Alpha-actinin-4 is essential for maintaining normal trophoblast proliferation and differentiation during early pregnancy.

BACKGROUND: Proper differentiation of trophoblasts in the human placenta is essential for a successful pregnancy, whereas abnormal regulation of this process may lead to adverse pregnancy outcomes, especially preeclampsia (PE). However, the underlying mechanism of trophoblast differentiation remains unclear. Previous studies have reported the involvement of alpha-actinin-4 (ACTN4) in the actin cytoskeleton dynamics and motility. Hence, we hypothesized that ACTN4 may act as an important regulator in the normal proliferation and differentiation of trophoblasts during early pregnancy. METHOD: To test this hypothesis, we collected villous tissues from women undergoing a legal pregnancy termination during 6-10 weeks of gestation and explanted them for cell culture and siRNA transfection. We also obtained placental tissues from PE patients and healthy pregnant women and isolated the primary cytotrophoblast (CTB) cells. The expression of ACTN4 in the CTBs of placental villi and during the differentiation of CTBs into STBs was detected by immunofluorescence, immunohistochemistry (IHC), and EdU proliferation assays. Besides, villous explant, Matrigel invasion, transwell migration assay, and Wound-healing assay were performed to identify the possible role of ACTN4 in the outgrowth of explants and the invasion, migration, and proliferation of cell column trophoblasts (CCTs). Western blot analysis was carried out to compare the protein expression level of AKT, Snail activities, and epithelial-to-mesenchymal transition (EMT) in the villi or HTR8/SVneo cells with ACTN4 knockdown. RESULTS: ACTN4 was highly expressed in CTB cells and interstitial extravillous trophoblast (iEVT) cells but not found in the syncytiotrophoblast (STB) cells in the first trimester villi. Downregulation of ACTN4 led to reduced trophoblast proliferation and explant outgrowth ex vivo, as well as iEVT invasion and migration in vitro due to disrupt of actin filaments organization. Such ACTN4 inhibition also decreased AKT and Snail activities and further impeded the EMT process. In addition, ACTN4 expression was found to be downregulated in the iEVTs from preeclamptic placentas. CONCLUSIONS: Our findings suggest that ACTN4 may act as an important regulator of trophoblast proliferation and differentiation during early pregnancy, and dysregulation of this protein may contribute to preeclampsia pathogenesis.

The actin binding protein α-actinin-2 expression is associated with dendritic spine plasticity and migrating granule cells in the rat dentate gyrus following pilocarpine-induced seizures.

α-actinin-2 (α-actn-2) is an F-actin-crosslinking protein, localized in dendritic spines. In vitro studies suggested that it is involved in spinogenesis, morphogenesis, actin organization, cell migration and anchoring of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptors in dendritic spines. However, little is known regarding its function in vivo. We examined the levels of α-actn-2 expression within the dentate gyrus (DG) during the development of chronic limbic seizures (epileptogenesis) induced by pilocarpine in rats. In this model, plasticity of the DG glutamatergic granule cells including spine loss, spinogenesis, morphogenesis, neo-synaptogenesis, aberrant migration, and alterations of NMDA receptors have been well characterized. We showed that α-actn-2 immunolabeling was reduced in the inner molecular layer at 1-2 weeks post-status epilepticus (SE), when granule cell spinogenesis and morphogenesis occur. This low level persisted at the chronic stage when new functional synapses are established. This decreased of α-actn-2 protein is concomitant with the recovery of drebrin A (DA), another actin-binding protein, at the chronic stage. Indeed, we demonstrated in cultured cells that in contrast to DA, α-actn-2 did not protect F-actin destabilization and DA inhibited α-actn-2 binding to F-actin. Such alteration could affect the anchoring of NR1 in dendritic spines. Furthermore, we showed that the expression of α-actn-2 and NR1 are co-down-regulated in membrane fractions of pilocarpine animals at chronic stage. Last, we showed that α-actn-2 is expressed in migrating newly born granule cells observed within the hilus of pilocarpine-treated rats. Altogether, our results suggest that α-actn-2 is not critical for the structural integrity and stabilization of granule cell dendritic spines. Instead, its expression is regulated when spinogenesis and morphogenesis occur and within migrating granule cells. Our data also suggest that the balance between α-actn-2 and DA expression levels may modulate NR1 anchoring within dendritic spines.

Differential regulation of Actn2 and Actn3 expression during unfolded protein response in C2C12 myotubes.

ACTN2 and ACTN3 encode sarcomeric α-actinin-2 and α-actinin-3 proteins, respectively, that constitute the Z-line in mammalian skeletal muscle fibers. In human ACTN3, a nonsense mutation at codon 577 that encodes arginine (R) produces the R577X polymorphism. Individuals having a homozygous 577XX genotype do not produce α-actinin-3 protein. The 577XX genotype reportedly occurs in sprint and power athletes in frequency lower than in the normal population, suggesting that α-actinin-3 deficiency diminishes fast-type muscle function. Among humans who carry 577R alleles, varying ACTN3 expression levels under certain conditions can have diverse effects on atheletic and muscle performance. However, the factors that regulate ACTN3 expression are unclear. Here we investigated whether the unfolded protein response (UPR) under endoplasmic reticulum (ER) stress regulates expression of Actn3 and its isoform Actn2 in mouse C2C12 myotubes. Among UPR-related transcription factors, XBP1 upregulated Actn2, whereas XBP1, ATF4 and ATF6 downregulated Actn3 promoter activity. Chemical induction of ER stress increased Actn2 mRNA levels, but decreased those for Actn3. ER stress also decreased α-actinin-3 protein levels, whereas levels of α-actinin-2 were unchanged. The intracellular composition of muscle contraction-related proteins was altered under ER stress, in that expression of parvalbumin (a fast-twitch muscle-specific protein) and troponin I type 1 (skeletal, slow) was suppressed. siRNA-induced suppression of Actn3 mimicked the inhibitory effect of ER stress on parvalbumin levels. Thus, endogenous expression levels of α-actinin-3 can be altered by ER stress, which may modulate muscle performance and athletic aptitudes, particularly in humans who carry ACTN3 577R alleles.

CTCF-induced upregulation of HOXA11-AS facilitates cell proliferation and migration by targeting miR-518b/ACTN4 axis in prostate cancer.

BACKGROUND: Testified as crucial participators in different types of human malignancies, long noncoding RNAs (lncRNAs) have been revealed to exert a significant effect on the complicated courses of tumor progression. Although existing literatures have revealed the oncogenic role of lncRNA homeobox A11 antisense RNA (HOXA11-AS) in multiple cancers, the underlying role of HOXA11-AS in prostate cancer (PCa) and its potential molecular mechanism remains poorly understood. AIM: To decipher the molecular performance of HOXA11-AS in PCa. METHODS: The expression of HOXA11-AS, miR-518b and actinin alpha 4 (ACTN4) was detected by a real-time quantitative polymerase chain reaction. Colony formation, EdU, flow cytometry, wound healing, and transwell assays were utilized to explore the biological role of HOXA11-AS in PCa. The interaction between RNAs (CCCTC-binding factor [CTCF], HOXA11-AS, miR-518b, and ACTN4) was tested via chromatin immunoprecipitation, luciferase reporter and RNA immunoprecipitation assays. RESULTS: HOXA11-AS in PCa cells was expressed at high levels. Silenced HOXA11-AS in PCa cells could lead to a significant elevation in the abilities of cell proliferation and migration whereas a remarkable declination in cell apoptosis capability. Subsequent molecular mechanism assays confirmed that HOXA11-AS bound with miR-518b and negatively regulates miR-518b expression. Besides, HOXA11-AS could regulate the expression of ACTN4 by sponging miR-518b. Moreover, rescued-function assays revealed that miR-518b inhibition or ACTN4 upregulation reversed the repressive effect of HOXA11-AS knockdown on PCa progression. Furthermore, CTCF was validated to activate HOXA11-AS transcription in PCa cells. CONCLUSIONS: CTCF-induced upregulation of HOXA11-AS facilitates PCa progression via miR-518b/ACTN4 axis, providing a new target for PCa treatment.

Readthrough of ACTN3 577X nonsense mutation produces full-length α-actinin-3 protein.

The ACTN3 gene encodes α-actinin-3 protein, which stabilizes the contractile apparatus at the Z-line in skeletal muscle cell fast fibers. A nonsense mutation of the arginine (R) at the codon for amino acid 577 of the ACTN3 gene generates a premature termination codon (PTC) and produces the R577X polymorphism in humans (X specifies translational termination). The ACTN3 577X genotype abolishes α-actinin-3 protein production due to targeted degradation of the mutant transcript by the cellular nonsense-mediated mRNA decay (NMD) system, which requires mRNA splicing. In humans, α-actinin-3 deficiency can decrease sprinting and power performance as well as skeletal muscle mass and strength. Here we investigated whether suppression of the in-frame PTC induced by treatment with the aminoglycosides gentamicin and G418 that promote termination codon readthrough could allow production of full-length α-actinin-3 protein from ACTN3 577X. We constructed expression plasmids encoding mature mRNA that lacks introns or pre-mRNA, which carries introns for the ACTN3 577X gene (X and Xpre, respectively) and transfected the constructs into HEK293 cells. Similar constructs for the ACTN3 577R gene were used as controls. HEK293 cells carrying the X gene, but not the Xpre gene, expressed exogenous truncated α-actinin-3 protein, indicating NMD-mediated suppression of exogenous Xpre expression. Cells treated with aminoglycosides produced exogenous full-length α-actinin-3 protein in X-transfected cells, but not in Xpre-transfected cells. The NMD inhibitor caffeine prevented suppression of Xpre expression and thereby induced production of full-length α-actinin-3 protein in the presence of aminoglycoside. Together these results indicate that the ACTN3 R577X polymorphism could be a novel target for readthrough therapy, which may affect athletic and muscle performance in humans.

Crystallization of Recombinant α-Actinin and Related Proteins.

When it comes to crystallization each protein is unique. It can never be predicted beforehand in which condition the particular protein will crystallize or even if it is possible to crystallize. Still, by following some simple checkpoints the chances of obtaining crystals are increased. The primary checkpoints are purity, stability, concentration, and homogeneity. High-quality protein crystals are needed. This protocol will allow an investigator to: clone, express, and crystallize a protein of interest.

Direct inhibition of ACTN4 by ellagic acid limits breast cancer metastasis via regulation of ß-catenin stabilization in cancer stem cells.

BACKGROUND: Pharmacology-based target identification has become a novel strategy leading to the discovery of novel pathological biomarkers. Ellagic acid (EA), a dietary polyphenol compound, exhibits potent anticancer activities; however, the underlying mechanisms remain unclear. The current study sought to determine the role and regulation of ACTN4 expression in human breast cancer metastasis and EA-based therapy. METHODS: The anti-metastasis ability of EA was validated by MMTV-PyMT mice and in vitro cell models. Drug affinity responsive target stability (DARTS) was utilized to identify ACTN4 as the direct target of EA. The metastatic regulated function of ACTN4 were assessed by cancer stem cells (CSCs)-related assays, including mammosphere formation, tumorigenic ability, reattachment differentiation, and signaling pathway analysis. The mechanisms of ACTN4 on ß-catenin stabilization were investigated by western blotting, co-immunoprecipitation and ubiquitination assays. The clinical significance of ACTN4 was based on human tissue microarray (TMA) analysis and The Cancer Genome Atlas (TCGA) database exploration. RESULTS: EA inhibited breast cancer growth and metastasis via directly targeting ACTN4 in vitro and in vivo, and was accompanied by a limited CSC population. ACTN4 knockdown resulted in the blockage of malignant cell proliferation, colony formation, and ameliorated metastasis potency. ACTN4-positive CSCs exhibited a higher ESA+ proportion, increased mammosphere-formation ability, and enhanced in vivo tumorigenesis ability. Mechanism exploration revealed that interruption of ACTN4/ß-catenin interaction will result in the activation of ß-catenin proteasome degradation. Increased ACTN4 expression was directly associated with the advanced cancer stage, an increased incidence of metastasis, and poor overall survival period. CONCLUSIONS: Taken together, our results suggest that ACTN4 plays an important role in breast CSCs-related metastasis and is a novel therapeutic target of EA treatment.

α Actinin 4 (ACTN4) Regulates Glucocorticoid Receptor-mediated Transactivation and Transrepression in Podocytes.

Glucocorticoids are a general class of steroids that possess renoprotective activity in glomeruli through their interaction with the glucocorticoid receptor. However, the mechanisms by which glucocorticoids ameliorate proteinuria and glomerular disease are not well understood. In this study, we demonstrated that α actinin 4 (ACTN4), an actin-cross-linking protein known to coordinate cytoskeletal organization, interacts with the glucocorticoid receptor (GR) in the nucleus of human podocytes (HPCs), a key cell type in the glomerulus critical for kidney filtration function. The GR-ACTN4 complex enhances glucocorticoid response element (GRE)-driven reporter activity. Stable knockdown of ACTN4 by shRNA in HPCs significantly reduces dexamethasone-mediated induction of GR target genes and GRE-driven reporter activity without disrupting dexamethasone-induced nuclear translocation of GR. Synonymous mutations or protein expression losses in ACTN4 are associated with kidney diseases, including focal segmental glomerulosclerosis, characterized by proteinuria and podocyte injury. We found that focal segmental glomerulosclerosis-linked ACTN4 mutants lose their ability to bind liganded GR and support GRE-mediated transcriptional activity. Mechanistically, GR and ACTN4 interact in the nucleus of HPCs. Furthermore, disruption of the LXXLL nuclear receptor-interacting motif present in ACTN4 results in reduced GR interaction and dexamethasone-mediated transactivation of a GRE reporter while still maintaining its actin-binding activity. In contrast, an ACTN4 isoform, ACTN4 (Iso), that loses its actin-binding domain is still capable of potentiating a GRE reporter. Dexamethasone induces the recruitment of ACTN4 and GR to putative GREs in dexamethasone-transactivated promoters, SERPINE1, ANGPLT4, CCL20, and SAA1 as well as the NF-κB (p65) binding sites on GR-transrepressed promoters such as IL-1ß, IL-6, and IL-8 Taken together, our data establish ACTN4 as a transcriptional co-regulator that modulates both dexamethasone-transactivated and -transrepressed genes in podocytes.

Triiodo-L-Thyronine Promotes the Maturation of Cardiomyocytes Derived From Rat Bone Marrow Mesenchymal Stem Cells.

Bone marrow mesenchymal stem cells (BMMSCs) can differentiate into cardiomyocytes and be used in cardiac tissue engineering for heart regeneration. However, the effective clinical application of cardiomyocytes derived from BMMSCs is limited because of their immature phenotype. The aim of this study was to investigate the potential of triiodo-L-thyronine (T3) to drive cardiomyocytes derived from BMMSCs to a more mature state. BMMSCs were divided into 3 groups: untreated controls, differentiated, and T3 treated. The differentiation potential was evaluated by immunofluorescence microscopy and flow cytometry. Data were represented as the numbers of cells positive for the troponin I (cTnI), α-actinin, GATA4, and the connexin-43 (Cx-43). The mRNA levels of these specific markers of cardiomyocytes were determined by quantitative real-time polymerase chain reaction. The levels of cardiomyocytes markers protein and octamer-binding transcription factor 4 (Oct-4) were determined by Western blot analyses. Our data demonstrate that T3 treatment leads to a significant increase in cells positive for cTnI, GATA4, Cx-43, and α-actinin. The mRNA and protein expression levels of these specific markers of cardiomyocytes were also increased after T3 treatment. At the same time, the protein expression level of Oct-4 was substantially downregulated in T3-treated cells. These results demonstrate that T3 treatment increases the differentiation of BMMSCs induced to cardiomyocytes and promotes their maturation.

ACTN4 and the pathways associated with cell motility and adhesion contribute to the process of lung cancer metastasis to the brain.

BACKGROUND: The aim of this study was to identify critical gene pathways that are associated with lung cancer metastasis to the brain. METHODS: The RNA-Seq approach was used to establish the expression profiles of a primary lung cancer, adjacent benign tissue, and metastatic brain tumor from a single patient. The expression profiles of these three types of tissues were compared to define differentially expressed genes, followed by serial-cluster analysis, gene ontology analysis, pathway analysis, and knowledge-driven network analysis. Reverse transcription-polymerase chain reaction (RT-PCR) was used to validate the expression of essential candidate genes in tissues from ten additional patients. RESULTS: Differential gene expression among these three types of tissues was classified into multiple clusters according to the patterns of their alterations. Further bioinformatic analysis of these expression profile data showed that the network of the signal transduction pathways related to actin cytoskeleton reorganization, cell migration, and adhesion was associated with lung cancer metastasis to the brain. The expression of ACTN4 (actinin, alpha 4), a cytoskeleton protein gene essential for cytoskeleton organization and cell motility, was significantly elevated in the metastatic brain tumor but not in the primary lung cancer tissue. CONCLUSIONS: The signaling pathways involved in the regulation of cytoskeleton reorganization, cell motility, and focal adhesion play a role in the process of lung cancer metastasis to the brain. The contribution of ACTN4 to the process of lung cancer metastasis to the brain could be mainly through regulation of actin cytoskeleton reorganization, cell motility, and focal adhesion.

Icariin promotes expression of junctophilin 2 and Ca2+ related function during cardiomyocyte differentiation of murine embryonic stem cells.

Junctophilin2 (JP2) is a critical protein associated with cardiogenesis. Icariin (ICA) facilitated the directional differentiation of murine embryonic stem (ES) cells into cardiomyocytes. However, little is known about the effects of ICA on JP2 during cardiac differentiation. Here, we explored whether ICA has effects on the expression and Ca2+ related function of JP2 during cardiomyocyte differentiation of ES cells in vitro. Embryonid bodies (EBs) formed by hanging drop were treated with 10(-7) mol/L ICA from day 5 to promote the cardiac differentiation. Percentage of beating EBs and number of beating area within EBs were monitored. Cardiomyocytes were purified by discontinuous percoll gradient centrifugation from EBs. The expression of JP2, α-actinin and troponin-T within EBs or isolated cardiomyocytes were analyzed by immunocytochemistry, western blot and flow cytometry. The transient Ca2+ release was characterized in cardiomyocytes treated with/without 10 mmol/L caffeine and 8 mmol/L Ca2+. Our results showed that ES cell-derived cardiomyocytes were well characterized with JP2 proteins. ICA promoted cardiomyocyte differentiation as indicated by an increased percentage of beating EBs and number of beating area within EBs. The expression of JP2, α-actinin and troponin-T were up-regulated both in EBs and isolated cardiomyocytes from EBs. Furthermore, ICA-induced JP2 expression was accompanied by a remarkable increase of the amplitude of Ca2+ transients in cardiomyocytes before/after caffeine and Ca2+ stimulating. In conclusion, ICA promotes in cardiac differentiation partly through regulating JP2 and improved the Ca2+ modulatory function of cardiomyocytes.

Alpha-actinin 4 is associated with cancer cell motility and is a potential biomarker in non-small cell lung cancer.

BACKGROUND: Differential expression and secretion of alpha-actinin 4 (ACTN4) in the lung cancer cell lines CL1-0 and CL1-5 have been reported in previous proteomic studies. The aim of this study is to investigate the functional properties of the ACTN4 protein in non-small-cell lung cancer (NSCLC) cells and evaluate its clinical importance. METHODS: We used RNA interference to knock down and overexpress ACTN4 protein to evaluate the effects of this intervention on cancer cell invasion and migration, as well as on microscopic cellular morphology. Furthermore, we examined by immunohistochemistry the expression of ACTN4 protein in tissue samples at different stages of lung cancer and compared the protein levels of ACTN4 in blood plasma samples from patients with histologically confirmed lung cancer and healthy controls. RESULTS: CL1-5 cell motility was significantly suppressed by the knockdown of ACTN4 protein. The morphology of CL1-5 cells changed from a predominantly mesenchymal-like shape into a globular shape in response to ACTN4 protein knockdown. A quantitative immunohistochemical assessment of lung cancer tissues revealed that ACTN4 protein level was considerably higher in cancerous tissues than in the adjacent normal ones, and the area under the receiver operating characteristic curve was 0.736 (p < 0.001). According to an enzyme-linked immunosorbent assay, the plasma levels of ACTN4 protein were significantly different between cancer patients and healthy controls, and the areas under the receiver operating characteristic curves were 0.828 and 0.909, respectively, for two independent cohorts (p < 0.001). CONCLUSIONS: We demonstrate that the knockdown of ACTN4 protein inhibited cell invasion and migration. These results suggest that ACTN4 is associated with lung cancer cell motility. Thus, the level of ACTN4 in cancerous tissue and plasma is related to the presence of lung cancer.

Expression patterns of sarcomeric α-actin, α-actinin and UCP2 in the myocardium of Kunming mice after exposure to c-terminal polypeptide of cardiotrophin-1.

Cardiotrophin-1 (CT-1) activates a distinct form of cardiac muscle cell hypertrophy in which the sarcomeric units are assembled in series. The aim of the study was to determine the expression pattern of sarcomeric contractile protein α-actin, specialized cytoskeletal protein α-actinin and mitochondrial uncoupling protein-2 (UCP2) in myocardial remodeling induced by chronic exposure to CT-1. Kunming mice were intraperitoneally injected with carboxy-terminal polypeptide (CP) of CT-1 (CT-1-CP, 500 µg·kg(-1)· day(-1)) for 1, 2, 3 and 4 week (s), respectively (4 groups obtained according to the injection time, n=10 each, with 5 males and 5 females in each group). Those injected with physiological saline for 4 weeks served as controls (n=10, with 5 males and 5 females). The heart tissues of mice were harvested at 1, 2, 3 or 4 week (s). Immunohistochemistry (IHC) and Western blotting (WB) were used to detect the distribution and expression of sarcomeric α-actin, α-actinin and mitochondrial UCP2 in myocardial tissues. IHC showed that α-actin was mainly distributed around the nuclei of cardiomyocytes, α-actinin concentrated around the striae and UCP2 scattered rather evenly in the plasma. The expression of α-actin was slightly greater than that of α-actinin and UCP2 in the control group (IHC: χ(2)=6.125; WB: F=0.249, P>0.05) and it gradually decreased after exposure to CT-1-CP. There was no significant difference in the expression of α-actin between the control group and the CT-1-CP-treated groups (χ (2)=7.386, P>0.05). But Western blotting revealed significant difference in the expression of α-actin between the control group and the 4-week CT-1-CP-treated group (F=2.912; q=4.203, P<0.05). Moreover, it was found that the expression of α-actinin increased stepwise with the exposure time in CT-1-CP-treated groups and differed significantly between CT-1-CP-treated groups and the control group (ICH: χ (2)=21.977; WB: F=50.388; P<0.01). The expression of UCP2 was initially increased (WB: control group vs. 1- or 2-week group, q values: 5.603 and 9.995, respectively, P<0.01) and then decreased (WB: control group vs. 3-week group, q=4.742, P<0.01; control group vs. 4-week group, q=0.558, P>0.05). It was suggested that long-term exposure to CT-1-CP could lead to the alteration in the expression of sarcomeric α-actin, α-actinin and mitochondrial UCP2. The different expressions of sarcomeric structure proteins and mitochondrial UCP2 may be involved in myocardial remodeling.

iPS cell-derived cardiogenicity is hindered by sustained integration of reprogramming transgenes.

BACKGROUND: Nuclear reprogramming inculcates pluripotent capacity by which de novo tissue differentiation is enabled. Yet, introduction of ectopic reprogramming factors may desynchronize natural developmental schedules. This study aims to evaluate the effect of imposed transgene load on the cardiogenic competency of induced pluripotent stem (iPS) cells. METHODS AND RESULTS: Targeted inclusion and exclusion of reprogramming transgenes (c-MYC, KLF4, OCT4, and SOX2) was achieved using a drug-inducible and removable cassette according to the piggyBac transposon/transposase system. Pulsed transgene overexpression, before iPS cell differentiation, hindered cardiogenic outcomes. Delayed in counterparts with maintained integrated transgenes, transgene removal enabled proficient differentiation of iPS cells into functional cardiac tissue. Transgene-free iPS cells generated reproducible beating activity with robust expression of cardiac α-actinin, connexin 43, myosin light chain 2a, α/ß-myosin heavy chain, and troponin I. Although operational excitation-contraction coupling was demonstrable in the presence or absence of transgenes, factor-free derivatives exhibited an expedited maturing phenotype with canonical responsiveness to adrenergic stimulation. CONCLUSIONS: A disproportionate stemness load, caused by integrated transgenes, affects the cardiogenic competency of iPS cells. Offload of transgenes in engineered iPS cells ensures integrity of cardiac developmental programs, underscoring the value of nonintegrative nuclear reprogramming for derivation of competent cardiogenic regenerative biologics.

Actin cable distribution and dynamics arising from cross-linking, motor pulling, and filament turnover.

The growth of fission yeast relies on the polymerization of actin filaments nucleated by formin For3p, which localizes at tip cortical sites. These actin filaments bundle to form actin cables that span the cell and guide the movement of vesicles toward the cell tips. A big challenge is to develop a quantitative understanding of these cellular actin structures. We used computer simulations to study the spatial and dynamical properties of actin cables. We simulated individual actin filaments as semiflexible polymers in three dimensions composed of beads connected with springs. Polymerization out of For3p cortical sites, bundling by cross-linkers, pulling by type V myosin, and severing by cofilin are simulated as growth, cross-linking, pulling, and turnover of the semiflexible polymers. With the foregoing mechanisms, the model generates actin cable structures and dynamics similar to those observed in live-cell experiments. Our simulations reproduce the particular actin cable structures in myoVΔ cells and predict the effect of increased myosin V pulling. Increasing cross-linking parameters generates thicker actin cables. It also leads to antiparallel and parallel phases with straight or curved cables, consistent with observations of cells overexpressing α-actinin. Finally, the model predicts that clustering of formins at cell tips promotes actin cable formation.

Sarcomere length nanometry in rat neonatal cardiomyocytes expressed with α-actinin-AcGFP in Z discs.

Nanometry is widely used in biological sciences to analyze the movement of molecules or molecular assemblies in cells and in vivo. In cardiac muscle, a change in sarcomere length (SL) by a mere ∼100 nm causes a substantial change in contractility, indicating the need for the simultaneous measurement of SL and intracellular Ca(2+) concentration ([Ca(2+)]i) in cardiomyocytes at high spatial and temporal resolution. To accurately analyze the motion of individual sarcomeres with nanometer precision during excitation-contraction coupling, we applied nanometry techniques to primary-cultured rat neonatal cardiomyocytes. First, we developed an experimental system for simultaneous nanoscale analysis of single sarcomere dynamics and [Ca(2+)]i changes via the expression of AcGFP in Z discs. We found that the averaging of the lengths of sarcomeres along the myocyte, a method generally used in today's myocardial research, caused marked underestimation of sarcomere lengthening speed because of the superpositioning of different timings for lengthening between sequentially connected sarcomeres. Then, we found that after treatment with ionomycin, neonatal myocytes exhibited spontaneous sarcomeric oscillations (cell-SPOCs) at partial activation with blockage of sarcoplasmic reticulum functions, and the waveform properties were indistinguishable from those obtained in electric field stimulation. The myosin activator omecamtiv mecarbil markedly enhanced Z-disc displacement during cell-SPOC. Finally, we interpreted the present experimental findings in the framework of our mathematical model of SPOCs. The present experimental system has a broad range of application possibilities for unveiling single sarcomere dynamics during excitation-contraction coupling in cardiomyocytes under various settings.

Development of podocyte injuries in Osborne-Mendel rats is accompanied by reduced expression of podocyte proteins.

Osborne-Mendel (OM) rats spontaneously develop glomerulopathy with progressive podocyte injury. Changes in protein expression levels in the foot processes of podocytes have been suggested to play an important role in the development of renal disease. The aim of this study was to investigate the temporal relationship between the expression of five podocyte proteins (nephrin, podocin, synaptopodin, α-actinin-4 and α3-integrin) and the development of podocyte injuries, proteinuria and glomerulosclerosis in OM rats. Male OM rats 5-20 weeks of age and age-matched Fischer 344 rats were used. Semiquantitative analysis of expression of the five podocyte proteins was performed by immunofluorescence labelling. Nephrin mRNA expression was determined by quantitative real-time reverse transcriptase polymerase chain reaction and nephrin protein expression was determined by mass spectrometry. Progressive reduction in expression of the podocyte proteins correlated with the progression of podocyte injuries, the development of proteinuria and the subsequent development of glomerulosclerosis. Nephrin mRNA expression and nephrin concentration also showed temporal decreases in OM rats. Altered expression of podocyte proteins preceded the development of proteinuria and glomerulosclerosis, suggesting that this event contributes to podocyte dysfunction and progression to glomerulosclerosis.

Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy.

BACKGROUND: Online label-free monitoring of in-vitro differentiation of stem cells remains a major challenge in stem cell research. In this paper we report the use of Raman micro-spectroscopy (RMS) to measure time- and spatially-resolved molecular changes in intact embryoid bodies (EBs) during in-vitro cardiogenic differentiation. METHODS: EBs formed by aggregation of human embryonic stem cells (hESCs) were cultured in defined medium to induce differentiation towards cardiac phenotype and maintained in purpose-built micro-bioreactors on the Raman microscope for 5days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24h intervals. RESULTS: The Raman spectra showed that the onset of spontaneous beating of EBs at day 7 coincided with an increase in the intensity of the Raman bands at 1340cm(-1), 1083cm(-1), 937cm(-1), 858cm(-1), 577cm(-1) and 482cm(-1). The spectral maps corresponding to these bands had a high positive correlation with the expression of the cardiac-specific α-actinin obtained by immuno-fluorescence imaging of the same EBs. The spectral markers obtained here are also in agreement with previous studies performed on individual live hESC-derived CMs. CONCLUSIONS: The intensity profile of these Raman bands can be used for label-free in-situ monitoring of EBs to estimate the efficacy of cardiogenic differentiation. GENERAL SIGNIFICANCE: As the acquisition of the time-course Raman spectra did not affect the viability or the differentiation potential of the hESCs, this study demonstrates the feasibility of using RMS for on-line non-invasive continuous monitoring of such processes inside bioreactor culture systems.

Expression of cardiac proteins in neonatal cardiomyocytes on PGS/fibrinogen core/shell substrate for Cardiac tissue engineering.

BACKGROUND: Heart failure due to myocardial infarction remains the leading cause of death worldwide owing to the inability of myocardial tissue regeneration. The aim of this study is to develop a core/shell fibrous cardiac patch having desirable mechanical properties and biocompatibility to engineer the infarcted myocardium. METHOD: We fabricated poly(glycerol sebacate)/fibrinogen (PGS/fibrinogen) core/shell fibers with core as elastomeric PGS provides suitable mechanical properties comparable to that of native tissue and shell as fibrinogen to promote cell-biomaterial interactions. The PGS/fibrinogen core/shell fibers and fibrinogen nanofibers were characterized by SEM, contact angle and tensile testing to analyze the fiber morphology, wettability, and mechanical properties of the scaffold. The cell-scaffold interactions were analyzed using isolated neonatal cardiomyocytes for cell proliferation, confocal analysis for the expression of marker proteins α-actinin, Troponin-T, ß-myosin heavy chain and connexin 43 and SEM analysis for cell morphology. RESULTS: We observed PGS/fibrinogen core/shell fibers had a Young's modulus of about 3.28 ± 1.7 MPa, which was comparable to that of native myocardium. Neonatal cardiomyocytes cultured on these scaffolds showed normal expression of cardiac specific marker proteins α-actinin, Troponin, ß-myosin heavy chain and connexin 43 to prove PGS/fibrinogen core/shell fibers have potential for cardiac tissue engineering. CONCLUSION: Results indicated that neonatal cardiomyocytes formed predominant gap junctions and expressed cardiac specific marker proteins on PGS/fibrinogen core/shell fibers compared to fibrinogen nanofibers, indicating PGS/fibrinogen core/shell fibers may serve as a suitable cardiac patch for the regeneration of infarcted myocardium.

Migfilin, α-parvin and ß-parvin are differentially expressed in ovarian serous carcinoma effusions, primary tumors and solid metastases.

OBJECTIVE: The objective of this study is to analyze the expression and clinical role of integrin-linked kinase (ILK), α-parvin, ß-parvin and migfilin in advanced-stage serous ovarian carcinoma. METHODS: Expression of these 4 proteins was investigated in 205 effusions and in 94 patient-matched solid lesions (33 primary tumors and 61 solid metastases) using immunohistochemistry. Protein expression was analyzed for association with clinicopathologic parameters and survival. RESULTS: ILK, α-parvin, ß-parvin and migfilin were expressed in tumor cells in 53%, 2%, 28% and 53% of effusions and 57%, 20%, 83% and 25% of solid lesions, respectively. Statistical analysis showed significantly higher α-parvin and ß-parvin expression in primary carcinomas (p=0.02 and p=0.001, respectively) and solid metastases (p=0.001 and p<0.001, respectively), compared to effusions, with opposite findings for migfilin (p=0.006 and p=0.008 for primary carcinomas and solid metastases, respectively). ILK expression was comparable at all anatomic sites. ß-Parvin expression in effusions was associated with better response to chemotherapy at diagnosis (p=0.014), with no other significant association with clinicopathologic parameters or survival. Expression in primary tumors and solid metastases was similarly unrelated to clinicopathologic parameters or survival. CONCLUSIONS: This study provides further evidence to our previous observations that the adhesion profile of ovarian serous carcinoma cells in effusions differs from their counterparts in primary carcinomas and solid metastases. ß-Parvin may be a novel marker of chemoresponse in metastatic ovarian carcinoma.