Organization Of Actin Cytoskeleton Research Articles

Screening for Novel Regulators of Wingless Signaling

The Wingless (Wg) pathway within Drosophila melanogaster is a well characterized intercellular signaling network that has been implicated in tissue specific cellular differentiation. However, the mechanism by which Wg signaling contributes to tissue specific changes in gene expression is still unclear. Previously, the Kennell Lab conducted a series of misexpression screens in order to identify novel regulators of Wg signaling. Wg signaling alters gene expression in various tissues through the action of the transcriptional coactivator Armadillo (Arm). As such, the Kennell lab was able to identify potential regulators of Wg signaling during eye development by measuring whether the alteration of a gene's expression using UAS enhancers had any effect on the development of an Arm‐dependent small‐eye phenotype. Once genes known to cause apoptosis independent of Wg signaling were discarded from the list of genes that caused suppression of the small‐eye phenotype, fifteen promising candidates remained. Of these identified genes, siz and CG5902 were selected for further in vivo characterization. The gene CG5902 has never before been characterized and the gene siz has been implicated in actin cytoskeleton organization and central nervous system development. The gene CG5902 was successfully subcloned into expression vectors and transfected into Kc167 cells in order to test Arm target gene expression using reporter gene assays. Additionally, siz and CG5902 knockdown strains of Drosophila melanogaster are currently being created in order to further characterize the effect of these genes on Arm target gene expression and eye development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Study of potential biomedical application of sol-gel derived Zn-doped SiO2-hydroxypropyl cellulose nanohybrids

A series of Zn-doped hybrid materials based on silica from tetraethoxysilane (TEOS) and hydroxypropyl cellulose (HPC) were prepared by a sol-gel route. The structure, morphology and thermal behavior of synthesized hybrids were characterized by infrared (IR) spectroscopy, ultraviolet-visible spectroscopy (UV–Vis), transmission electron microscopy (TEM) and differential thermal analysis with thermogravimetric analysis (DTA/TG). The obtained materials were investigated for a potential biomedical application. The antibacterial properties of hybrids were investigated by measuring the inhibition zones formed around the materials containing different zinc content in presence of reference strains of Gram-positive and Gram-negative bacteria. The biocompatibility tests showed no cytotoxicity and genotoxicity, as well as no changes in actin cytoskeleton organization for hybrids with Zn content below 5 wt%.

NOTCH Signaling Is Activated through Mechanical Strain in Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Skeletal development and remodeling of adult bone are critically controlled by activated NOTCH signaling in genetically modified mice. It is yet unclear whether NOTCH signaling is activated by mechanical strain sensed by bone cells. We found that expression of specific NOTCH target genes is induced after in vivo tibial mechanical loading in wild-type mice. We further applied mechanical strain through cyclic stretching in human bone marrow-derived mesenchymal stromal cells (BMSCs) in vitro by using a bioreactor system and detected upregulation of NOTCH target gene expression. Inhibition of the NOTCH pathway in primary BMSCs as well as telomerase-immortalized human BMSCs (hMSC-TERT) through the gamma-secretase inhibitor GSI XII blocked mechanotransduction and modulated actin cytoskeleton organization. Short-hairpin RNA gene silencing identified NOTCH2 as the key receptor mediating NOTCH effects on hMSC-TERT cells. Our data indicate a functional link between NOTCH activation and mechanotransduction in human BMSCs. We suggest that NOTCH signaling is an important contributor to molecular mechanisms that mediate the bone formation response to mechanical strain.

Depletion of β3-adrenergic receptor induces left ventricular diastolic dysfunction via potential regulation of energy metabolism and cardiac contraction

Left ventricular diastolic dysfunction (LVDD) is a central perturbation in heart failure with preserved ejection fraction, and there are currently no effective remedies to improve LVDD in clinical practice. The β3-adrenergic receptor (ADRB3) was reported to play protective effects on inhibiting myocardial fibrosis in response to hemodynamic stress. However, the effects of ADRB3 on LVDD and its underlying mechanisms are still undefined. In the current study, the role of ADRB3 in LVDD was identified in ADRB3-knockout mice. Echocardiography parameters showed that depletion of ADRB3 had little effect on cardiac systolic function but obviously led to cardiac diastolic dysfunction in vivo. Proteomics (including the global proteome, phosphorylated and acetylated proteome) and bioinformatics analysis (including GO analysis, KEGG pathway analysis, GO-Tree network, Pathway-Act network, and protein-protein interaction network) were performed on cardiac specimens of ADRB3-KO mice and wild-type mice. The results showed that the cardiac energy metabolism (especially the citrate cycle), actin cytoskeleton organization, and cardiac muscle contraction (related to mitogen-activated protein kinase, toll-like receptor, and ErbB signalling pathway) were potential core mechanisms underlying ADRB3-KO–induced LVDD. In addition, the protein-protein interaction network indicated that the core proteins associated with ADRB3-KO–induced LVDD were FGG, ALDH1A1, FGA, APOC3, SLC4A1, SERPINF2, HP, CTNNB1, and TKT. In conclusion, the absence of ADRB3 leads to LVDD, which is potentially associated with the regulation of cardiac energy metabolism, actin cytoskeleton organization, and cardiac muscle contraction.

A Comparison of sun, ovate, fs8.1 and Auxin Application on Tomato Fruit Shape and Gene Expression.

Elongated tomato fruit shape is the result of the action of the fruit shape genes possibly in coordination with the phytohormone auxin. To investigate the possible link between auxin and the fruit shape genes, a series of auxin (2,4-D) treatments were performed on the wild-type and the fruit shape near-isogenic lines (NILs) in Solanum pimpinellifolium accession LA1589 background. Morphological and histological analyses indicated that auxin application approximately 3 weeks before anthesis led to elongated pear-shaped ovaries and fruits, which was mainly attributed to the increase of ovary/fruit proximal end caused by the increase of both cell number and cell size. Fruit shape changes caused by SUN, OVATE and fs8.1 were primarily due to the alterations of cell number along different growth axes. Particularly, SUN caused elongation by extending cell number along the entire proximal-distal axis, whereas OVATE caused fruit elongation in the proximal area, which was most similar to the effect of auxin on ovary shape. Expression analysis of flower buds at different stages in fruit shape NILs indicated that SUN had a stronger impact on the transcriptome than OVATE and fs8.1. The sun NIL differentially expressed genes were enriched in several biological processes, such as lipid metabolism, ion transmembrane and actin cytoskeleton organization. Additionally, SUN also shifted the expression of the auxin-related genes, including those involved in auxin biosynthesis, homeostasis, signal transduction and polar transport, indicating that SUN may regulate ovary/fruit shape through modifying the expression of auxin-related genes very early during the formation of the ovary in the developing flower.

Endothelial Differentiation G Protein-Coupled Receptor 5 Plays an Important Role in Induction and Maintenance of Pluripotency.

Direct reprogramming of human somatic cells toward induced pluripotent stem cells holds great promise for regenerative medicine and basic biology. We used a high‐throughput small interfering RNA screening assay in the initiation phase of reprogramming for 784 genes belonging to kinase and phosphatase families and identified 68 repressors and 22 effectors. Six new candidates belonging to the family of the G protein‐coupled receptors (GPCRs) were identified, suggesting an important role for this key signaling pathway during somatic cell‐induced reprogramming. Downregulation of one of the key GPCR effectors, endothelial differentiation GPCR5 (EDG5), impacted the maintenance of pluripotency, actin cytoskeleton organization, colony integrity, and focal adhesions in human embryonic stem cells, which were associated with the alteration in the RhoA‐ROCK‐Cofilin‐PAXILLIN‐actin signaling pathway. Similarly, downregulation of EDG5 during the initiation stage of somatic cell‐induced reprogramming resulted in alteration of cytoskeleton, loss of human‐induced pluripotent stem cell colony integrity, and a significant reduction in partially and fully reprogrammed cells as well as the number of alkaline phosphatase positive colonies at the end of the reprogramming process. Together, these data point to an important role of EDG5 in the maintenance and acquisition of pluripotency. Stem Cells 2019;37:318–331

SHROOM2 inhibits tumor metastasis through RhoA\u2013ROCK pathway-dependent and -independent mechanisms in nasopharyngeal carcinoma

SHROOM2 is a key mediator of RhoA–ROCK pathway that regulates cell motility and actin cytoskeleton organization. However, the functions of SHROOM2 beyond RhoA/ROCK signaling remain poorly understood. Here, we report that SHROOM2 not only participates in RhoA–ROCK-induced stress fiber formation and focal adhesion, but also had an unanticipated role in suppressing epithelial-to-mesenchymal transition (EMT) and tumor metastasis. Depletion of SHROOM2 in nasopharyngeal carcinoma (NPC) cells enhances mesenchymal characteristics and reduces epithelial markers, concomitant with increased motility, enabling the development of invasion and tumor metastasis, which are largely ROCK-independent, as ROCK inhibitor Y-27632 did not cause EMT phenotype; furthermore, combination of ROCK inhibition and SHROOM2 depletion resulted in the most robust increases in cell migration and invasion, indicating that SHROOM2 and ROCK work synergistically rather than epistatic. Analysis of clinical samples suggested that SHROOM2 is downregulated in NPC and the expression of SHROOM2 in metastatic NPC was even lower than in the primary tumors. Our findings uncover a non-canonical role of SHROOM2 as a potent antagonist for EMT and NPC metastasis.

Neutral endopeptidase inhibitors blunt kidney fibrosis by reducing myofibroblast formation.

Kidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides have been demonstrated to have cyclic guanosine monophosphate (cGMP)-dependent anti-fibrotic properties likely due to interference with pro-fibrotic tissue growth factor β (TGF-β) signaling. However, in vivo, natriuretic peptides are rapidly degraded by neutral endopeptidases (NEP). In a unilateral ureteral obstruction (UUO) mouse model for kidney fibrosis we assessed the anti-fibrotic effects of SOL1, an orally active compound that inhibits NEP and endothelin-converting enzyme (ECE). Mice (n=10 per group) subjected to UUO were treated for 1 week with either solvent, NEP-/ECE-inhibitor SOL1 (two doses), reference NEP-inhibitor candoxatril or the angiotensin II receptor type 1 (AT1)-antagonist losartan. While NEP-inhibitors had no significant effect on blood pressure, they did increase urinary cGMP levels as well as endothelin-1 (ET-1) levels. Immunohistochemical staining revealed a marked decrease in renal collagen (∼55% reduction, P<0.05) and α-smooth muscle actin (α-SMA; ∼40% reduction, P<0.05). Moreover, the number of α-SMA positive cells in the kidneys of SOL1-treated groups inversely correlated with cGMP levels consistent with a NEP-dependent anti-fibrotic effect. To dissect the molecular mechanisms associated with the anti-fibrotic effects of NEP inhibition, we performed a 'deep serial analysis of gene expression (Deep SAGE)' transcriptome and targeted metabolomics analysis of total kidneys of all treatment groups. Pathway analyses linked increased cGMP and ET-1 levels with decreased nuclear receptor signaling (peroxisome proliferator-activated receptor [PPAR] and liver X receptor/retinoid X receptor [LXR/RXR] signaling) and actin cytoskeleton organization. Taken together, although our transcriptome and metabolome data indicate metabolic dysregulation, our data support the therapeutic potential of NEP inhibition in the treatment of kidney fibrosis via cGMP elevation and reduced myofibroblast formation.

3,4,5-Tricaffeoylquinic acid induces adult neurogenesis and improves deficit of learning and memory in aging model senescence-accelerated prone 8 mice.

Caffeoylquinic acid (CQA) is a natural polyphenol with evidence of antioxidant and neuroprotective effects and prevention of deficits in spatial learning and memory. We studied the cognitive-enhancing effect of 3,4,5-tricaffeoylquinic acid (TCQA) and explored its cellular and molecular mechanism in the senescence-accelerated mouse prone 8 (SAMP8) model of aging and Alzheimer’s disease as well as in human neural stem cells (hNSCs). Mice were fed with 5 mg/kg of TCQA for 30 days and were tested in the Morris water maze (MWM). Brain tissues were collected for immunohistochemical detection of bromodeoxyuridine (BrdU) to detect activated stem cells and newborn neurons. TCQA-treated SAMP8 exhibited significantly improved cognitive performance in MWM compared to water-treated SAMP8. TCQA-treated SAMP8 mice also had significantly higher numbers of BrdU+/glial fibrillary acidic protein (GFAP+) and BrdU+/Neuronal nuclei (NeuN+) cells in the dentate gyrus (DG) neurogenic niche compared with untreated SAMP8. In hNSCs, TCQA induced cell cycle arrest at G0/G1, actin cytoskeleton organization, chromatin remodeling, neuronal differentiation, and bone morphogenetic protein signaling. The neurogenesis promoting effect of TCQA in the DG of SAMP8 mice might explain the cognition-enhancing influence of TCQA observed in our study, and our hNSCs in aggregate suggest a therapeutic potential for TCQA in aging-associated diseases.

Cdc42: A Novel Regulator of Insulin Secretion and Diabetes-Associated Diseases.

Cdc42, a member of the Rho GTPases family, is involved in the regulation of several cellular functions including cell cycle progression, survival, transcription, actin cytoskeleton organization and membrane trafficking. Diabetes is a chronic and metabolic disease, characterized as glycometabolism disorder induced by insulin deficiency related to β cell dysfunction and peripheral insulin resistance (IR). Diabetes could cause many complications including diabetic nephropathy (DN), diabetic retinopathy and diabetic foot. Furthermore, hyperglycemia can promote tumor progression and increase the risk of malignant cancers. In this review, we summarized the regulation of Cdc42 in insulin secretion and diabetes-associated diseases. Organized researches indicate that Cdc42 is a crucial member during the progression of diabetes, and Cdc42 not only participates in the process of insulin synthesis but also regulates the insulin granule mobilization and cell membrane exocytosis via activating a series of downstream factors. Besides, several studies have demonstrated Cdc42 as participating in the pathogenesis of IR and DN and even contributing to promote cancer cell proliferation, survival, invasion, migration, and metastasis under hyperglycemia. Through the current review, we hope to cast light on the mechanism of Cdc42 in diabetes and associated diseases and provide new ideas for clinical diagnosis, treatment, and prevention.

Novel sulphamoylated 2-methoxy estradiol derivatives inhibit breast cancer migration by disrupting microtubule turnover and organization

BackgroundThe estrogen metabolite 2-methoxyestradiol (2ME2) and a number of synthesised derivatives have been shown to bind to microtubules thereby arresting cancer cells in mitosis which leads to apoptosis. In interphase cells, microtubules play an important role in the delivery of proteins to subcellular locations including the focal adhesions. In fact, focal adhesion dynamics and cell migration are in part regulated by microtubules. We hypothesised that novel 2ME2 derivatives can alter cell migration by influencing microtubule dynamics in interphase cells. In this report we describe 2ME2 derivatives that display anti-migratory capabilities in a metastatic breast cancer cell line through their effects on the microtubule network resulting in altered focal adhesion signalling and RhoA activity.MethodsCell migration was assayed using wound healing assays. To eliminate mitosis blockage and cell rounding as a confounding factor cell migration was also assessed in interphase blocked cells. Fluorescence confocal microscopy was used to visualise microtubule dynamics and actin cytoskeleton organisation while western blot analysis was performed to analyse focal adhesion signalling and RhoA activation.Results2ME2 derivatives, ESE-one and ESE-15-one, inhibited cell migration in cycling cells as expected but equally diminished migration in cells blocked in interphase. While no significant effects were observed on the actin cytoskeleton, focal adhesion kinase activity was increased while RhoA GTPase activity was inhibited after exposure to either compound. Microtubule stability was increased as evidenced by the increased length and number of detyrosinated microtubules while at the same time clear disorganisation of the normal radial microtubule organisation was observed including multiple foci.ConclusionsESE-15-one and ESE-one are potent migration inhibitors of metastatic breast cancer cells. This ability is coupled to alterations in focal adhesion signalling but more importantly is associated with severe disorganisation of microtubule dynamics and polarity. Therefore, these compounds may offer potential as anti-metastatic therapies.

Yeast-model-based study identified myosin- and calcium-dependent calmodulin signalling as a potential target for drug intervention in chorea-acanthocytosis.

ABSTRACTChorea-acanthocytosis (ChAc) is a rare neurodegenerative disease associated with mutations in the human VPS13A gene. The mechanism of ChAc pathogenesis is unclear. A simple yeast model was used to investigate the function of the single yeast VSP13 orthologue, Vps13. Vps13, like human VPS13A, is involved in vesicular protein transport, actin cytoskeleton organisation and phospholipid metabolism. A newly identified phenotype of the vps13Δ mutant, sodium dodecyl sulphate (SDS) hypersensitivity, was used to screen a yeast genomic library for multicopy suppressors. A fragment of the MYO3 gene, encoding Myo3-N (the N-terminal part of myosin, a protein involved in the actin cytoskeleton and in endocytosis), was isolated. Myo3-N protein contains a motor head domain and a linker. The linker contains IQ motifs that mediate the binding of calmodulin, a negative regulator of myosin function. Amino acid substitutions that disrupt the interaction of Myo3-N with calmodulin resulted in the loss of vps13Δ suppression. Production of Myo3-N downregulated the activity of calcineurin, a protein phosphatase regulated by calmodulin, and alleviated some defects in early endocytosis events. Importantly, ethylene glycol tetraacetic acid (EGTA), which sequesters calcium and thus downregulates calmodulin and calcineurin, was a potent suppressor of vps13Δ. We propose that Myo3-N acts by sequestering calmodulin, downregulating calcineurin and increasing activity of Myo3, which is involved in endocytosis and, together with Osh2/3 proteins, functions in endoplasmic reticulum-plasma membrane contact sites. These results show that defects associated with vps13Δ could be overcome, and point to a functional connection between Vps13 and calcium signalling as a possible target for chemical intervention in ChAc. Yeast ChAc models may uncover the underlying pathological mechanisms, and may also serve as a platform for drug testing.This article has an associated First Person interview with the first author of the paper.

Intracellular Galectin-9 Controls Dendritic Cell Function by Maintaining Plasma Membrane Rigidity

Extracellular Galectins constitute a novel mechanism of membrane protein organisation at the cell surface. Although Galectins are also highly expressed intracellularly, their cytosolic functions are poorly understood. Here, we investigated the role of Galectin-9 in dendritic cell (DC) surface organisation and function. By combining functional, super-resolution and atomic force microscopy experiments to analyse membrane stiffness, we identified intracellular Galectin-9 to be indispensable for plasma membrane integrity and structure in DCs. Galectin-9 knockdown studies revealed intracellular Galectin-9 to directly control cortical membrane structure via modulating Rac1 activity, providing the underlying mechanism of Galectin-9-dependent actin cytoskeleton organisation. Consequent to its role in maintaining plasma membrane structure, phagocytosis studies revealed that Galectin-9 was essential for C-type lectin receptor-mediated pathogen uptake by human DCs. This was confirmed by the impaired phagocytic capacity of Galectin-9-null murine DCs. Together, this study demonstrates a novel role for intracellular Galectin-9 in modulating DC function, which may be evolutionary conserved.

Tropomyosin Tpm 2.1 loss induces glioblastoma spreading in soft brain-like environments.

The brain is a very soft tissue. Glioblastoma (GBM) brain tumours are highly infiltrative into the surrounding healthy brain tissue and invasion mechanisms that have been defined using rigid substrates therefore may not apply to GBM dissemination. GBMs characteristically lose expression of the high molecular weight tropomyosins, a class of actin-associating proteins and essential regulators of the actin stress fibres and focal adhesions that underpin cell migration on rigid substrates. Here, we investigated how loss of the high molecular weight tropomyosins affects GBM on soft matrices that recapitulate the biomechanical architecture of the brain. We find that Tpm 2.1 is down-regulated in GBM grown on soft substrates. We demonstrate that Tpm 2.1 depletion by siRNA induces cell spreading and elongation in soft 3D hydrogels, irrespective of matrix composition. Tpm 1.7, a second high molecular weight tropomyosin is also down-regulated when cells are cultured on soft brain-like surfaces and we show that effects of this isoform are matrix dependent, with Tpm 1.7 inducing cell rounding in 3D collagen gels. Finally, we show that the absence of Tpm 2.1 from primary patient-derived GBMs correlates with elongated, mesenchymal invasion. We propose that Tpm 2.1 down-regulation facilitates GBM colonisation of the soft brain environment. This specialisation of the GBM actin cytoskeleton organisation that is highly suited to the soft brain-like environment may provide novel therapeutic targets for arresting GBM invasion.

Contribution of the Rho-kinase to Systemic Sclerosis and Behçet's Disease.

The small GTPase Rho family and its effectors, Rho-kinases (ROCK) play essential roles in the actin cytoskeleton organization and coordinate a broad range of cellular functions, such as inflammatory responses, cell contractility, migration, adhesion, proliferation, and apoptosis. The goal of this work is to review existing literature about systemic sclerosis and Behçet's disease in relation to ROCK. There are some evidence that ROCK expression is elevated in patients with systemic sclerosis and Behçet's disease. Rho/ROCK gene polymorphisms have been shown to be associated with these disorders. Endothelial function is also impaired in these autoimmune diseases. Rho/Rho-kinase pathway might have a crucial role in endothelial, vascular, and fibrotic pathologies. Dysregulation in the Rho/ROCK pathway may represent a common pathogenic mechanism in multiple autoimmune disorders. Current evidence indicate that Rho/ROCK genes might be risk factors, and can contribute to susceptibility and development of systemic sclerosis and Behçet's disease. These studies may also provide important insights into the future development or use of potential novel therapeutic approaches, such as selective Rho-kinase inhibitors, for the treatment of patients with systemic sclerosis and Behçet's disease.

Phenotypic spectrum associated with SPECC1L pathogenic variants: new families and critical review of the nosology of Teebi, Opitz GBBB, and Baraitser-Winter syndromes

The SPECC1L protein plays a role in adherens junctions involved in cell adhesion, actin cytoskeleton organization, microtubule stabilization, spindle organization and cytokinesis. It modulates PI3K-AKT signaling and controls cranial neural crest cell delamination during facial morphogenesis. SPECC1L causative variants were first identified in individuals with oblique facial clefts. Recently, causative variants in SPECC1L were reported in a pedigree reported in 1988 as atypical Opitz GBBB syndrome. Six families with SPECC1L variants have been reported thus far. We report here eight further pedigrees with SPECC1L variants, including a three-generation family, and a further individual of a previously published family. We discuss the nosology of Teebi and GBBB, and the syndromes related to SPECC1L variants. Although the phenotype of individuals with SPECC1L mutations shows overlap with Opitz syndrome in its craniofacial anomalies, the canonical laryngeal malformations and male genital anomalies are not observed. Instead, individuals with SPECCL1 variants have branchial fistulae, omphalocele, diaphragmatic hernias, and uterus didelphis. We also point to the clinical overlap of SPECC1L syndrome with mild Baraitser-Winter craniofrontofacial syndrome: they share similar dysmorphic features (wide, short nose with a large tip, cleft lip and palate, blepharoptosis, retrognathia, and craniosynostosis), although intellectual disability, neuronal migration defect, and muscular problems remain largely specific to Baraitser-Winter syndrome. In conclusion, we suggest that patients with pathogenic variants in SPECC1L should not be described as “dominant (or type 2) Opitz GBBB syndrome”, and instead should be referred to as “SPECC1L syndrome” as both disorders show distinctive, non overlapping developmental anomalies beyond facial communalities.

A novel non-sulphamoylated 2-methoxyestradiol derivative causes detachment of breast cancer cells by rapid disassembly of focal adhesions

Background2-Methoxyestradiol (2ME2) is an estradiol metabolite with well documented antiproliferative properties in many cancer cell lines. However, it is rapidly metabolised in vivo which limits its clinical application. Therefore, more stable derivatives with potentially improved clinical features have been designed by our group. Here we describe an estrone-like derivative of 2ME2, namely EE-15-one, that unlike other derivatives which induce cell cycle arrest, induces a rapid loss of cell–substrate adhesion through the inactivation and disassembly of focal adhesions.MethodsTo assess the effect of 2-ethyl-estra-1,3,5 (10),15-tetraen-3-ol-17-one (EE-15-one) on breast cancer cell lines, cell survival was quantified. The effect of EE-15-one on cell attachment was assessed by measuring cell adhesion and cell rounding via light microscopy. Effects on focal adhesion dynamics and actin cytoskeleton organisation were visualised by immunofluorescence while focal adhesion signalling was assessed by western blot. Cell death was quantified using a lactate dehydrogenase activity (LDH) assay. To investigate specificity towards cell–substrate over cell–cell contact inhibition, EE-15-one effects on 3D cell cultures were assessed.ResultsCell survival assays show an almost complete loss of cells within 24 h of EE-15-one exposure in contrast to published sulphamoylated 2ME2 derivatives. Cell loss is linked to rapid detachment and adhesion inhibition. Focal adhesion size and number are rapidly diminished while actin fibres became severed and disappeared within 2 h post exposure. These changes were not due to cell necrosis as LDH activity only slightly increased after 24 h. Cells grown in cell–cell adhesion dependent spheroids did not respond to EE-15-one exposure suggesting that EE-15-one specifically inhibits cell–substrate adhesions but not cell–cell adhesions and does not directly impact the actin cytoskeleton.ConclusionWe show that a novel 2ME2 derivative, EE-15-one, induces rapid loss of focal adhesion function leading to cell–substrate detachment through interference with integrin-based cell–substrate adhesions, but not cadherin dependent cell–cell adhesions. Therefore, EE-15-one is the first 2ME2 derivative that has an alternative mode of action to the antimitotic activity of 2ME2. As such EE-15-one shows potential as a lead compound for further development as an inhibitor of cell–substrate adhesion which is essential for metastatic dissemination.

Recessive mutations in the neuronal isoforms of DST, encoding dystonin, lead to abnormal actin cytoskeleton organization and HSAN type VI.

Hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders, characterized by a progressive sensory neuropathy often complicated by ulcers and amputations, with variable motor and autonomic involvement. Several pathways have been implicated in the pathogenesis of neuronal degeneration in HSAN, while recent observations point to an emerging role of cytoskeleton organization and function. Here, we report novel biallelic mutations in the DST gene encoding dystonin, a large cytolinker protein of the plakin family, in an adult form of HSAN type VI. Affected individuals harbored the premature termination codon variant p.(Lys4330*) in trans with the p.(Ala203Glu) change affecting a highly conserved residue in an isoform-specific N-terminal region of dystonin. Functional studies showed defects in actin cytoskeleton organization and consequent delayed cell adhesion, spreading and migration, while recombinant p.Ala203Glu dystonin loses the ability to bind actin. Our data aid in the clinical and molecular delineation of HSAN-VI and suggest a central role for cell-motility and cytoskeletal defects in its pathogenesis possibly interfering with the neuronal outgrowth and guidance processes.

The Impact of ESCRT on Aβ1-42 Induced Membrane Lesions in a Yeast Model for Alzheimer's Disease.

Aβ metabolism plays a pivotal role in Alzheimer’s disease. Here, we used a yeast model to monitor Aβ42 toxicity when entering the secretory pathway and demonstrate that processing in, and exit from the endoplasmic reticulum (ER) is required to unleash the full Aβ42 toxic potential. Consistent with previously reported data, our data suggests that Aβ42 interacts with mitochondria, thereby enhancing formation of reactive oxygen species and eventually leading to cell demise. We used our model to search for genes that modulate this deleterious effect, either by reducing or enhancing Aβ42 toxicity, based on screening of the yeast knockout collection. This revealed a reduced Aβ42 toxicity not only in strains hampered in ER-Golgi traffic and mitochondrial functioning but also in strains lacking genes connected to the cell cycle and the DNA replication stress response. On the other hand, increased Aβ42 toxicity was observed in strains affected in the actin cytoskeleton organization, endocytosis and the formation of multivesicular bodies, including key factors of the ESCRT machinery. Since the latter was shown to be required for the repair of membrane lesions in mammalian systems, we studied this aspect in more detail in our yeast model. Our data demonstrated that Aβ42 heavily disturbed the plasma membrane integrity in a strain lacking the ESCRT-III accessory factor Bro1, a phenotype that came along with a severe growth defect and enhanced loading of lipid droplets. Thus, it appears that also in yeast ESCRT is required for membrane repair, thereby counteracting one of the deleterious effects induced by the expression of Aβ42. Combined, our studies once more validated the use of yeast as a model to investigate fundamental mechanisms underlying the etiology of neurodegenerative disorders.

The effect of cyclic tensile force on the actin cytoskeleton organization and morphology of human periodontal ligament cells

To explore Girdin/Akt pathway protein expression and morphology change by cyclic tension in the periodontal ligament cells. Human periodontal ligament cells were exposed to cyclic tension force at 4000 μstrain and 0.5 Hz for 6 h though a four-point bending system. Cyclic tension force upregulated F-actin, Girdin and Akt expression in hPDL. In transmission electron microscope assay showed that there are more and bigger mitochondria, more and longer cynapses, more cellular organisms after tension force stimulation than control. The actin filament was changed to be regular lines and pointed to poles of cells. However, we found that the Girdin-depleted cells are small and there are more micro-organisms including more lysosomes and matrix vesicles than control. These finding suggest that the STAT3/Girdin/Akt pathway in PDL to response to mechanical stimulation as well, and Girdin may play a significant role in triggering cell proliferation and migration during orthodontic treatment. It provided an insight into the molecular basis for development of a vitro cell model in studying orthodontic treatment.