Alterations In Actin Cytoskeleton Research Articles

Altered actin cytoskeleton in ageing eggs of starfish affects fertilization process

Integrity of oocytes is of pivotal interest in the medical and zootechnical practice of in vitro fertilization. With time, oocytes undergo deterioration in quality, and ageing oocytes often exhibit compromised competence in fertilization and the subsequent embryonic development. With ageing oocytes and eggs of starfish (Astropecten aranciacus), we addressed the issue by examining changes of the subcellular structure and their performance at fertilization. Ageing eggs were simulated in two different experimental paradigms: i) oocytes were overmatured by 6 hours stimulation with 1-methyladenine (1-MA); ii) oocytes were removed from the gonad and maintained in seawater for 24 or 48 h before applying the hormonal stimulation (1-MA, 70 min). These eggs were compared with normally matured eggs (stimulated after isolation from the gonad with 1-MA for 70 min) with respect to the sperm-induced intracellular Ca2+ signaling and the structural changes of the egg surface. The cytoskeletal and ultrastructural differences in these eggs were assessed by confocal and transmission electron microscopy, respectively. In the two categories of ageing eggs, we have found remarkable structural modifications of the actin cytoskeleton and the cortical vesicles beneath the plasma membrane. At fertilization, these ageing eggs manifested an altered pattern of intracellular Ca2+ release, aberrant actin dynamics, and increased rate of polyspermy often despite full elevation of the fertilization envelope. Taken together, our results highlight the importance of spatio-temporal regulation of the actin cytoskeleton in the cortex of the eggs, and we postulate that the status of the actin cytoskeleton is one of the major determinants of the oocyte quality that ensures successful monospermic fertilization.

Disruption of podocyte cytoskeletal biomechanics by dasatinib leads to nephrotoxicity

Nephrotoxicity is a critical adverse event that leads to discontinuation of kinase inhibitor (KI) treatment. Here we show, through meta-analyses of FDA Adverse Event Reporting System, that dasatinib is associated with high risk for glomerular toxicity that is uncoupled from hypertension, suggesting a direct link between dasatinib and podocytes. We further investigate the cellular effects of dasatinib and other comparable KIs with varying risks of nephrotoxicity. Dasatinib treated podocytes show significant changes in focal adhesions, actin cytoskeleton, and morphology that are not observed with other KIs. We use phosphoproteomics and kinome profiling to identify the molecular mechanisms of dasatinib-induced injury to the actin cytoskeleton, and atomic force microscopy to quantify impairment to cellular biomechanics. Furthermore, chronic administration of dasatinib in mice causes reversible glomerular dysfunction, loss of stress fibers, and foot process effacement. We conclude that dasatinib induces nephrotoxicity through altered podocyte actin cytoskeleton, leading to injurious cellular biomechanics.

Cadmium induces actin cytoskeleton alterations and dysfunction in Neuro-2a cells.

Cadmium (Cd) is considered a possible etiological factor in neurodegenerative diseases. However, the exact mechanism by which Cd induces neurotoxicity is not well elucidated. In this study, Neuro-2a cells were treated with 0, 10, 20, and 40 μM cadmium chloride for 24 hours to investigate the effects of Cd on the cytoskeleton of nerve cells. MTT assay and ELISA assay were used to examine cell viability and release of lactate dehydrogenase (LDH) from cells, respectively. Results showed that Cd reduced cell viability and increased the release of LDH in a dose-dependent manner (P < 0.05). The morphology of treated cell was damaged as indicated by cell collapse and dimensionality reduction. Moreover, the axonal spines and normal features of Cd-treated neurons disappeared. We checked the ultrastructure of Neuro-2a cells and found that Cd-induced swelling, membrane damage, overflow of cytoplasm contents, and cell fragmentation. Damaged mitochondria, expanded endoplasmic reticulum, and abnormal microfilaments were found in Cd-treated cells rather than in untreated cells. Compared with the control group, the relative release of glutamate in the supernatant after Cd treatment was reduced, indicating that Cd exposure could reduce the release of glutamate by inhibiting the function of nerve-2a cells. Cd decreased the mRNA and protein expression levels of cytoskeletal proteins including DBN, SYP, and TAU, which might promote cytoskeleton alterations in Cd-treated cells. In conclusion, Cd-induced actin cytoskeleton alterations and dysfunction of cultured neurons. The results of the present study provide new insights for the investigation of Cd-induced neurotoxicity.

Stimuli-responsive polymer-doxorubicin conjugate: Antitumor mechanism and potential as nano-prodrug

Polymer-drug conjugates has significantly improved the anti-tumor efficacy of chemotherapeutic drugs and alleviated their side effects. N-(1,3-dihydroxypropan-2-yl) methacrylamide (DHPMA) copolymer was synthesized via RAFT polymerization and polymer-doxorubicin (DOX) (diblock pDHPMA-DOX) were formed by conjugation, resulting in a self-aggregation-induced nanoprodrug with a favorable size of 21 nm and great stability. The nanoprodrug with a molecular weight (MW) of 95 kDa released drugs in response to tumor microenvironmental pH variations and they were enzymatically hydrolyzed into low MW segments (45 kDa). The nanoprodrug was transported through the endolysosomal pathway, released the drug into the cytoplasm and some was localized in the mitochondria, resulting in disruption of the cellular actin cytoskeleton. Cellular apoptosis was also associated with reduction in the mitochondrial potential caused by the nanoprodrug. Notably, the nanoprodrug had a significantly prolonged blood circulation time with an elimination half time of 9.8 h, displayed high accumulation within tumors, and improved the in vivo therapeutic efficacy against 4T1 xenograft tumors compared to free DOX. The tumor xenograft immunohistochemistry study clearly indicated tumor inhibition was through the inhibition of cell proliferation and antiangiogenic effects. Our studies demonstrated that the diblock pDHPMA-DOX nanoprodrug with a controlled molecular structure is promising to alleviate adverse effects of free DOX and have a great potential as an efficient anticancer agent. Statement of SignificanceIn this work, we prepared a biodegradable diblock DHPMA polymer-doxorubicin conjugate via one-pot of RAFT polymerization and conjugate chemistry. The conjugate-based nanoprodrug was internalized by endocytosis to intracellularly release DOX and further induce disruption of mitochondrial functions, actin cytoskeleton alterations and cellular apoptosis. The nanoprodrug with a high molecular weight (MW) (95 kDa) showed a long blood circulation time and achieved high accumulation into tumors. The nanoprodrug was degraded into low MW (∼45 kDa) products below the renal threshold, which ensured its biosafety. Additionally, the multi-stimuli-responsive nanoprodrug demonstrated an enhanced antitumor efficacy against 4T1 breast tumors and alleviated side effects, showing a great potential as an efficient and safe anticancer agent.

Specific and Targetable Interactions with the Bone Marrow Microenvironment Govern Outcome in Imatinib-Resistant Chronic Myeloid Leukemia

Mutations within BCR-ABL1, the oncogene giving rise to chronic myeloid leukaemia (CML), can lead to resistance to tyrosine kinase inhibitors and some are associated with clinically more aggressive disease and worse outcome (Branford et al., 2003).This phenomenon was faithfully recapitulated in our murine transduction/transplantation model of BCR-ABL1+ CML, in which recipients of bone marrow transduced with the imatinib-resistant BCR-ABL1 point mutant T315I (or Y253F) had shortened survival, compared to wild-type BCR-ABL1. Consistent with the situation in humans we found an increase in the number of blast-like cells and alterations of the immunophenotypic composition of cells in the bone marrow of murine recipients of BCR-ABL1T315I+ leukaemia-initiating cells (LIC).To understand the interaction between BCR-ABL1+ LIC and the bone marrow microenvironment (BMM), a complex entity of cellular and acellular constituents, which may influence the course of the disease, we used a combination of confocal and 2-photon intravital microscopy of the murine calvarium. BCR-ABL1T315I + Lin- c-Kit+ Sca-1+ (LKS) cells, which contain the leukaemic stem cell (LSC) fraction in this model, homed closer to osteoblastic cells thanLKS cells expressing native BCR-ABL1 which suggests a differential homing location of BCR-ABL1T315I+ LSC. Indeed, in a transwell migration assay we demonstrated faster migration of BCR-ABL1T315I+ compared toBCR-ABL1+ cells. Other experiments revealed an altered actin cytoskeleton and striking differences in focal adhesion kinase (FAK), a component of integrin mediated focal adhesion sites, in BCR-ABL1T315I+ compared to wildtype BCR-ABL1+ cells. Additionally, in in vivo and ex vivo studies we showed that integrin β3 was overexpressed in BCR-ABL1T315I+ compared to wildtype BCR-ABL1+ cells, and that further overexpression of integrin β3 significantly reduced the tumor load and prolonged the survival of mice with BCR-ABL1+, but especially BCR-ABL1T315I+ CML.Further, we found another component of the focal adhesion complex, namely integrin linked kinase (ILK) and its phosphorylated form (pS243) to be more highly expressed in BCR-ABL1T315I+ compared to BCR-ABL1+ wildtype cells. ShRNA-mediated knockdown of ILK in BCR-ABL1T315I+ LIC significantly reduced the tumor load and prolonged survival in recipient mice. Inhibition of ILK by the use of a commercially available (non clinical grade) inhibitor (Cpd22) in combination with the tyrosine kinase inhibitor ponatinib prolonged the survival of mice compared to treatment with ponatinib alone, further confirming an important role of ILK in BCR-ABL1T315I+ disease.Fibronectin is a known interaction partner of integrins in the extracellular matrix of the BMM and, as previously reported, its deposition in the extracellular matrix can be regulated by ILK. We found that the deposition of fibronectin in BCR-ABL1T315I + disease was decreased compared to BCR-ABL1+ wildtypecells. Intrafemoral or intravenous administration of fibronectin, however, led to a significant prolongation of survival in 70% of mice with BCR-ABL1T315I + CML, and knockdown of ILK in BCR-ABL1T315I + LIC led to a replenishment of fibronectin levels in the BMM.In confirmation of these murine data, bone sections of patients with BCR-ABL1T315I+ CML revealed decreased levels of fibronectin,and Western blot analysis of human BCR-ABL1T315I+ CML cellsshowed increased levels of ILK and pS243ILK compared to the BCR-ABL1+ controls.In summary, these data suggest that interactions with the BMM via the integrin β3/ILK-mediated signaling pathway may influence leukaemic progression and clinical outcome in BCR-ABL1T315I+ imatinib-resistant CML. Targeting the interaction of BCR-ABL1T315I+ leukemia cells with the BMM in the form of ILK inhibition or administration of fibronectin may offer a beneficial, innovative, adjunct treatment strategy for patients with BCR-ABL1T315I+ CML and, as to be tested, possibly also other leukaemias. DisclosuresKumar:European Patent No. 16187926.7: Patents & Royalties: European Patent No. 16187926.7-1401 “FIBRONECTIN FOR USE IN THE TREATMENT OF LEUKEMIA” . Nicolini:Sun Pharma Ltd: Consultancy; Incyte Biosciences: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Krause:Glycomimetics Inc. until 06/2016: Consultancy, Research Funding; Merck KgGA in future: Research Funding; Glycotope: Consultancy; European Patent: Patents & Royalties: European Patent No. 16187926.7-1401 “FIBRONECTIN FOR USE IN THE TREATMENT OF LEUKEMIA” .

The Ric-8A/Gα13/FAK signalling cascade controls focal adhesion formation during neural crest cell migration in Xenopus.

Ric-8A is a pleiotropic guanine nucleotide exchange factor involved in the activation of various heterotrimeric G-protein pathways during adulthood and early development. Here, we sought to determine the downstream effectors of Ric-8A during the migration of the vertebrate cranial neural crest (NC) cells. We show that the Gα13 knockdown phenocopies the Ric-8A morphant condition, causing actin cytoskeleton alteration, protrusion instability, and a strong reduction in the number and dynamics of focal adhesions. In addition, the overexpression of Gα13 is sufficient to rescue Ric-8A-depleted cells. Ric-8A and Gα13 physically interact and colocalize in protrusions of the cells leading edge. The focal adhesion kinase FAK colocalizes and interacts with the endogenous Gα13, and a constitutively active form of Src efficiently rescues the Gα13 morphant phenotype in NC cells. We propose that Ric-8A-mediated Gα13 signalling is required for proper cranial NC cell migration by regulating focal adhesion dynamics and protrusion formation.

Biological safety and tissue distribution of (16-mercaptohexadecyl)trimethylammonium bromide-modified cationic gold nanorods

The exceptionally high cellular uptake of gold nanorods (GNRs) bearing cationic surfactants makes them a promising tool for biomedical applications. Given the known specific toxic and stress effects of some preparations of cationic nanoparticles, the purpose of this study was to evaluate, in an in vitro and in vivo in mouse, the potential harmful effects of GNRs coated with (16-mercaptohexadecyl)trimethylammonium bromide (MTABGNRs). Interestingly, even after cellular accumulation of high amounts of MTABGNRs sufficient for induction of photothermal effect, no genotoxicity (even after longer-term accumulation), induction of autophagy, destabilization of lysosomes (dominant organelles of their cellular destination), alterations of actin cytoskeleton, or in cell migration could be detected in vitro. In vivo, after intravenous administration, the majority of GNRs accumulated in mouse spleen followed by lungs and liver. Microscopic examination of the blood and spleen showed that GNRs interacted with white blood cells (mononuclear and polymorphonuclear leukocytes) and thrombocytes, and were delivered to the spleen red pulp mainly as GNR-thrombocyte complexes. Importantly, no acute toxic effects of MTABGNRs administered as 10 or 50 μg of gold per mice, as well as no pathological changes after their high accumulation in the spleen were observed, indicating good tolerance of MTABGNRs by living systems.

Chronophin activation is necessary in Doxorubicin-induced actin cytoskeleton alteration

Although doxorubicin (Dox)-induced oxidative stress is known to be associated with cytotoxicity, the precise mechanism remains unclear. Genotoxic stress not only generates free radicals, but also affects actin cytoskeleton stability. We showed that Dox-induced RhoA signaling stimulated actin cytoskeleton alterations, resulting in central stress fiber disruption at early time points and cell periphery cortical actin formation at a later stage, in HeLa cells. Interestingly, activation of a cofilin phosphatase, chronophin (CIN), was initially evoked by Dox-induced RhoA signaling, resulting in a rapid phosphorylated cofilin turnover leading to actin cytoskeleton remodeling. In addition, a novel interaction between CIN and 14-3-3ζ was detected in the absence of Dox treatment. We demonstrated that CIN activity is quite contrary to 14-3-3ζ binding, and the interaction leads to enhanced phosphorylated cofilin levels. Therefore, initial CIN activation regulation could be critical in Dox-induced actin cytoskeleton remodeling through RhoA/cofilin signaling.

Downregulation of Profilin-1 Expression Attenuates Cardiomyocytes Hypertrophy and Apoptosis Induced by Advanced Glycation End Products in H9c2 Cells.

Cardiomyocytes hypertrophy and apoptosis induced by advanced glycation end products (AGEs) is the crucial pathological foundation contributing to the onset and development of diabetic cardiomyopathy (DCM). However, the mechanism remains poorly understood. Here, we report that profilin-1 (PFN-1), a well-known actin-binding protein, serves as a potent regulator in AGEs-induced cardiomyocytes hypertrophy and apoptosis. PFN-1 was upregulated in AGEs-treated H9c2 cells, which was associated with increased cardiomyocytes hypertrophy and apoptosis. Silencing PFN-1 expression remarkably attenuated AGEs-induced H9c2 cell hypertrophy and apoptosis. Mechanistically, AGEs increased PFN-1 expression through elevating ROS production and RhoA and ROCK2 expression. Consequently, elevated PFN-1 promoted actin cytoskeleton disorganization. When either ROS production/ROCK activation was blocked or cells were treated with Cytochalasin D (actin depolymerizer), H9c2 cells were protected against AGEs-induced cardiac myocyte abnormalities, concomitantly with downregulated expression of PFN-1 and improved actin cytoskeleton alteration. Collectively, these data suggest that PFN-1 may play an important role in AGEs-induced hypertrophy and apoptosis in H9c2 cells.

Extracellular matrix 1 (ECM1) regulates the actin cytoskeletal architecture of aggressive breast cancer cells in part via S100A4 and Rho-family GTPases.

ECM1 overexpression is an independent predictor of poor prognosis in primary breast carcinomas, however the mechanisms by which ECM1 affects tumor progression have not been completely elucidated. ECM1 was silenced in the triple-negative breast cancer cell lines Hs578T and MDAMB231 using siRNA and the cells were evaluated for changes in morphology, migration, invasion and adhesion. Actin cytoskeleton alterations were evaluated by fluorescent staining and levels of activated Rho GTPases by pull down assays. ECM1 downregulation led to significantly diminished cell migration (p=0.0005 for Hs578T and p=0.02 for MDAMB231) and cell adhesion (p<0.001 for Hs578T and p=0.01 for MDAMB231). Cell invasion (matrigel) was reduced only in the Hs578T cells (p<0.01). Silencing decreased the expression of the prometastatic molecules S100A4 and TGFβR2 in both cell lines and CD44 in Hs578T cells. ECM1-silenced cells also exhibited alterations in cell shape and showed bundles of F-actin across the cell (stress fibers) whereas NT-siRNA treated cells showed peripheral membrane ruffling. Downregulation of ECM1 was also associated with an increased F/G actin ratio, when compared to the cells transfected with NT siRNA (p<0.001 for Hs578T and p<0.00035 for MDAMB231) and a concomitant decline of activated Rho A in the Hs578T cells. Re-expression of S100A4 in ECM1-silenced cells rescued the phenotype in the Hs578T cells but not the MDAMB231 cells. We conclude that ECM1 is a key player in the metastatic process and regulates the actin cytoskeletal architecture of aggressive breast cancer cells at least in part via alterations in S100A4 and Rho A.

Pleiotropic roles of the matricellular protein Sparc in tendon maturation and ageing.

Acute and chronic tendinopathies remain clinically challenging and tendons are predisposed to degeneration or injury with age. Despite the high prevalence of tendon disease in the elderly, our current understanding of the mechanisms underlying the age-dependent deterioration of tendon function remains very limited. Here, we show that Secreted protein acidic and rich in cysteine (Sparc) expression significantly decreases in healthy-aged mouse Achilles tendons. Loss of Sparc results in tendon collagen fibrillogenesis defects and Sparc−/− tendons are less able to withstand force in comparison with their respective wild type counterparts. On the cellular level, Sparc-null and healthy-aged tendon-derived cells exhibited a more contracted phenotype and an altered actin cytoskeleton. Additionally, an elevated expression of the adipogenic marker genes PPARγ and Cebpα with a concomitant increase in lipid deposits in aged and Sparc−/− tendons was observed. In summary, we propose that Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties favors lipid accretion in tendons.

Self-incompatible-like response induces alterations of actin cytoskeleton and cytosolic calcium in theCitruspollen tube of ‘Banpeiyu’ and ‘Hyuganatsu’

Self-incompatible-like response induces alterations of actin cytoskeleton and cytosolic calcium in the<i>Citrus</i>pollen tube of ‘Banpeiyu’ and ‘Hyuganatsu’

R-Ras Inhibits VEGF-Induced p38MAPK Activation and HSP27 Phosphorylation in Endothelial Cells

R-Ras is a Ras family small GTPase that is highly expressed in mature functional blood vessels in normal tissues. It inhibits pathological angiogenesis and promotes vessel maturation and stabilization. Previous studies suggest that R-Ras affects cellular signaling in endothelial cells, pericytes and smooth-muscle cells to regulate vessel formation and remodeling in adult tissues. R-Ras suppresses VEGF-induced endothelial permeability and vessel sprouting while promoting normalization of pathologically developing vessels in mice. It attenuates VEGF receptor-2 (VEGFR2) activation by inhibiting internalization of the receptor upon VEGF ligand binding, leading to significant reduction of VEGFR2 autophosphorylation. Here, we show that R-Ras strongly suppresses the VEGF-dependent activation of stress-activated protein kinase-2/p38 mitogen-activated protein kinase (SAPK2/p38MAPK) and the phosphorylation of downstream heat-shock protein 27 (HSP27), a regulator of actin cytoskeleton organization, in endothelial cells. The suppression of p38MAPK activation and HSP27 phosphorylation by R-Ras concurred with altered actin cytoskeleton architecture, reduced membrane protrusion and inhibition of endothelial cell migration toward VEGF. Silencing of endogenous R-Ras by RNA interference increased membrane protrusion and cell migration stimulated by VEGF, and these effects were offset by p38MAPK inhibitor SB203580. These results suggest that R-Ras regulates angiogenic activities of endothelial cells in part via inhibition of the p38MAPK-HSP27 axis of VEGF signaling.

衝撃波照射によるアクチンフィラメントの構造変化

衝撃波照射によるアクチンフィラメントの構造変化

State of actin cytoskeleton and development of slow-frozen and vitrified rabbit pronuclear zygotes

This study was focused on the effect of cryopreservation on the state of actin cytoskeleton and development of rabbit pronuclear zygotes. Zygotes were collected from superovulated females and immediately used for 1) slow-freezing in a solution containing 1.5 M 1,2-propanediol and 0.2 M sucrose, or 2) vitrification in a solution containing 42.0% (v/v) of ethylene glycol, 18.0% (w/v) of dextran and 0.3 M sucrose as cryoprotectants. After thawing or warming, respectively, zygotes were evaluated for 1) actin distribution, 2) in vitro or 3) in vivo development to blastocyst. Comparing actin filaments distribution, a significantly higher number of vitrified zygotes with actin distributed in cell border was observed (55 ± 7.7 vs. 74 ± 6.1% for slow-frozen vs. vitrified, respectively). After 24 and 72 h of in vitro development, significant differences in the cleavage and morula rate among the groups were observed (9 ± 2.4 and 3 ± 1.3 vs. 44 ± 3.0 and 28 ± 2.7% for slow-frozen vs. vitrified, respectively). None of the slow-frozen zygotes reached the blastocyst stage, in contrast to the vitrified counterparts (11 ± 1.9%). Under in vivo culture conditions, a significant difference in blastocyst rate was observed between vitrified and fresh embryos (6 ± 1.5 vs. 35 ± 4.4% respectively). Our results showed that alterations in actin cytoskeleton and deteriorated development are more evident in slow-frozen than vitrified pronuclear zygotes. Vitrification method seems to be a more effective option for rabbit zygotes cryopreservation, although pronuclear zygotes manipulation per se resulted in a notable decrease in embryo development.

Dissecting the Mechanisms of Doxorubicin and Oxidative Stress-Induced Cytotoxicity: The Involvement of Actin Cytoskeleton and ROCK1.

We have recently reported that ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has superior anti-apoptotic and pro-survival effects than antioxidants against doxorubicin, a chemotherapeutic drug. Although oxidative stress is the most widely accepted mechanism, our studies suggest that ROCK1-dependent actin cytoskeleton remodeling plays a more important role in mediating doxorubicin cytotoxicity on MEFs. To further explore the contributions of ROCK1-dependent actin cytoskeleton remodeling in response to stress, this study investigates the mechanistic differences between the cytotoxic effects of doxorubicin versus hydrogen peroxide (H2O2), with a focus on cytoskeleton alterations, apoptosis and necrosis induction. We found that both types of stress induce caspase activation but with different temporal patterns and magnitudes in MEFs: H2O2 induces the maximal levels (2 to 4-fold) of activation of caspases 3, 8, and 9 within 4 h, while doxorubicin induces much higher maximal levels (15 to 25-fold) of caspases activation at later time points (16–24 h). In addition, necrosis induced by H2O2 reaches maximal levels within 4 h while doxorubicin-induced necrosis largely occurs at 16–24 h secondary to apoptosis. Moreover, both types of stress induce actin cytoskeleton remodeling but with different characteristics: H2O2 induces disruption of stress fibers associated with cytosolic translocation of phosphorylated myosin light chain (p-MLC) from stress fibers, while doxorubicin induces cortical F-actin formation associated with cortical translocation of p-MLC from central stress fibers. Furthermore, N-acetylcysteine (an antioxidant) is a potent suppressor for H2O2-induced cytotoxic effects including caspase activation, necrosis, and cell detachment, but shows a much reduced inhibition on doxorubicin-induced changes. On the other hand, ROCK1 deficiency is a more potent suppressor for the cytotoxic effects induced by doxorubicin than by H2O2. These results support the notion that doxorubicin induces caspase activation, necrosis, and actin cytoskeleton alterations largely through ROCK1-dependent and oxidative stress-independent pathways.

Chronic kidney disease: Actin cytoskeleton alterations in podocytes: a therapeutic target for chronic kidney disease.

Chronic kidney disease: Actin cytoskeleton alterations in podocytes: a therapeutic target for chronic kidney disease

Distinct impact of targeted actin cytoskeleton reorganization on mechanical properties of normal and malignant cells

The actin cytoskeleton is substantially modified in cancer cells because of changes in actin-binding protein abundance and functional activity. As a consequence, cancer cells have distinctive motility and mechanical properties, which are important for many processes, including invasion and metastasis. Here, we studied the effects of actin cytoskeleton alterations induced by specific nucleation inhibitors (SMIFH2, CK-666), cytochalasin D, Y-27632 and detachment from the surface by trypsinization on the mechanical properties of normal Vero and prostate cancer cell line DU145. The Young's modulus of Vero cells was 1300±900Pa, while the prostate cancer cell line DU145 exhibited significantly lower Young's moduli (600±400Pa). The Young's moduli exhibited a log-normal distribution for both cell lines. Unlike normal cells, cancer cells demonstrated diverse viscoelastic behavior and different responses to actin cytoskeleton reorganization. They were more resistant to specific formin-dependent nucleation inhibition, and reinforced their cortical actin after detachment from the substrate. This article is part of a Special Issue entitled: Mechanobiology.

Abstract 138: Cigarette Smoke and Monocytes Induce Endothelial Dysfunction

Cigarette smoke induces endothelial cell (EC) dysfunction. Circulating monocytes (HM) adhere to a damaged endothelium and migrate into the intima, differentiate to macrophages and contribute to atherosclerotic plaque growth. We demonstrated that HM exposed to cigarette smoke condensate (CSC) increase the transmigration of HM through EC. Thus, we hypothesized that CSC may affect HM/EC interaction. We analyzed the effects on EC of CSC (30 μg/ml) alone and/or of medium conditioned by HM incubated without (CM) or with CSC (CM/CSC). CSC, CM or CM/CSC did not affect EC vitality (MTT assay). CM and CM/CSC increased the expression (measured by RT-PCR) of adhesion molecule ICAM1 (36-fold and 90-fold, p&lt;0.01 vs control or CM, respectively). The effect on VCAM1 was even more dramatic (63-fold vs control for CM, p&lt;0.01 and 725-fold vs control, p&lt;0.01 with CM/CSC). In addition, CM and CM/CSC enhanced the expression of matrix metalloproteinase (MMP)-3 (+60% vs control, p&lt;0.01; +200% vs control, p&lt;0.01, respectively) and MMP-9 (+70% vs control, p&lt;0.01; +140% vs control, p&lt;0.01, respectively). MMP-1 and MMP-2 were not affected. Then, we used the iCELLigence System to analyze EC biological status (cell number, adhesion, viability, and morphology). Only the addition of CM/CSC to confluent EC monolayer dramatically modified the pattern of the electric flow, indicative of modifications in EC morphology. Indeed, we observed an altered actin cytoskeleton organization by immunofluorescence studies performed with FITC-phalloidin. In particular, CM/CSC caused the shrinking of the EC cytoplasm by about 50% (p&lt;0.01 vs control or CM). Moreover, CM/CSC altered sub-cellular localization of beta-catenin promoting its trafficking from membrane into the cytosol and the nucleus. In contrast, EC morphology was not affected by exposure to CSC or CM. Finally, CM and CM/CSC reduced (by 40% and 60%, p&lt;0.05, respectively) the expression of the small signaling G protein Rac 1 GTPase involved in cytoskeletal remodeling. In conclusion, these results indicate that monocytes, in the presence of cigarette smoke, secrete some soluble factors that affect endothelium morphology, thus increasing its permeability and facilitating monocytes transmigration through the endothelial monolayer.

Endothelial alpha-parvin controls integrity of developing vasculature and is required for maintenance of cell-cell junctions.

Angiogenesis and vessel integrity depend on the adhesion of endothelial cells (ECs) to the extracellular matrix and to adjacent ECs. The focal adhesion protein α-parvin (α-pv) is essential for vascular development. However, the role of α-pv in ECs in vivo is not known. To determine the function of α-pv in ECs during vascular development in vivo and the underlying mechanisms. We deleted the α-pv gene specifically in ECs of mice to study its role in angiogenesis and vascular development. Here, we show that endothelial-specific deletion of α-pv in mice results in late embryonic lethality associated with hemorrhages and reduced vascular density. Postnatal-induced EC-specific deletion of α-pv leads to retinal hypovascularization because of reduced vessel sprouting and excessive vessel regression. In the absence of α-pv, blood vessels display impaired VE-cadherin junction morphology. In vitro, α-pv-deficient ECs show reduced stable adherens junctions, decreased monolayer formation, and impaired motility, associated with reduced formation of integrin-mediated cell-extracellular matrix adhesion structures and an altered actin cytoskeleton. Endothelial α-pv is essential for vessel sprouting and for vessel stability.