 0.001 μg/mL, and limit of quantitation ranged from 0.005 to 0.025 μg/mL. The developed method was successfully applied to 323 actual plasma samples submitted by Korea central physical examination center of military manpower administration in 2016, and is expected to contribute to the rapid and accurate disposition of military service.

Ectodomain shedding regulates functions of many membrane proteins through the cleavage of their juxtamembrane region mainly by a disintegrin and metalloproteinase family proteinases. Tumor necrosis factor-alpha converting enzyme (TACE) is known to be responsible for phorbol myristate acetate (PMA)-induced shedding of various membrane proteins. How PMA regulates TACE-dependent shedding and how TACE exhibits substrate specificity without proteolysis of other membrane proteins are questionable. Here, we show that TACE can interact with an actin-binding protein, filamin, through 20th filamin repeat. We found that the interaction between TACE and filamin was increased by PMA treatment. In addition, loss of filamin or specific disruption of TACE-filamin interaction inhibited ectodomain shedding of representative TACE substrates, CD44 and amyloid protein precursor. From these data, we suggest that filamin may work as a scaffold that can recruit TACE and its substrates in a PMA-dependent manner to achieve substrate specificity for TACE.

Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbβ3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbβ3 activation, but it activates αIIbβ3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbβ3 is tightly regulated by the topology of β3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin β3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-β TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.

Additional file 1: Figure S1. Surface localization of Epi-GV fusion protein. (A) Diagram of Epi-GV construct with C-terminal Strep tag (Epi-GV-strep). (B) Epi-GV-strep construct (or MOCK) was transfected to 293F cells with a trace amount of EGFP cDNA as a transfection marker, and its surface expression was analyzed by flow cytometry. Representative dot plots are shown. Figure S2. PMA-induced nuclear localization of epithin/PRSS14 intracellular domain. (A) 427 cells were transfected with two siRNAs against epithin/PRSS14, starved for serum, and stimulated with PMA or not. Cells were then stained with anti-N antibody (green) and DAPI (blue). Representative images are shown. Scale bars, 20 μm. (B) The computational method for determining EICD-positive pixels is illustrated. If the mean fluorescence of a particular pixel and the 8 pixels surrounding it in the nuclear image is greater than the threshold described in Methods section, the pixel was considered as an EICD-positive pixel. (C) Representative anti-N antibody-stained original image and binary image with epithin/PRSS14 positive pixels indicated in white are shown. (D) In the experiment described in A, normalized EICD nuclear localization (the sum of the fluorescence intensity of EICD-positive pixels in each cell divided by the mean fluorescence of nuclear region of the cell) was calculated and represented as bar graph. At least 57 cells from three microscopic fields were used for each condition. The error bar indicates SEM. ****p

Microtubules are a major cytoskeletal component of neurites, and the regulation of microtubule stability is essential for neurite morphogenesis. βPix (ARHGEF7) is a guanine nucleotide exchange factor for the small GTPases Rac1 and Cdc42, which modulate the organization of actin filaments and microtubules. βPix is expressed as alternatively spliced variants, including the ubiquitous isoform βPix-a and the neuronal isoforms βPix-b and βPix-d, but the function of the neuronal isoforms remains unclear. Here, we reveal the novel role of βPix neuronal isoforms in regulating tubulin acetylation and neurite outgrowth. At DIV4, hippocampal neurons cultured from βPix neuronal isoform knockout (βPix-NIKO) mice exhibit defects in neurite morphology and tubulin acetylation, a type of tubulin modification which often labels stable microtubules. Treating βPix-NIKO neurons with paclitaxel, which stabilizes the microtubules, or reintroducing either neuronal βPix isoform to the KO neurons overcomes the impairment in neurite morphology and tubulin acetylation, suggesting that neuronal βPix isoforms may promote microtubule stabilization during neurite development. βPix-NIKO neurons also exhibit lower phosphorylation levels for Stathmin1, a microtubule-destabilizing protein, at Ser16. Expressing either βPix neuronal isoform in the βPix-NIKO neurons restores Stathmin1 phosphorylation levels, with βPix-d having a greater effect than βPix-b. Furthermore, we find that the recovery of neurite length and Stathmin1 phosphorylation via βPix-d expression requires PAK kinase activity. Taken together, our study demonstrates that βPix-d regulates the phosphorylation of Stathmin1 in a PAK-dependent manner and that neuronal βPix isoforms promote tubulin acetylation and neurite morphogenesis during neuronal development.

Background Epithin/PRSS14, a type II transmembrane serine protease, is an emerging target of cancer therapy because of its critical roles in tumor progression and metastasis. In many circumstances, the protease, through its ectodomain shedding, exists as a soluble form and performs its proteolytic functions in extracellular environments increasing cellular invasiveness. The seemingly functional integrity of the soluble form raises the question of why the protease is initially made as a membrane-associated protein. Results In this report, we show that the epithin/PRSS14 intracellular domain (EICD) can be released from the membrane by the action of signal peptide peptidase-like 2b (SPPL2b) after ectodomain shedding. The EICD preferentially localizes in the nucleus and can enhance migration, invasion, and metastasis of epithelial cancer when heterologously expressed. Unbiased RNA-seq analysis and subsequent antibody arrays showed that EICD could control the gene expression of chemokines involved in cell motility, by increasing their promoter activities. Finally, bioinformatics analysis provided evidence for the clinical significance of the intramembrane proteolysis of epithin/PRSS14 by revealing that the poor survival of estrogen receptor (ER)-negative breast cancer patients with high epithin/PRSS14 expression is further worsened by high levels of SPPL2b. Conclusions These results show that ectodomain shedding of epithin/PRSS14 can initiate a unique and synchronized bidirectional signal for cancer metastasis: extracellularly broadening proteolytic modification of the surrounding environment and intracellularly reprogramming the transcriptome for metastatic conversion. Clinically, this study also suggests that the intracellular function of epithin/PRSS14 should be considered for targeting this protease for anti-cancer treatment.

βPix is a guanine nucleotide exchange factor for the Rho family small GTPases, Rac1 and Cdc42. It is known to regulate focal adhesion dynamics and cell migration. However, the in vivo role of βPix is currently not well understood. Here, we report the production and characterization of βPix-KO mice. Loss of βPix results in embryonic lethality accompanied by abnormal developmental features, such as incomplete neural tube closure, impaired axial rotation, and failure of allantois-chorion fusion. We also generated βPix-KO mouse embryonic fibroblasts (MEFs) to examine βPix function in mouse fibroblasts. βPix-KO MEFs exhibit decreased Rac1 activity, and defects in cell spreading and platelet-derived growth factor (PDGF)-induced ruffle formation and chemotaxis. The average size of focal adhesions is increased in βPix-KO MEFs. Interestingly, βPix-KO MEFs showed increased motility in random migration and rapid wound healing with elevated levels of MLC2 phosphorylation. Taken together, our data demonstrate that βPix plays essential roles in early embryonic development, cell spreading, and cell migration in fibroblasts.

Objective— Angiogenesis, the process of building complex vascular structures, begins with sprout formation on preexisting blood vessels, followed by extension of the vessels through proliferation and migration of endothelial cells. Based on the potential therapeutic benefits of preventing angiogenesis in pathological conditions, many studies have focused on the mechanisms of its initiation as well as control. However, how the extension of vessels is terminated remains obscure. Thus, we investigated the negative regulation mechanism. Approach and Results— We report that increased intracellular calcium can induce dephosphorylation of the endothelial receptor tyrosine kinase Tie2. The calcium-mediated dephosphorylation was found to be dependent on Tie2–calmodulin interaction. The Tyr1113 residue in the C-terminal end loop of the Tie2 kinase domain was mapped and found to be required for this interaction. Moreover, mutation of this residue into Phe impaired both the Tie2-calmodulin interaction and calcium-mediated Tie2 dephosphorylation. Furthermore, expressing a mutant Tie2 incapable of binding to calmodulin or inhibiting calmodulin function in vivo causes unchecked growth of the vasculature in Xenopus . Specifically, knockdown of Tie2 in Xenopus embryo retarded the sprouting and extension of intersomitic veins. Although human Tie2 expression in the Tie2-deficient animals almost completely rescued the retardation, the Tie2(Y1113F) mutant caused overgrowth of intersomitic veins with strikingly complex and excessive branching patterns. Conclusions— We propose that the calcium/calmodulin-dependent negative regulation of Tie2 can be used as an inhibitory signal for vessel growth and branching to build proper vessel architecture during embryonic development.

PAK-interacting guanine nucleotide exchange factor (βPix; also known as Arhgef7) has been implicated in many actin-based cellular processes, including spine morphogenesis in neurons. However, the molecular mechanisms by which βPix controls spine morphology remain elusive. Previously, we have reported the expression of several alternative spliced βPix isoforms in the brain. Here, we report a novel finding that the b isoform of βPix (βPix-b) mediates the regulation of spine and synapse formation. We found that βPix-b, which is mainly expressed in neurons, enhances spine and synapse formation through preferential localization at spines. In neurons, glutamate treatment efficiently stimulates Rac1 GEF activity of βPix-b. The glutamate stimulation also promotes Src-mediated phosphorylation of βPix-b in both an AMPA receptor- and NMDA receptor-dependent manner. Tyrosine 598 (Y598) of βPix-b is identified as the major Src-mediated phosphorylation site. Finally, Y598 phosphorylation of βPix-b enhances its Rac1 GEF activity that is critical for spine and synapse formation. In conclusion, we provide a novel mechanism by which βPix-b regulates activity-dependent spinogenesis and synaptogenesis via Src-mediated phosphorylation. Published version

Microtubule dynamics are important for axon growth during development as well as axon regeneration after injury. We have previously identified HDAC5 as an injury-regulated tubulin deacetylase that functions at the injury site to promote axon regeneration. However, the mechanisms involved in the spatial control of HDAC5 activity remain poorly understood. Here we reveal that HDAC5 interacts with the actin binding protein filamin A via its C-terminal domain. Filamin A plays critical roles in HDAC5-dependent tubulin deacetylation because, in cells lacking filamin A, the levels of acetylated tubulin are elevated markedly. We found that nerve injury increases filamin A axonal expression in a protein synthesis-dependent manner. Reducing filamin A levels or interfering with the interaction between HDAC5 and filamin A prevents injury-induced tubulin deacetylation as well as HDAC5 localization at the injured axon tips. In addition, neurons lacking filamin A display reduced axon regeneration. Our findings suggest a model in which filamin A local translation following axon injury controls localized HDAC5 activity to promote axon regeneration.

Cardiovascular disease, which is caused by unregulated platelet aggregation, is one of the main causes of deaths worldwide. Many studies have focused on natural products with antiplatelet effects as a safe alternative therapy to prevent the disease. In this context, an in-house chemical library was screened to find natural products capable of inhibiting the interaction between platelet integrin αIIbβ3 and fibrinogen, which is an essential step in platelet aggregation. On the basis of the screening results, indothiazinone, an alkaloid found in microbial cultures, was identified as a potential antiplatelet agent. Specifically, indothiazinone treatment significantly inhibited the binding of fibrinogen to Chinese hamster ovary cells expressing integrin αIIbβ3. It also restricted thrombin- and adenosine diphosphate-dependent spreading of human platelets on a fibrinogen matrix. More importantly, surface plasmon resonance and molecular dynamics studies suggested that indothiazinone suppressed talin-induced activation of integrin αIIbβ3 presumably by inhibiting talin-integrin interaction. In conclusion, these results suggest that indothiazinone can be used as a lead compound for the development of antiplatelet drugs with a novel mode of action.

Vimentin, an intermediate filament protein induced during epithelial-to-mesenchymal transition, is known to regulate cell migration and invasion. However, it is still unclear how vimentin controls such behaviors. In this study, we aimed to find a new integrin regulator by investigating the H-Ras-mediated integrin suppression mechanism. Through a proteomic screen using the integrin β3 cytoplasmic tail protein, we found that vimentin might work as an effector of H-Ras signaling. H-Ras converted filamentous vimentin into aggregates near the nucleus, where no integrin binding can occur. In addition, an increase in the amount of vimentin filaments accessible to the integrin β3 tail enhanced talin-induced integrin binding to its ligands by inducing integrin clustering. In contrast, the vimentin head domain, which was found to bind directly to the integrin β3 tail and compete with endogenous vimentin filaments for integrin binding, induced nuclear accumulation of vimentin filaments and reduced the amount of integrin-ligand binding. Finally, we found that expression of the vimentin head domain can reduce cell migration and metastasis. From these data, we suggest that filamentous vimentin underneath the plasma membrane is involved in increasing integrin adhesiveness, and thus regulation of the vimentin-integrin interaction might control cell adhesion.