Actin Skeleton Research Articles

Identification of entry inhibitors of Ebola virus pseudotyped vectors from a myxobacterial compound library

Myxobacteria produce secondary metabolites many of which were described to have various biological effects including anti-fungal, anti-bacterial and anti-viral activity. The majority of these metabolites are novel scaffolds with unique modes-of-action and hence might be potential leads for drug discovery. Here, we tested a myxobacterial natural product library for compounds with inhibitory activity against Ebola virus (EBOV). The assay was performed with a surrogate system using Ebola envelope glycoprotein (GP) pseudotyped lentiviral vectors. EBOV specificity was proven by counter-screening with vesicular stomatitis virus G protein pseudotyped vectors. Two compounds were identified that preferentially inhibited EBOV GP mediated cell entry: Chondramides that act on the actin skeleton but might be too toxic and noricumazole A, a potassium channel inhibitor, which might constitute a novel pathway to inhibit Ebola virus cell entry.

Effect of arsenic trioxide on cortical neuronal migration of rats and its potential mechanism

Objective To explore the effect of arsenic trioxide (As2O3) on the migration of neurons and the potential mechanism through the establishment of primary neuron culture from the brains of neonatal rats. Methods Brain tissues were selected from SD neonatal rats for primary neuron calture.The cells were divided into 4 groups based on the addition of As2O3: normal control group, 1 μmol/L As2O3 group, 10 μmol/L As2O3 group and 20 μmol/L As2O3 group.The primary neurons were treated with different concentrations of As2O3 and cultured for 24 hours.Boyden chamber assay was used to detect the effect of As2O3 on neuronal migration.Immunofluorescence laser confocal microscope was used to observe the structure of actin. Results In the control group, the cultured neurons showed a regular pattern of distribution.In the 3 groups treated with As2O3, the distribution of neurons was loose and disordered, which was most obvious in the 20 μmol/L As2O3 group.The results showed that the higher concentration of As2O3, more difficult it was for the neurons to survive.The number of neuronal migration was 64.6±4.3 for normal control group, 63.0±7.0 for 1 μmol/L As2O3 group, 54.8±3.6 for 10 μmol/L As2O3 group, and 21.6±3.9 for 20 μmol/L As2O3 group.The results showed that As2O3 might inhibit the migration of primary neurons in a dose-dependent manner (F=49.31, P<0.001). The normal actin skeleton was destroyed under the laser confocal microscope in 10 μmol/L As2O3 group and 20 μmol/L As2O3 group, while they remained unaffected in normal control group and 1 μmol/L As2O2 group. Conclusion As2O3 exposure can reduce the neuron migration in a dose-independent manner probably through disrupting the organization of acting cytoskeleton. Key words: Arsenic trioxide; Neuron migration; Actin cytoskeleton

The effect of curcumin in intestinal cells

Curcumin, a major component of the Curcuma species, contains antioxidant and anti‐inflammatory properties. Although it was found to induce apoptosis in cancer cells, the functional role of curcumin as well as its molecular mechanism in anti‐inflammatory response, particularly in intestinal cells, has been less investigated. The intestine epithelial barrier is the first barrier and the most important location for the substrate coming from the lumen of the gut. We administered curcumin treatment in the human intestinal epithelial cell lines, T84 and Caco‐2. We examined endoplasmic reticulum (ER) stress response by thapsigargin, qPCR of XBP1 and BiP, electrophysiology by wild‐type cholera toxin in the cells. In this study, we showed that curcumin treatment reduces ER stress and thereby decreases inflammatory response in human intestinal epithelial cells. In addition, curcumin confers protection without damaging the membrane tight junction or actin skeleton change in intestine epithelial cells. Therefore, curcumin treatment protects the gut from bacterial invasion via reduction of ER stress and anti‐inflammatory response in intestinal epithelial cells. Taken together, our data demonstrate the important role of curcumin in protecting the intestine by modulating ER stress and inflammatory response post intoxication.Support or Funding InformationNational Research of Korea ( 2015R1C1A1A02037579)

The oculocerebrorenal syndrome of Lowe: an update.

The oculocerebrorenal syndrome of Lowe is a rare X-linked multisystemic disorder characterized by the triad of congenital cataracts, intellectual disability, and proximal renal tubular dysfunction. Whereas the ocular manifestations and severe muscular hypotonia are the typical first diagnostic clues apparent at birth, the manifestations of incomplete renal Fanconi syndrome are often recognized only later in life. Other characteristic features are progressive severe growth retardation and behavioral problems, with tantrums. Many patients develop a debilitating arthropathy. Treatment is symptomatic, and the life span rarely exceeds 40 years. The causative oculocerebrorenal syndrome of Lowe gene (OCRL) encodes the inositol polyphosphate 5-phosphatase OCRL-1. OCRL variants have not only been found in classic Lowe syndrome, but also in patients with a predominantly renal phenotype classified as Dent disease type 2 (Dent-2). Recent data indicate that there is a phenotypic continuum between Dent-2 disease and Lowe syndrome, suggesting that there are individual differences in the ability to compensate for the loss of enzyme function. Extensive research has demonstrated that OCRL-1 is involved in multiple intracellular processes involving endocytic trafficking and actin skeleton dynamics. This explains the multi-organ manifestations of the disease. Still, the mechanisms underlying the wide phenotypic spectrum are poorly understood, and we are far from a causative therapy. In this review, we provide an update on clinical and molecular genetic findings in Lowe syndrome and the cellular and physiological functions of OCRL-1.

Abstract P162: Nicotine via Cd36, Cox2 and Oxidative Stress Promotes Podocyte Injury: Link Between E-cigarettes and Renal Disease Progression?

Cigarette smoking (CS) accounts for 175,000 annual CV deaths in US. CKD is a major CV risk factor. Epidemiologically link between CS proteinuria and progression of diabetic and of hypertensive nephropathy is documented. We showed that Nicotine (NIC) in concentrations achieved in CS and E- smokers a)Increased proteinuria (100%) renal Nox4 &amp; Nitrotyrosine in diabetic db/db mice (AJP ‘10) and b) Promoted in human macrophages O 2 - production and foam cell formation associated with upregulation of B scavenger receptor CD36 and oxLDL uptake (AJP’13). Podocytes (POD) are vulnerable to diabetes and to hypertension; POD injury results in detachment and glomerulosclerosis. We demonstrated in human POD NIC receptors α2, 3,4 and β3 Methods: We treated human POD with NIC, 100nmol/L, a concentration attained in serum of CS and E-Cigarettes smokers, and determined O 2 - production with lucigening; some POD were pre-incubated with Hexametonium: blocker of NIC receptors; DPI: inhibitor of NADPH; Catalase: inhibitor of H2O2 and AICAR: activator of AMPK, a suppressor of NADPH. By Western Blot (WB) we determined in Control and NIC exposed POD, CD36, COX2 known to induce POD injury (JASN ‘08) and Synaptopodin (Synpo) a stabilizer of POD actin skeleton. In POD incubated with 50ug/ml oxLDL we measured POD apoptosis (APOPT) by flow cytometry. Stat. ANOVA- Bonferonni’s- Scheffe’s. Results: NIC increased POD O 2 - production 225% (200 to 450 CPM/ug); Synpo expression was reduced 50% (1to 0.5) (p&lt; 0.05). DPI and AICAR prevented Synpo reduction and increase in O 2 - (p&lt;0.05). NIC upregulated expression of COX2 300% (0.5-1.5) and CD36 40 % (1-1.4) (p&lt;0.05). DPI, Catalase, and Hexametonium inhibited CD36 and COX2 upregulation. The COX inhibitor NS-398 prevented O 2 - production and CD36 upregulation .POD incubation with oxLDL plus NIC significantly increased POD oxLDL uptake and APOPT 10% over baseline (2%),(p&lt;0.05) both inhibited by CD36-SiRNA. Conclusion: NIC activated in human POD a reciprocal interplay between NADPH oxidases and COX2 that increased O2- production, reduced Synpo and upregulated the B scavenger receptor CD36 that, via increased uptake of oxidized LDL, promoted podocyte apoptosis. We surmise that these novel findings implicate NIC as a risk factor for CVD and CKD progression.

Abstract 4042: Kruppel-like factor 5 promotes breast cancer proliferation, migration and invasion partially through upregulating the transcription of TNFα-induced protein 2

Abstract The Krupple-like factor 5 (KLF5) transcription factor is highly expressed in high-grade and basal-like TNBC breast cancers. Our previous studies reported that KLF5 promotes breast cancer cell proliferation and survival through target genes, such as FGF-BP and mPGES1. However, the mechanism by which KLF5 promotes cell migration and invasion is still not completely understood. In this study, we demonstrated that TNFAIP2, a TNFα-induced gene, is a KLF5 direct downstream target gene. The expression of the TNFAIP2 is highly correlated with the expression of the KLF5 protein in breast epithelial cell lines at both mRNA and protein levels. Manipulation of KLF5 expression positively alters TNFAIP2 mRNA and protein expression levels in multiple breast cell lines. KLF5 binds to TNFAIP2 gene promoter and activates its transcription. Functionally, TNFAIP2 promotes cell proliferation and xenograft growth in NOD-SCID mice. Importantly, knockdown of either KLF5 or TNFAIP2 significantly reduces cancer cell migration and invasion. Overexpression of TNFAIP2 rescues KLF5 knockdown-induced migration suppression in HCC1937 cells. TNFAIP2 interacts with two small GTPase Cdc42 and Rac1, increases their activities, and changes actin skeleton and cell morphyology. Finally, KLF5 and TNFAIP2 promote cell migration and invasion through activating Cdc42 and Rac1. These findings collectively suggest that TNFAIP2 is a KLF5 direct target gene and both KLF5 and TNFAIP2 promotes TNBC cell migration and invasion through Cdc42 and Rac1. Citation Format: Lin Jia, Rong Liu, Ceshi Chen. Kruppel-like factor 5 promotes breast cancer proliferation, migration and invasion partially through upregulating the transcription of TNFα-induced protein 2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4042. doi:10.1158/1538-7445.AM2015-4042

A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria

In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin.

Wolbachia utilize host actin for efficient maternal transmission in Drosophila melanogaster.

Wolbachia pipientis is a ubiquitous, maternally transmitted bacterium that infects the germline of insect hosts. Estimates are that Wolbachia infect nearly 40% of insect species on the planet, making it the most prevalent infection on Earth. The bacterium, infamous for the reproductive phenotypes it induces in arthropod hosts, has risen to recent prominence due to its use in vector control. Wolbachia infection prevents the colonization of vectors by RNA viruses, including Drosophila C virus and important human pathogens such as Dengue and Chikungunya. Here we present data indicating that Wolbachia utilize the host actin cytoskeleton during oogenesis for persistence within and transmission between Drosophila melanogaster generations. We show that phenotypically wild type flies heterozygous for cytoskeletal mutations in Drosophila profilin (chic221/+ and chic1320/+) or villin (qua6-396/+) either clear a Wolbachia infection, or result in significantly reduced infection levels. This reduction of Wolbachia is supported by PCR evidence, Western blot results and cytological examination. This phenotype is unlikely to be the result of maternal loading defects, defects in oocyte polarization, or germline stem cell proliferation, as the flies are phenotypically wild type in egg size, shape, and number. Importantly, however, heterozygous mutant flies exhibit decreased total G-actin in the ovary, compared to control flies and chic221 heterozygous mutants exhibit decreased expression of profilin. Additionally, RNAi knockdown of profilin during development decreases Wolbachia titers. We analyze evidence in support of alternative theories to explain this Wolbachia phenotype and conclude that our results support the hypothesis that Wolbachia utilize the actin skeleton for efficient transmission and maintenance within Drosophila.

The longevity of tor1Δ, sch9Δ, and ras2Δ mutants depends on actin dynamics in Saccharomyces cerevisiae.

Recent studies have revealed the role of actin dynamics in the regulation of yeast aging. Although the target of rapamycin (TOR) complex, serine/threonine kinase Sch9, and Ras2 have been shown to play important roles in aging for a long time, the relationship between these regulators and actin has not yet been reported. In this study we investigated the roles of actin polarization in tor1Δ, sch9Δ, and ras2Δ mutant cells. We found that the actin structures in tor1Δ, sch9Δ, and ras2Δ mutant cells were more dynamic than those in the wild type. Destruction of the actin structures with jasplakinolide decreased the life span of tor1Δ, sch9Δ, and ras2Δ mutants. Furthermore, deletion of SLA1 in tor1Δ, sch9Δ, and ras2Δ mutants inhibited the actin dynamics and life span. In addition, we found that the actin cytoskeleton of the long-lived mutant sch9Δ, depended on the transcription factors RIM15 and MSN2/4, but not GIS1, while the actin skeleton of the tor1Δ and ras2Δ mutants depended on RIM15 as expected. Our data suggest that the longevity of tor1Δ, sch9Δ, and ras2Δ mutants is dependent on actin dynamics.

Suspensor Development in Gagea Lutea (L.) Ker Gawl., with Emphasis on the Cytoskeleton

Abstract The study used fluorescence microscopy to examine changes in cytoskeleton configuration during development of the embryo suspensor in Gagea lutea and to describe them in tandem with the development of the embryo proper. During the early phase of embryo suspensor development, tubulin and actin filaments were observed in the cytoplasm of the basal cell from the micropylar to the chalazal ends of the cell. Around the nucleus of the basal cell were clusters of numerous microtubules. These accumulations of tubulin arrays congregated near the nucleus surface; numerous bundles of microtubules radiated from the nucleus envelope. At this time, microfil-aments formed a delicate network in the cytoplasm of the basal cell. In the fully differentiated embryo suspensor, microtubules were observed at the chalazal end of the basal cell. Numerous bundles of microtubules were visualized in the cytoplasm adjacent to the wall separating the basal cell from the embryo proper. Microfilaments formed a dense network which uniformly filled the basal cell cytoplasm. There were some foci of F-actin material in the vicinity of the nucleus surface and at the chalazal end of the basal cell. In all studied phases of embryo suspensor development a prominent cortical network of actin and tubulin skeleton was observed in embryo proper cells.

Curcumin utilizes the anti-inflammatory response pathway to protect the intestine against bacterial invasion.

BACKGROUND/OBJECTIVESCurcumin, a major component of the Curcuma species, contains antioxidant and anti-inflammatory properties. Although it was found to induce apoptosis in cancer cells, the functional role of curcumin as well as its molecular mechanism in anti-inflammatory response, particularly in intestinal cells, has been less investigated. The intestine epithelial barrier is the first barrier and the most important location for the substrate coming from the lumen of the gut.SUBJECTS/METHODSWe administered curcumin treatment in the human intestinal epithelial cell lines, T84 and Caco-2. We examined endoplasmic reticulum (ER) stress response by thapsigargin, qPCR of XBP1 and BiP, electrophysiology by wild-type cholera toxin in the cells.RESULTSIn this study, we showed that curcumin treatment reduces ER stress and thereby decreases inflammatory response in human intestinal epithelial cells. In addition, curcumin confers protection without damaging the membrane tight junction or actin skeleton change in intestine epithelial cells. Therefore, curcumin treatment protects the gut from bacterial invasion via reduction of ER stress and anti-inflammatory response in intestinal epithelial cells.CONCLUSIONSTaken together, our data demonstrate the important role of curcumin in protecting the intestine by modulating ER stress and inflammatory response post intoxication.

Novel mechanisms of surfactants against Candida albicans growth and morphogenesis

Candida albicans is a common opportunistic fungal pathogen, causing not only superficial mucosal infections but also life-threatening systemic candidiasis in immune-compromised individuals. Surfactants are a kind of amphiphilic compounds implemented in a wide range of applications. Although their antimicrobial activity has been characterized, their effect on C. albicans physiology remains to be elucidated. In this study, we investigated the inhibitory effect of two representative surfactants, cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), on C. albicans growth and morphogenesis. Both surfactants exhibited inhibitory effect on C. albicans growth. This effect was not attributed to plasma membrane (PM) damage, but was associated with mitochondrial dysfunction. Excitingly, the surfactants, especially CTAB, showed strong inhibitory effect on hyphal development (IC50=0.183ppm for CTAB and 6.312ppm for SDS) and biofilms (0.888ppm for CTAB and 76.092ppm for SDS). Actin staining and Hwp1-GFP localization further revealed that this inhibition is related to abnormal organization of actin skeleton and subsequent defect in polarized transport of hyphae-related factors. This study sheds a novel light on the antimicrobial mechanisms of surfactants, and suggests these agents as potential drugs against C. albicans hyphae-related infections in clinical practice.

A recurrent activating PLCG1 mutation in cardiac angiosarcomas increases apoptosis resistance and invasiveness of endothelial cells.

Primary cardiac angiosarcomas are rare tumors with unfavorable prognosis. Pathogenic driver mutations are largely unknown. We therefore analyzed a collection of cases for genomic aberrations using SNP arrays and targeted next-generation sequencing (tNGS) of oncogenes and tumor-suppressor genes. Recurrent gains of chromosome 1q and a small region of chromosome 4 encompassing KDR and KIT were identified by SNP array analysis. Repeatedly mutated genes identified by tNGS were KDR with different nonsynonymous mutations, MLL2 with different nonsense mutations, and PLCG1 with a recurrent nonsynonymous mutation (R707Q) in the highly conserved autoinhibitory SH2 domain in three of 10 cases. PLCγ1 is usually activated by Y783 phosphorylation and activates protein kinase C and Ca(2+)-dependent second messengers, with effects on cellular proliferation, migration, and invasiveness. Ectopic expression of the PLCγ1-R707Q mutant in endothelial cells revealed reduced PLCγ1-Y783 phosphorylation with concomitant increased c-RAF/MEK/ERK1/2 phosphorylation, increased IP3 amounts, and increased Ca(2+)-dependent calcineurin activation compared with ectopic expressed PLCγ1-wild-type. Furthermore, cofilin, whose activation is associated with actin skeleton reorganization, showed decreased phosphorylation, and thus activation after expression of PLCγ1-R707Q compared with PLCγ1-wild-type. At the cellular level, expression of PLCγ1-R707Q in endothelial cells had no influence on proliferation rate, but increased apoptosis resistance and migration and invasiveness in in vitro assays. Together, these findings indicate that the PLCγ1-R707Q mutation causes constitutive activation of PLCγ1 and may represent an alternative way of activation of KDR/PLCγ1 signaling besides KDR activation in angiosarcomas, with implications for VEGF/KDR targeted therapies.

ROCK-dependent ATP5D modulation contributes to the protection of notoginsenoside NR1 against ischemia-reperfusion-induced myocardial injury.

Cardiac ischemia-reperfusion (I/R) injury remains a challenge for clinicians, which initiates with energy metabolism disorder. The present study was designed to investigate the protective effect of notoginsenoside R1 (NR1) on I/R-induced cardiac injury and underlying mechanism. Male Sprague-Dawley rats were subjected to 30-min occlusion of the left coronary anterior descending artery followed by reperfusion with or without NR1 pretreatment (5 mg·kg(-1)·h(-1)). In vitro, H9c2 cells were cultured under oxygen and glucose deprivation/reoxygenation conditions after NR1 (0.1 mM), Rho kinase (ROCK) inhibitor Y-27632 (10 μM), or RhoA/ROCK activator U-46619 (10 nM) administration. Myocardial infarct size, myocardial histology, and cardiac function were evaluated. Myofibril and mitochondria morphology were observed by transmission electron microscopy. F-actin and apoptosis were determined by immunofluorescence and TUNEL staining. ATP and AMP content were assessed by ELISA. Phosphorylated-AMP-activated protein kinase, ATP synthase subunits, apoptosis-related molecules, and the level and activity of ROCK were determined by Western blot analysis. We found that NR1 pretreatment ameliorated myocardial infarction, histological injury, and cardiac function induced by I/R. Furthermore, similar to the effect of Y-27632, NR1 improved H9c2 cell viability, maintained actin skeleton and mitochondria morphology, and attenuated apoptosis induced by oxygen and glucose deprivation/reoxygenation. Importantly, NR1 prevented energy abnormity, inhibited the expression and activation of ROCK, and restored the expression of the mitochondrial ATP synthase δ-subunit both in vivo and in vitro, whereas U-46619 suppressed the effect of NR1. These results prove NR1 as an agent able to prevent I/R-induced energy metabolism disorder via inhibiting ROCK and enhancing mitochondrial ATP synthase δ-subunits, which at least partially contributes to its protection against cardiac I/R injury.

High glucose-induced hyperosmolarity impacts proliferation, cytoskeleton remodeling and migration of human induced pluripotent stem cells via aquaporin-1

Background and objective: Hyperglycemia leads to adaptive cell responses in part due to hyperosmolarity. In endothelial and epithelial cells, hyperosmolarity induces aquaporin-1 (AQP1) which plays a role in cytoskeletal remodeling, cell proliferation and migration. Whether such impairments also occur in human induced pluripotent stem cells (iPS) is not known. We therefore investigated whether high glucose-induced hyperosmolarity impacts proliferation, migration, expression of pluripotency markers and actin skeleton remodeling in iPS cells in an AQP1-dependent manner. Methods and results: Human iPS cells were generated from skin fibroblasts by lentiviral transduction of four reprogramming factors (Oct4, Sox2, Klf4, c-Myc). After reprogramming, iPS cells were characterized by their adaptive responses to high glucose-induced hyperosmolarity by incubation with 5.5mmol/L glucose, high glucose (HG) at 30.5mM, or with the hyperosmolar control mannitol (HM). Exposure to either HG or HM increased the expression of AQP1. AQP1 co-immunoprecipitated with β-catenin. HG and HM induced the expression of β-catenin. Under these conditions, iPS cells showed increased ratios of F-actin to G-actin and formed increased tubing networks. Inhibition of AQP1 with small interfering RNA (siRNA) reverted the inducing effects of HG and HM. Conclusions: High glucose enhances human iPS cell proliferation and cytoskeletal remodeling due to hyperosmolarity-induced upregulation of AQP1.

MiR-206 inhibits cell migration through direct targeting of the actin-binding protein Coronin 1C in triple-negative breast cancer

Patients with triple-negative breast cancer (TNBC) have an overall poor prognosis, which is primarily due to a high metastatic capacity of these tumors. Novel therapeutic approaches to target the signaling pathways that promote metastasis are desirable, in order to improve the outcome for these patients. A loss of function of a microRNA, miR-206, is related to increased metastasis potential in breast cancers but the mechanism is not known. In this study, we show that miR-206 was decreased in TNBC clinical tumor samples and cell lines whereas one of its predicted targets, actin-binding protein CORO1C, was increased. Expression of miR-206 significantly reduced proliferation and migration while repressing CORO1C mRNA and protein levels. We demonstrate that miR-206 interacts with the 3'-untranslated region (3'-UTR) of CORO1C and regulates this gene post-transcriptionally. This post-transcriptional regulation was dependent on two miR-206-binding sites within the 3'-UTR of CORO1C and was relieved by mutations of corresponding sites. Further, silencing of CORO1C reduced tumor cell migration and affected the actin skeleton and cell morphology, similar to miR-206 expression, but did not reduce proliferation. In accordance with this, overexpression of CORO1C rescued the inhibitory effect of miR-206 on cell migration. Our findings suggest that miR-206 represses tumor cell migration through direct targeting of CORO1C in TNBC cells which modulates the actin filaments. This pathway is a novel mechanism that offers a mechanistic basis through which the metastatic potential of TNBC tumors could be targeted.

Tracking Single Serotonin Transporter Molecules at the Endoplasmic Reticulum and Plasma Membrane

Transmembrane proteins are synthesized and folded in the endoplasmic reticulum (ER), an interconnected network of flattened sacs or tubes. Up to now, this organelle has eluded a detailed analysis of the dynamics of its constituents, mainly due to the complex three-dimensional morphology within the cellular cytosol, which precluded high-resolution, single-molecule microscopy approaches. Recent evidences, however, pointed out that there are multiple interaction sites between ER and the plasma membrane, rendering total internal reflection microscopy of plasma membrane proximal ER regions feasible. Here we used single-molecule fluorescence microscopy to study the diffusion of the human serotonin transporter at the ER and the plasma membrane. We exploited the single-molecule trajectories to map out the structure of the ER close to the plasma membrane at subdiffractive resolution. Furthermore, our study provides a comparative picture of the diffusional behavior in both environments. Under unperturbed conditions, the majority of proteins showed similar mobility in the two compartments; at the ER, however, we found an additional 15% fraction of molecules moving with 25-fold faster mobility. Upon degradation of the actin skeleton, the diffusional behavior in the plasma membrane was strongly influenced, whereas it remained unchanged in the ER.

Abstract P4-07-12: miR-206 inhibits cell migration through direct targeting of the actin-binding protein coronin 1C in triple-negative breast cancer

Abstract Patients with triple-negative breast cancer (TNBC) have an overall poor prognosis, which is primarily due to a high metastatic capacity of these tumors. Novel therapeutic approaches to target the signaling pathways that promote metastasis are desirable, in order to improve the outcome of TNBC patients. A loss of function of a microRNA, miR-206, is related to increased metastasis potential in breast cancers but the mechanism remains to be elucidated. In this study, we show that miR-206 was decreased in TNBC clinical tumor samples and cell lines whereas one of its predicted targets, actin-binding protein CORO1C, was increased. Expression of miR-206 significantly reduced proliferation by inducing a G1-S cell cycle arrest and migration and also repressed CORO1C mRNA and protein levels. We demonstrate that miR-206 interacts with the 3’-untranslated region (3’-UTR) of CORO1C and regulates this gene post-transcriptionally. Further, silencing of CORO1C reduced tumor cell migration and affected the actin skeleton and cell morphology, similar to miR-206 expression, but did not reduce proliferation. Our findings suggest that miR-206 targets CORO1C in TNBC cells, thereby affecting the actin filaments which results in a repressed tumor cell migration. This pathway is a novel mechanism offering a mechanistic basis whereby the metastatic potential of TNBC tumors could be targeted. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-12.

Hippocampal subregion-specific microRNA expression during epileptogenesis in experimental temporal lobe epilepsy

Since aberrant miRNA expression has been implicated in numerous brain diseases, we studied miRNA expression and miRNA regulation of important signaling pathways during temporal lobe epileptogenesis in order to identify possible targets for epilepsy therapy.The temporal profile of miRNA expression was analyzed in three brain regions (CA1; dentate gyrus, DG; parahippocampal cortex, PHC) associated with epileptogenesis in a rat model for temporal lobe epilepsy. Tissue was obtained after electrically-induced status epilepticus (SE) at 1day (n=5), 1week (n=5) and 3-4months (n=5), and compared with control tissue (n=10) using the Exiqon microRNA arrays which contain capture probes targeting all miRNAs for rat (p<0.01, and a 1.5 fold up- or downregulation). Expression of three blood plasma miRNAs from the same group of rats was also investigated in rats in order to determine whether plasma miRNAs could serve as potential biomarkers of the epileptogenic process. Molecular pathways potentially altered by the expression of multiple miRNAs were identified using a web-based algorithm, DIANA.In CA1 and DG, more upregulated than downregulated miRNAs were present during each stage after SE. The highest numbers of upregulated miRNAs were encountered during the chronic stage in the DG. In PHC, a high number of downregulated miRNAs were detected. Key pathways involved, based upon quantitatively altered miRNA expression were: axon guidance, MAPK signaling pathway, focal adhesion, TGFβ, ErbB-, Wnt- and mTOR signaling, and regulation of actin skeleton. Expression of plasma miRNAs was differentially regulated after induction of SE.This study identified several signaling pathways possibly involved in temporal lobe epileptogenesis, not previously indicated by RNA microarray studies. These include miRNAs that regulate the ErbB and Wnt pathways and focal adhesion, which may represent interesting new targets for therapeutic interventions.

Entry of human rhinovirus 89 via ICAM-1 into HeLa epithelial cells is inhibited by actin skeleton disruption and by bafilomycin

HRV89, a major-group rhinovirus, uses intercellular adhesion molecule 1 (ICAM-1) for cell entry, while minor-group HRV2 uses the LDL receptor for clathrin-mediated endocytosis. Entry of HRV89 into HeLa epithelial cells was found to be inefficient, and infectious virus was still detected on the plasma membrane after 3 h of incubation with the cells. Endocytosis, and consequently infection, of HRV89 but not of HRV2, was almost completely blocked by the actin-polymerization inhibitor cytochalasin D, while the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on infection with either virus. Cytochalasin D also inhibited major-group HRV infection of rhabdomyosarcoma cells expressing ICAM-1 when the time available for uncoating was limited to 30 min. Although cholesterol depletion strongly inhibited HRV89 infection of HeLa cells, it only slightly affected HRV89 endocytosis, indicating that a lipid raft environment was not essential for virus uptake. The sodium-proton exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) significantly reduced cell entry and infection by HRV89 only at a concentration that also inhibited HRV2 infection and Alexa 488-transferrin entry. These data rule out classical macropinocytosis as an infectious entry pathway of HRV89 in HeLa cells. Notably, the proton ATPase inhibitor bafilomycin strongly affected cell entry of both viruses, suggesting a role for submembraneous pH in rhinovirus endocytosis.