F-actin Cytoskeletal Organization Research Articles

Effects of decellularized extracellular matrix derived from Jagged1-treated human dental pulp stem cells on biological responses of stem cells isolated from apical papilla.

Objective: Indirect Jagged1 immobilization efficiently activates canonical Notch signaling in human dental pulp stem cells (hDPSCs). This study aimed to investigate the characteristics of the Jagged1-treated hDPSC-derived decellularized extracellular matrix (dECM) and its biological activity on odonto/osteogenic differentiation of stem cells isolated from apical papilla (SCAPs). Methods: Bioinformatic database of Jagged1-treated hDPSCs was analyzed using NetworkAnalyst. hDPSCs seeded on the Jagged1 immobilized surface were maintained with normal or osteogenic induction medium (OM) followed by decellularization procedure, dECM-N, or dECM-OM, respectively. SCAPs were reseeded on each dECM with either the normal medium or the OM. Cell viability was determined by MTT assay. Characteristics of dECMs and SCAPs were evaluated by SEM, EDX, immunofluorescent staining, and alcian blue staining. Mineralization was assessed by alizarin red S, Von Kossa, and alkaline phosphatase staining. Statistical significance was considered at p < 0.05. Results: RNA-seq database revealed upregulation of several genes involved in ECM organization, ECM–receptor interaction, and focal adhesion in Jagged1-treated hDPSCs. Immobilized Jagged1 increased the osteogenesis of the hDPSC culture with OM. dECMs showed fibrillar-like network structure and maintained major ECM proteins, fibronectin, type I-collagen, and glycosaminoglycans. A decrease in calcium and phosphate components was observed in dECMs after the decellularized process. Cell viability on dECMs did not alter by 7 days. Cell attachment and f-actin cytoskeletal organization of SCAPs proliferated on Jagged1-treated dECMs were comparable to those of the control dECMs. SCAPs exhibited significantly higher mineralization on dECM-N in OM and markedly enhanced on dECM-OM with normal medium or OM conditions. Conclusion: Jagged1-treated hDPSC-derived dECMs are biocompatible and increase odonto/osteogenic differentiation of SCAPs. The results suggested the potential of Jagged1 dECMs, which could be further developed into ECM scaffolds for application in regenerative medicine.

Studies on the secondary metabolism of Rosellinia and Dematophora strains (Xylariaceae) from Iran

The xylariaceous genus Dematophora has recently been resurrected and segregated from Rosellinia based on a molecular phylogeny and morphological characters. This was an important taxonomic change because Dematophora in the current sense contains several important pathogens, while Rosellinia is limited to mainly saprotrophic species that have an endophytic stage in their life cycle and may even have beneficial effects on the host plants. During our ongoing work on the functional biodiversity of the Xylariales, we have encountered new strains of rosellinoid Xylariaceae from Iran and have studied their mycelial cultures for secondary metabolites in an attempt to establish further chemotaxonomic affinities. In the process, we isolated and identified 13 compounds, of which rosellisteroid (1), the cichorine derivative 2, and the alkaloid 3 are new. Out of these, nine were tested for their antimicrobial affinities with cytochalasin E (6) exhibiting weak activity against Schizosaccharomyces pombe. The cytotoxicity of three cytochalasin derivatives was examined and their effects on the F-actin cytoskeletal organization studied by fluorescence microscopy using fluorescent phalloidin. Cytochalasin E (6) and Δ6,12-cytochalasin E (7) showed strong and irreversible action on actin, while cytochalasin K (8) exhibited weaker, reversible effects.

Regulation of mesenchymal stem cell osteogenic potential via microfluidic manipulation of microcarrier surface curvature

Microsphere-carriers have gained great interests as three-dimensional substrates for cultivation/expansion of tissue cells, required by cell-based therapy. However, how the microsphere curvature affects the cell proliferation/differentiation as well as the underlying signaling pathway of stem cells, and how to consequently regulate those cellular functionalities via manipulating microcarrier surface curvature, still remain to be explored. The current study was thus designed to develop a microfluidic manipulation technology to precisely control poly (lactic-co-glycolic) acid (PLGA) microsphere surface curvature, and subsequently to investigate the cellular responses and responding pathways of rat bone mesenchymal stem cells (BMSCs) cultured on these microspheres of predetermined curvature. A microfluidic device was developed to produce mono-distributed PLGA microspheres of diameters ranging from 52 µm to 250 µm, corresponding to curvatures (κ) from 1/26 µm−1 to 1/125 µm−1. BMSCs attachment and proliferation was evaluated on them and the one of κ = 1/82.5 µm−1 was shown to provide the most suitable microenvironment for cells to grow and undergo osteogenic differentiation. It was even found that F-actin cytoskeletal organization, nuclear distortion and expression of Lamin A were significantly enhanced by cells on the microcarriers of κ = 1/82.5 µm−1. Furthermore, a long non-coding RNA named lnc-LMNA, was found in this study to be the key factor associated with Lamin A to regulate osteogenic differentiation of BMSCs on spherical substrates. The current study thus provides a smart manipulation technology via microfluidic-manufacturing microcarriers to regulate cell functionalities, thereby enhancing desired therapeutic outcomes of cell-based regeneration or repair.

Discovery of Spilanthol Endoperoxide as a Redox Natural Compound Active against Mammalian Prx3 and Chlamydia trachomatis Infection.

Chlamydia trachomatis (Ct) is a bacterial intracellular pathogen responsible for a plethora of diseases ranging from blindness to pelvic inflammatory diseases and cervical cancer. Although this disease is effectively treated with antibiotics, concerns for development of resistance prompt the need for new low-cost treatments. Here we report the activity of spilanthol (SPL), a natural compound with demonstrated anti-inflammatory properties, against Ct infections. Using chemical probes selective for imaging mitochondrial protein sulfenylation and complementary assays, we identify an increase in mitochondrial oxidative state by SPL as the underlying mechanism leading to disruption of host cell F-actin cytoskeletal organization and inhibition of chlamydial infection. The peroxidation product of SPL (SPL endoperoxide, SPLE), envisioned to be the active compound in the cellular milieu, was chemically synthesized and showed more potent anti-chlamydial activity. Comparison of SPL and SPLE reactivity with mammalian peroxiredoxins, demonstrated preferred reactivity of SPLE with Prx3, and virtual lack of SPL reaction with any of the reduced Prx isoforms investigated. Cumulatively, these findings support the function of SPL as a pro-drug, which is converted to SPLE in the cellular milieu leading to inhibition of Prx3, increased mitochondrial oxidation and disruption of F-actin network, and inhibition of Ct infection.

NC1-peptide regulates spermatogenesis through changes in cytoskeletal organization mediated by EB1.

During the epithelial cycle of spermatogenesis, different sets of cellular events take place across the seminiferous epithelium in the testis. For instance, remodeling of the blood-testis barrier (BTB) that facilitates the transport of preleptotene spermatocytes across the immunological barrier and the release of sperms at spermiation take place at the opposite ends of the epithelium simultaneously at stage VIII of the epithelial cycle. These cellular events are tightly coordinated via locally produced regulatory biomolecules. Studies have shown that collagen α3 (IV) chains, a major constituent component of the basement membrane, release the non-collagenous (NC) 1 domain, a 28-kDa peptide, designated NC1-peptide, from the C-terminal region, via the action of MMP-9 (matrix metalloproteinase 9). NC1-peptide was found to be capable of inducing BTB remodeling and spermatid release across the epithelium. As such, the NC1-peptide is an endogenously produced biologically active peptide which coordinates these cellular events across the epithelium in stage VIII tubules. Herein, we used an animal model, wherein NC1-peptide cloned into the pCI-neo mammalian expression vector was overexpressed in the testis, to better understanding the molecular mechanism by which NC1-peptide regulated spermatogenic function. It was shown that NC1-peptide induced considerable downregulation on a number of cell polarity and planar cell polarity (PCP) proteins, and studies have shown these polarity and PCP proteins modulate spermatid polarity and adhesion via their effects on microtubule (MT) and F-actin cytoskeletal organization across the epithelium. More important, NC1-peptide exerted its effects by downregulating the expression of microtubule (MT) plus-end tracking protein (+TIP) called EB1 (end-binding protein 1). We cloned the full-length EB1 cDNA for its overexpression in the testis, which was found to block the NC1-peptide-mediated disruptive effects on cytoskeletal organization in Sertoli cell epithelium and pertinent Sertoli cell functions. These findings thus illustrate that NC1-peptide is working in concert with EB1 to support spermatogenesis.

Non-Redundant and Overlapping Oncogenic Readouts of Non-Canonical and Novel Colorectal Cancer KRAS and NRAS Mutants.

RAS oncogene family members are molecular switches of signaling pathways that control cell growth, proliferation, differentiation, and survival. In colorectal cancer, Kirsten-RAS (KRAS) and neuroblastoma-RAS (NRAS) are the commonly mutated isoforms. Activating mutations in RAS result in cellular transformation independent of upregulated epidermal growth factor receptor (EGFR)-initiated signaling. The present study characterized the functional consequences of non-canonical/novel KRAS and NRAS mutants identified in a targeted next-generation sequencing study of colorectal cancer specimens from Filipino patients. In vitro assays in NIH3T3 cells showed that similar to the canonical KRAS G12D mutant, overexpression of KRAS G12S, A59T, and Y137C, but not NRAS G12D and NRAS A11V, confer higher proliferation and migration rates. HCT116 cells transfected with the novel NRAS A11V and the canonical NRAS G12D, but not the KRAS mutants, display enhanced resistance to apoptosis. All four non-canonical/novel KRAS and NRAS mutants induce gross changes in F-actin cytoskeletal organization and cellular morphology of NIH3T3 cells. Only KRAS G12S and KRAS A59T appear to deregulate extracellular signal-regulated kinase (ERK) and its downstream target ETS transcription factor ELK1 (ELK1). Elucidation of differential effector engagement responsible for the variable phenotypic readouts of the mutants is warranted. If validated by mouse studies and clinical correlates, these can have wider implications in choosing treatment options.

EXTH-19. IBRUTINIB MODULATES BLOOD-BRAIN BARRIER INTEGRITY TO ENHANCE CHEMOTHERAPY PERMEABILITY

Abstract BACKGROUND The blood-brain barrier (BBB) poses a major treatment challenge for malignant brain tumors. Ibrutinib is an FDA approved Bruton’s tyrosine kinase (BTK) and bone marrow x-linked tyrosine kinase (BMX) inhibitor indicated for the treatment of B-cell lymphomas. BMX is highly expressed in endothelial cells and is suggested to regulate cytoskeletal organization and barrier function in epithelial cells. We hypothesize that inhibition of BMX by ibrutinib in brain endothelial cells will disrupt the integrity of the BBB to increase drug permeability. METHODS Protein expression and phosphorylation of BMX was confirmed in immortalized mouse brain endothelial cells (bEnd.3). Endothelial barrier integrity was evaluated with increasing doses of ibrutinib (1–30μM) for 24hrs via the ACEA xCELLigence system (impedance) and the ECIS TEER24 system (resistance). Paracellular permeability of doxorubicin was measured by fluorescent spectrophotometry. Junctional protein expression of VE-Cadherin and F-actin cytoskeletal organization were detected by fluorescent staining. Ibrutinib’s effect on efflux pumps, ABCB1 and ABCG2, was performed by flow cytometry using specific substrates, valspodar and FTC, respectively. RESULTS Ibrutinib decreased endothelial cell impedance in a dose-dependent manner over 24hrs, with transient decrease by 46% at 30min with an optimal dose of 1μM, and partial recovery by 4hrs. TEER was decreased by 60% at 3hrs. Ibrutinib enhanced doxorubicin permeability by 30%; attributed to the transient disorganized nature of actin cytoskeletal filaments 2hrs post treatment. No effects on VE-cadherin expression or localization were observed. Ibrutinib successfully inhibited activity of ABCB1 and ABCG2 in a dose-dependent manner. CONCLUSION Our results suggest ibrutinib causes transient disruption of the brain endothelial monolayer due to cytoskeletal disorganization and ABC transporter inhibition to enhance permeability of doxorubicin. Further studies using ibrutinib and cytotoxic agents are warranted in glioma bearing animals. We propose that using ibrutinib as an adjunctive therapy may enhance drug delivery to patients with malignant brain tumors.

Freezing Responses in DMSO-Based Cryopreservation of Human iPS Cells: Aggregates Versus Single Cells.

Inadequate preservation methods of human induced pluripotent stem cells (hiPSCs) have impeded efficient reestablishment of cell culture after the freeze-thaw process. In this study, we examined roles of the cooling rate, seeding temperature, and difference between cell aggregates (3-50 cells) and single cells in controlled rate freezing of hiPSCs. Intracellular ice formation (IIF), post-thaw membrane integrity, cell attachment, apoptosis, and cytoskeleton organization were evaluated to understand the different freezing responses between hiPSC single cells and aggregates, among cooling rates of 1, 3, and 10°C/min, and between seeding temperatures of -4°C and -8°C. Raman spectroscopy images of ice showed that a lower seeding temperature (-8°C) did not affect IIF in single cells, but significantly increased IIF in aggregates, suggesting higher sensitivity of aggregates to supercooling. In the absence of IIF, Raman images showed greater variation of dimethyl sulfoxide concentration across aggregates than single cells, suggesting cryoprotectant transport limitations in aggregates. The ability of cryopreserved aggregates to attach to culture substrates did not correlate with membrane integrity for the wide range of freezing parameters, indicating inadequacy of using only membrane integrity-based optimization metrics. Lower cooling rates (1 and 3°C/min) combined with higher seeding temperature (-4°C) were better at preventing IIF and preserving cell function than a higher cooling rate (10°C/min) or lower seeding temperature (-8°C), proving the seeding temperature range of -7°C to -12°C from literature to be suboptimal. Unique f-actin cytoskeletal organization into a honeycomb-like pattern was observed in postpassage and post-thaw colonies and correlated with successful reestablishment of cell culture.

Cofilin Regulates Nuclear Architecture through a Myosin-II Dependent Mechanotransduction Module

Structural features of the nucleus including shape, size and deformability impact its function affecting normal cellular processes such as cell differentiation and pathological conditions such as tumor cell migration. Despite the fact that abnormal nuclear morphology has long been a defining characteristic for diseases such as cancer relatively little is known about the mechanisms that control normal nuclear architecture. Mounting evidence suggests close coupling between F-actin cytoskeletal organization and nuclear morphology however, mechanisms regulating this coupling are lacking. Here we identify that Cofilin/ADF-family F-actin remodeling proteins are essential for normal nuclear structure in different cell types. siRNA mediated silencing of Cofilin/ADF provokes striking nuclear defects including aberrant shapes, nuclear lamina disruption and reductions to peripheral heterochromatin. We provide evidence that these anomalies are primarily due to Rho kinase (ROCK) controlled excessive contractile myosin-II activity and not to elevated F-actin polymerization. Furthermore, we demonstrate a requirement for nuclear envelope LINC (linker of nucleoskeleton and cytoskeleton) complex proteins together with lamin A/C for nuclear aberrations induced by Cofilin/ADF loss. Our study elucidates a pivotal regulatory mechanism responsible for normal nuclear structure and which is expected to fundamentally influence nuclear function.

Three-dimensional direct cell bioprinting for tissue engineering.

Bioprinting is a relatively new technology where living cells with or without biomaterials are printed layer-by-layer in order to create three-dimensional (3D) living structures. In this article, novel bioprinting methodologies are developed to fabricate 3D biological structures directly from computer models using live multicellular aggregates. Multicellular aggregates made out of at least two cell types from fibroblast, endothelial and smooth muscle cells are prepared and optimized. A novel bioprinting approach is proposed in order to continuously extrude cylindrical multicellular aggregates through the bioprinter's glass microcapillaries. The multicellular aggregates are first aspirated into a capillary and then compressed to form a continuous cylindrical multicellular bioink. To overcome surface tension-driven droplet formation, the required compression ratio is calculated. Based on the developed bioprinting strategies, multicellular aggregates and their support structures are bioprinted to form 3D tissue constructs with predefined shapes. The effect of the bioprinting process was examined for fusion, cell viability at different compression ratios, and f-actin cytoskeletal organization. The results show that the bioprinted 3D constructs fuse rapidly and have high cell viability after printing. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2530-2544, 2017.

SND1 Acts Downstream of TGFβ1 and Upstream of Smurf1 to Promote Breast Cancer Metastasis.

SND1 is an AEG-1/MTDH/LYRIC-binding protein that is upregulated in numerous human cancers, where it has been assigned multiple functional roles. In this study, we report its association with the TGFβ1 signaling pathway, which promotes epithelial-mesenchymal transition (EMT) in breast cancer. SND1 was upregulated in breast cancer tissues, in particular in primary invasive ductal carcinomas. Transcriptional activation of the SND1 gene was controlled by the TGFβ1/Smad pathway, specifically by activation of the Smad2/Smad3 complex. The SND1 promoter region contained several Smad-specific recognition domains (RD motifs), which were recognized and bound by the Smad complex that enhanced the transcriptional activation of SND1. We found that SND1 promoted expression of the E3 ubiquitin ligase Smurf1, leading to RhoA ubiquitination and degradation. RhoA degradation in breast cancer cells disrupted F-actin cytoskeletal organization, reduced cell adhesion, increased cell migration and invasion, and promoted metastasis. Overall, our results define a novel role for SND1 in regulating breast tumorigenesis and metastasis.

The organization of the secretory machinery in chromaffin cells as a major factor in modeling exocytosis

The organization of cytoplasm in excitable cells was a largely ignored factor when mathematical models were developed to understand intracellular calcium and secretory behavior. Here we employed a combination of fluorescent evanescent and transmitted light microscopy to explore the F-actin cytoskeletal organization in the vicinity of secretory sites in cultured bovine chromaffin cells. This technique and confocal fluorescent microscopy show chromaffin granules associated with the borders of cortical cytoskeletal cages forming an intricate tridimensional network. Furthermore, the overexpression of SNAP-25 in these cells also reveals the association of secretory machinery clusters with the borders of these cytoskeletal cages. The importance of these F-actin cage borders is stressed when granules appear to interact and remain associated during exocytosis visualized in acridin orange loaded vesicles. These results will prompt us to propose a model of cytoskeletal cages, where the secretory machinery is associated with its borders. Both the calcium level and the secretory response are enhanced in this geometrical arrangement when compared with a random distribution of the secretory machinery that is not restricted to the borders of the cage.

F-actin cytoskeletal organization in intervertebral disc health and disease

The cytoskeleton, which in most cell types, including the intervertebral disc described here, comprises microfilaments, microtubules and intermediate filaments, plays important functions in many fundamental cellular events, including cell division, motility, protein trafficking and secretion. The cytoskeleton is also critical for communication; for example, alterations to the architecture of the F-actin (filamentous actin) cytoskeletal networks can affect communication between the cells and the extracellular matrix, potentially compromising tissue homoeostasis. Although there are limited studies to date, this paper aims to review current knowledge on F-actin cytoskeletal element organization in intervertebral disc cells, how F-actin differs with pathology and its implications for mechanotransduction.