Actin Cytoskeleton Arrangement Research Articles

Bioinformatics analysis of PSAT1 loss identifies downstream pathways regulated in EGFR mutant NSCLC and a selective gene signature for predicting the risk of relapse.

The majority of malignant tumors exhibit an altered metabolic phenotype that ultimately provides the required energy and molecular precursors necessary for unregulated cell division. Within this, phosphoserine aminotransferase 1 (PSAT1) is involved in de novo serine biosynthesis and its activity promotes various biochemical processes, including one-carbon metabolism. It also directly generates α-ketoglutarate (α-KG), a Kreb cycle intermediate and epigenetic-regulating metabolite. Prior studies examining PSAT1 depletion have identified individual affected downstream pathways, such as GSK3β and E2F, in several cancer types, including non-small-cell lung cancer (NSCLC). However, global gene expression examination in response to PSAT1 loss, particularly in EGFR mutant NSCLC, has not been unexplored. Transcriptional profiling of EGFR mutant NSCLC cells with or without stable knock-down of PSAT1 identified differentially expressed genes (DEGs) enriched in several metabolic pathways required for cell division, including amino acid and nucleotide biosynthesis. Supplementation studies involving non-essential amino acids, nucleosides and α-KG partially restored defects in anchorage-independent growth due to the knockdown of PSAT1. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analysis identified potential impacts on actin cytoskeleton arrangement and β-catenin activity, which were rescued by PSAT1 re-expression. Finally, a comparative analysis of PSAT1 DEGs against transcripts enriched in patient EGFR mutant lung tumors identified a gene signature that is associated with overall and relapse-free survival (RFS) and was able to distinguish low or high-risk populations for RFS in early-stage EGFR mutant NSCLC. Overall, investigating genes altered by PSAT1 loss confirmed known PSAT1-regulated cellular pathways, identified a previously unknown role in the mediation of cytoskeleton arrangement in EGFR mutant NSCLC cells and allowed for the characterization of a gene signature with putative predictive potential for RFS in early-stage disease.

Methylated dialkylphosphate metabolites of the organophosphate pesticide malathion modify actin cytoskeleton arrangement and cell migration via activation of Rho GTPases Rac1 and Cdc42

The non-cholinergic molecular targets of organophosphate (OP) compounds have recently been investigated to explain their role in the generation of non-neurological diseases, such as immunotoxicity and cancer. Here, we evaluated the effects of malathion and its dialkylphosphate (DAP) metabolites on the cytoskeleton components and organization of RAW264.7 murine macrophages as non-cholinergic targets of OP and DAPs toxicity. All OP compounds affected actin and tubulin polymerization. Malathion, dimethyldithiophosphate (DMDTP) dimethylthiophosphate (DMTP), and dimethylphosphate (DMP) induced elongated morphologies and the formation of pseudopods rich in microtubule structures, and increased filopodia formation and general actin disorganization in RAW264.7 cells and slightly reduced stress fibers in the human fibroblasts GM03440, without significantly disrupting the tubulin or vimentin cytoskeleton. Exposure to DMTP and DMP increased cell migration in the wound healing assay but did not affect phagocytosis, indicating a very specific modification in the organization of the cytoskeleton. The induction of actin cytoskeleton rearrangement and cell migration suggested the activation of cytoskeletal regulators such as small GTPases. We found that DMP slightly reduced Ras homolog family member A activity but increased the activities of Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) from 5 min to 2 h of exposure. Chemical inhibition of Rac1 with NSC23766 reduced cell polarization and treatment with DMP enhanced cell migration, but Cdc42 inhibition by ML-141 completely inhibited the effects of DMP. These results suggest that methylated OP compounds, especially DMP, can modify macrophage cytoskeleton function and configuration via activation of Cdc42, which may represent a potential non-cholinergic molecular target for OP compounds.

Regulation of actin cytoskeleton via photolithographic micropatterning

Actin cytoskeleton plays crucial roles in various cellular functions. Extracellular matrix (ECM) can modulate cell morphology by remodeling the internal cytoskeleton. To define how geometry of ECM regulates the organization of actin cytoskeleton, we plated individual NIH 3T3 cells on micropatterned substrates with distinct shapes and sizes. It was found that the stress fibers could form along the nonadhesive edges of T-shaped pattern, but were absent from the opening edge of V-shaped pattern, indicating that the organization of actin cytoskeleton was dependent on the mechanical environment. Furthermore, a secondary actin ring was observed on 50[Formula: see text][Formula: see text]m circular pattern while did not appear on 30[Formula: see text][Formula: see text]m and 40[Formula: see text][Formula: see text]m pattern, showing a size-dependent organization of actin cytoskeleton. Finally, osteoblasts, MDCK and A549 cells exhibited distinct organization of actin cytoskeleton on T-shaped pattern, suggesting a cell-type specificity in arrangement of actin cytoskeleton. Together, our findings brought novel insight into the organization of actin cytoskeleton on micropatterned environments.

Berberine inhibits glioma cell migration and invasion by suppressing TGF-β1/COL11A1 pathway

Glioma is a clinically heterogeneous disease with a poor prognosis. Berberine (BBR), as a multi-target anti-tumor alkaloid, has the ability to penetrate the blood-brain barrier and shows cytotoxicity to glioma cells. In previous studies, we demonstrated that berberine inhibits glioma cell proliferation by inhibiting mutant p53 protein and promoting mitochondrial damage. In addition, berberine has been shown to reduce collagen accumulation in pulmonary fibrosis, diabetic nephropathy and arthritis. However, its effect on collagen in cancer needs to be further elucidated. In this study, we proved that the collagen XI alpha 1 chain (COL11A1) is highly expressed in glioma cell lines and associated with migration and invasion of glioma cells. Knocking down COL11A1 caused decreased expression of MMPs. Berberine could inhibit the migration and invasion of glioma cells by suppressing the TGF-β1/COL11A1 pathway and changes actin cytoskeleton arrangement. Nude mouse subcutaneous xenografts model also showed that berberine inhibited the expression of COL11A1 in vivo. Collectively, berberine that targets COL11A1 to inhibit glioma migration and invasion, may serve as a promising candidate for the development of anti-glioma drugs in the future.

Soy Isoflavones Accelerate Glial Cell Migration via GPER-Mediated Signal Transduction Pathway.

Soybean isoflavones, such as genistein, daidzein, and its metabolite, S-equol, are widely known as phytoestrogens. Their biological actions are thought to be exerted via the estrogen signal transduction pathway. Estrogens, such as 17β-estradiol (E2), play a crucial role in the development and functional maintenance of the central nervous system. E2 bind to the nuclear estrogen receptor (ER) and regulates morphogenesis, migration, functional maturation, and intracellular metabolism of neurons and glial cells. In addition to binding to nuclear ER, E2 also binds to the G-protein-coupled estrogen receptor (GPER) and activates the nongenomic estrogen signaling pathway. Soybean isoflavones also bind to the ER and GPER. However, the effect of soybean isoflavone on brain development, particularly glial cell function, remains unclear. We examined the effects of soybean isoflavones using an astrocyte-enriched culture and astrocyte-derived C6 clonal cells. Isoflavones increased glial cell migration. This augmentation was suppressed by co-exposure with G15, a selective GPER antagonist, or knockdown of GPER expression using RNA interference. Isoflavones also activated actin cytoskeleton arrangement via increased actin polymerization and cortical actin, resulting in an increased number and length of filopodia. Isoflavones exposure increased the phosphorylation levels of FAK (Tyr397 and Tyr576/577), ERK1/2 (Thr202/Tyr204), Akt (Ser473), and Rac1/cdc42 (Ser71), and the expression levels of cortactin, paxillin and ERα. These effects were suppressed by knockdown of the GPER. Co-exposure of isoflavones to the selective RhoA inhibitor, rhosin, selective Cdc42 inhibitor, casin, or Rac1/Cdc42 inhibitor, ML-141, decreased the effects of isoflavones on cell migration. These findings indicate that soybean isoflavones exert their action via the GPER to activate the PI3K/FAK/Akt/RhoA/Rac1/Cdc42 signaling pathway, resulting in increased glial cell migration. Furthermore, in silico molecular docking studies to examine the binding mode of isoflavones to the GPER revealed the possibility that isoflavones bind directly to the GPER at the same position as E2, further confirming that the effects of the isoflavones are at least in part exerted via the GPER signal transduction pathway. The findings of the present study indicate that isoflavones may be an effective supplement to promote astrocyte migration in developing and/or injured adult brains.

Magnetic nanoparticles of Ga‐substituted ε‐Fe2O3 for biomedical applications: Magnetic properties, transverse relaxivity, and effects of silica‐coated particles on cytoskeletal networks

Magnetic nanoparticles of ε-Fe1.76 Ga0.24 O3 with the volume-weighted mean size of 17 nm were prepared by thermal treatment of a mesoporous silica template impregnated with metal nitrates and were coated with silica shell of four different thicknesses in the range 6-24 nm. The bare particles exhibited higher magnetization than the undoped compound, 22.4 Am2 kg-1 at 300 K, and were characterized by blocked state with the coercivity of 1.2 T at 300 K, being thus the very opposite of superparamagnetic iron oxides. The relaxometric study of the silica-coated samples at 0.47 T revealed promising properties for MRI, specifically, transverse relaxivity of 89-168 s-1 mmol(f.u.)-1 L depending on the shell thickness was observed. We investigated the effects of the silica-coated nanoparticles on human A549 and MCF-7 cells. Cell viability, proliferation, cell cycle distribution, and the arrangement of actin cytoskeleton were assessed, as well as formation and maturation of focal adhesions. Our study revealed that high concentrations of silica-coated particles with larger shell thicknesses of 16-24 nm interfere with the actin cytoskeletal networks, inducing thus morphological changes. Consequently, the focal adhesion areas were significantly decreased, resulting in impaired cell adhesion.

Localization Microscopy of Actin Cytoskeleton in Human Platelets.

Here, we measure the actin cytoskeleton arrangement of different morphological states of human platelets using a new protocol for photo-switching of rhodamine class fluorophores. A new medium composition was established for imaging the cytoskeleton using Alexa Fluor 488 conjugated to phalloidin. Morphological states of platelets bound to a glass substrate are visualized and quantified by two-dimensional localization microscopy at nanoscopic resolution. Marker-less drift correction yields localization of individual Alexa 488 conjugated to phalloidin with a positional accuracy of 12 nm.

Cortactin expression in nasal polyps of Aspirin-Exacerbated Respiratory Disease (AERD) patients

PurposeThe term aspirin-exacerbated respiratory disease (AERD) refers to a combination of asthma, chronic rhinosinusitis with nasal polyposis (CRSwNP), and acute respiratory tract reactions to nonsteroidal anti-inflammatory drugs. AERD has now been included among the CRSwNP endotypes, and is considered one of the most aggressive in terms of disease recurrence.Cortactin is a multi-domain protein with a part in several cellular mechanisms involving actin assembly and cytoskeleton arrangement. Cortactin seems to have a role in inflammatory responses and to be implicated in human airway secretion and contraction mechanisms.The novel aim of the present study was to examine cortactin expression in nasal polyps of a consecutive cohort of AERD patients and in nasal mucosa of a control group of patients. Materials and methodsCortactin expression was assessed immunohistochemically in nasal polyps from 18 consecutive AERD patients who underwent endoscopic sinus surgery and in nasal mucosa of 19 patients without chronic rhinosinusitis. ResultsConcomitant allergy was found in 11 AERD patients, most of them male (8 cases; p = 0.02). Cortactin expression in nasal polyps was definitely high (+3) in 17 out of 18 cases, in both epithelial cells (cytoplasmic and membranous immunoreactivity) and activated fibroblasts. A higher cortactin expression was seen in female than in male AERD patients (p = 0.05). ConclusionsGiven this preliminary evidence of cortactin upregulation in the polyps of AERD patients, prospective studies could further investigate the role of cortactin in the biology of AERD, and the potential role of cortactin-targeted approaches in integrated AERD treatments.

Calreticulin is required for calcium homeostasis and proper pollen tube tip growth in Petunia

Main conclusionCalreticulin is involved in stabilization of the tip-focused Ca2+gradient and the actin cytoskeleton arrangement and function that is required for several key processes drivingPetuniapollen tube tip growth.Although the precise mechanism is unclear, stabilization of a tip-focused calcium (Ca2+) gradient seems to be critical for pollen germination and pollen tube growth. We hypothesize that calreticulin (CRT), a Ca2+-binding/buffering chaperone typically residing in the lumen of the endoplasmic reticulum (ER) of eukaryotic cells, is an excellent candidate to fulfill this role. We previously showed that in Petunia pollen tubes growing in vitro, CRT is translated on ER membrane-bound ribosomes that are abundant in the subapical zone of the tube, where CRT’s Ca2+-buffering and chaperone activities might be particularly required. Here, we sought to determine the function of CRT using small interfering RNA (siRNA) to, for the first time in pollen tubes growing in vitro, knockdown expression of a gene. We demonstrate that siRNA-mediated post-transcriptional silencing of Petunia hybrida CRT gene (PhCRT) expression strongly impairs pollen tube growth, cytoplasmic zonation, actin cytoskeleton organization, and the tip-focused Ca2+ gradient. Moreover, reduction of CRT alters the localization and disturbs the structure of the ER in abnormally elongating pollen tubes. Finally, cytoplasmic streaming is inhibited, and most of the pollen tubes rupture. Our data clearly show an interplay between CRT, Ca2+ gradient, actin-dependent cytoplasmic streaming, organelle positioning, and vesicle trafficking during pollen tube elongation. Thus, we suggest that CRT functions in Petunia pollen tube growth by stabilizing Ca2+ homeostasis and acting as a chaperone to assure quality control of glycoproteins passing through the ER.

Low-Dose Endothelial Monocyte-Activating Polypeptide-II Increases Blood-Tumor Barrier Permeability by Activating the RhoA/ROCK/PI3K Signaling Pathway.

Previous studies have demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) can increase blood-tumor barrier (BTB) permeability via both paracellular and transcellular pathways. In addition, we revealed that the RhoA/Rho kinase (ROCK) signaling pathway is involved in EMAP-II-induced BTB opening. This study further investigated the exact mechanisms by which the RhoA/ROCK signaling pathway affects EMAP-II-induced BTB hyperpermeability. In an in vitro BTB model, low-dose EMAP-II significantly activated phosphatidylinositol-3-kinase (PI3K) in rat brain microvascular endothelial cells (RBMECs) at 0.75h. Pretreatment with RhoA inhibitor C3 exoenzyme or ROCK inhibitor Y-27632 completely blocked EMAP-II-induced activation of PI3K. PKC-α/β inhibitor GÖ6976 pretreatment caused no change in EMAP-II-induced activation of PI3K. Besides, pretreatment with LY294002, a specific inhibitor of PI3K, did not affect EMAP-II-induced activation of PKC-α/β. Furthermore, LY294002 pretreatment significantly diminished EMAP-II-induced changes in BTB permeability, phosphorylation of myosin light chain and cofilin, expression and distribution of tight junction-associated protein ZO-1, and actin cytoskeleton arrangement in RBMECs. In summary, this study demonstrates that low-dose EMAP-II can increase BTB permeability by activating the RhoA/ROCK/PI3K signaling pathway.

Low-Dose Endothelial Monocyte-Activating Polypeptide-II Induces Blood-Tumor Barrier Opening Via the cAMP/PKA/Rac1 Pathway.

Previous studies have demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood-tumor barrier (BTB) hyperpermeability via both paracellular and transcellular pathways. In a recent study, we revealed that cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent signaling pathway is involved in EMAP-II-induced BTB hyperpermeability. This study further investigated the exact mechanisms through which the cAMP/PKA-dependent signaling pathway affects EMAP-II-induced BTB hyperpermeability. In an in vitro BTB model, low-dose EMAP-II (0.05 nM) induced a significant decrease in Rac1 activity in rat brain microvascular endothelial cells (RBMECs). Pretreatment with forskolin to elevate intracellular cAMP concentration completely blocked EMAP-II-induced inactivation of Rac1. Besides, pretreatment with 6Bnz-cAMP to activate PKA partially attenuated EMAP-II-induced Rac1 inactivation. Moreover, 6Bnz-cAMP pretreatment significantly diminished EMAP-II-induced changes in BTB permeability, myosin light chain (MLC) phosphorylation, expression and distribution of ZO-1, and actin cytoskeleton arrangement in RBMECs. These effects of 6Bnz-cAMP were completely blocked in the presence of NSC-23766 (the specific inhibitor of Rac1). In conclusion, this study demonstrates that low-dose EMAP-II induces BTB hyperpermeability via the cAMP/PKA/Rac1 signaling pathway.

Functions for the cAMP/Epac/Rap1 Signaling Pathway in Low-Dose Endothelial Monocyte-Activating Polypeptide-II-Induced Opening of Blood-Tumor Barrier.

Previous studies have demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood-tumor barrier (BTB) hyperpermeability via both paracellular and transcellular pathways. In a recent study, we revealed that cAMP/PKA-dependent and cAMP/PKA-independent signaling pathways are both involved in EMAP-II-induced BTB hyperpermeability. The present study further investigated the exact mechanisms through which the cAMP/PKA-independent signaling pathway affects EMAP-II-induced BTB hyperpermeability. In an in vitro BTB model, low-dose EMAP-II (0.05nM) induced a significant decrease in Rap1 activity in RBMECs. Pretreatment with forskolin to elevate intracellular cAMP concentration completely blocked EMAP-II-induced Rap1 inactivation. Epac/Rap1 activation by 8-pCPT-2'-O-Me-cAMP significantly prevented EMAP-II-induced activation of RhoA/ROCK. Furthermore, 8-pCPT-2'-O-Me-cAMP pretreatment significantly inhibited EMAP-II-induced decreases in TEER and increases in HRP flux. Pretreatment also significantly prevented EMAP-II-induced changes in MLC phosphorylation, actin cytoskeleton arrangement, and expression and distribution of ZO-1 in RBMECs. This study demonstrates that the cAMP/Epac/Rap1 signaling cascade is a crucial pathway in EMAP-II-induced BTB hyperpermeability.

Role of cAMP-dependent protein kinase A activity in low-dose endothelial monocyte-activating polypeptide-II-induced opening of blood-tumor barrier.

Our previous studies demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) can selectively increase the permeability of blood-tumor barrier (BTB). In addition, low-dose EMAP-II significantly decreases the cyclic adenosine monophosphate (cAMP) concentration and the protein kinase A (PKA) expression level in tumor tissues in the rat C6 glioma model. In this study, an in vitro BTB model was used to investigate the potential role of cAMP/PKA signaling cascade in EMAP-II-induced BTB hyperpermeability. Our data revealed that low-dose EMAP-II (0.05nM) induced a significant decrease in total intracellular cAMP concentration and PKA activity in rat brain microvascular endothelial cells (RBMECs). Pretreatment with forskolin to increase intracellular cAMP nearly completely blocked the EMAP-II-induced decrease in transendothelial electric resistance and increase in horseradish peroxidase flux across the BTB. Similar pretreatment completely prevented the EMAP-II-induced changes in RhoA/Rho kinase activity, expression and distribution of tight junction-associated protein ZO-1, and myosin light chain phosphorylation, as well as actin cytoskeleton arrangement in RBMECs. Pretreatment with 6Bnz-cAMP to activate PKA significantly attenuated these EMAP-II-induced alterations in RBMECs. In summary, our present study demonstrates that the cAMP/PKA signaling cascade works as a crucial signaling pathway in EMAP-II-induced BTB hyperpermeability.

Effect of Spilanthes acmella hydroethanolic extract activity on tumour cell actin cytoskeleton

Numerous natural products have pharmacological activity such that many biologically active compounds have led to the development of cancer chemotherapy drugs. Spilanthes acmella (Asteraceae) is widely cultivated in the State of Pará, Brazil, being employed in folk medicine for its anti-inflammatory, antimicrobial, antioxidant, analgesic, insecticide, and larvicidal properties. However, its cytotoxicity and influence on actin cytoskeleton organisation in tumour cell lines are practically nonexistent. We have verified the cytotoxicity of a hydroethanolic extract of the inflorescence of S. acmella, and examined its effects on the cytoskeleton of tumour cells. Decreasing concentrations of the extract (250, 500 and 1,000 µg/mL) were given to cultures of neoplastic cells (HEp-2). Cytotoxicity was assessed by the MTT test, and the influence on cytoskeleton organisation was examined by fluorescence microscopy. The IC50 of the hydroethanolic extract was 513 µg/mL, confirming the data obtained from the MTT assay that gave high cytotoxicity. The actin cytoskeleton arrangement of HEp2 cells at 500 and 1,000 µg/mL showed depolymerisation of the filaments, causing loss of morphology and consequently compromising cell adhesion.

Hypoxia-inducible factor 1-induced G protein-coupled receptor 35 expression is an early marker of progressive cardiac remodelling

G protein-coupled receptor 35 (GPR35) has been characterized to be one of the genes that are up-regulated in human heart failure. Since mechanisms controlling GPR35 expression are not known, we investigated the regulation of GPR35 gene and protein expression in cardiac myocytes and in the mouse models of cardiac failure. In cardiac myocytes, GPR35 gene expression was found to be exceptionally sensitive to hypoxia and induced by hypoxia-inducible factor-1 (HIF-1) activation. HIF-1-dependent regulation was established by genetic (HIF-1/VP16, Inhibitory Per/Arnt/Sim domain protein) and chemical [desferrioxamine (DFO)] modulation of the HIF-1 pathway and further confirmed by mutation analysis of the GPR35 promoter and by demonstrating direct binding of endogenous HIF-1 to the gene promoter. Hypoxia increased the number and density of GPR35 receptors on the cardiomyocyte cell membranes. Chemical GPR35 agonist Zaprinast caused GPR35 activation and receptor internalization in cardiac myocytes. In addition, overexpressed GPR35 disrupted actin cytoskeleton arrangement and caused morphological changes in cultured cardiomyocytes. GPR35 gene and protein expressions were also induced in mouse models of cardiac failure; the acute phase of myocardial infarction and during the compensatory and decompensatory phase of pressure-load induced cardiac hypertrophy. Cardiac expression of GPR35 is regulated by hypoxia through activation of HIF-1. The expression of GPR35 in mouse models of cardiac infarction and pressure load suggests that GPR35 could be used as an early marker of progressive cardiac failure.

Atomic Force Microscopy Study of the Effects of Water‐Soluble Fullerenes on the Elasticity of Living Plant Cells

In this work, atomic force microscopy (AFM) was employed to characterize the elastic properties of a living suspension of Nicotiana tabacum L. cv. Bright Yellow (BY-2) cells and to investigate the changes in plant-cell elasticity that were induced by water-soluble C70 fullerene derivatives. The results revealed different effects of the three fullerene derivatives that had different numbers of carboxylic groups on the cell elasticity. BY-2 cells that were repressed by dimalonic-acid-modified C70 fullerenes (DiF70) and trimalonic-acid-modified C70 fullerenes (TriF70) showed a clear decrease in their Young's modulus. However, the Young's modulus of cells that were treated with tetramalonic-acid-modified C70 fullerenes (TetraF70) increased. Disruption of the actin cytoskeleton arrangement was observed following treatment with DiF70 and TriF70, but not with TetraF70. Moreover, the fullerene-induced cell-elasticity change was consistent with the change in cell-proliferation rate. This work provides a new approach and valuable information for the study of the biological effect of nanomaterials on plant cells.

Comparative Cytotoxicity Study of Water-Soluble Carbon Nanoparticles on Plant Cells

This study provides a parallel cytotoxicity study of water-soluble single-walled carbon nanotubes and fullerene C70(C(COOH)2)4-8 on Nicotiana tabacum BY-2 cells. After BY-2 cells were treated with single-walled carbon nanotubes or C70(C(COOH)2)4-8, the cells adopted a shrunken morphology and exhibited lower proliferation rate. Fluorescence imaging of the treated cells revealed increased reactive oxygen species generation, abnormal mitochondrial distribution, decreased mitochondrial activity, and impaired actin cytoskeleton arrangement. However, no obvious cells death was induced by either single-walled carbon nanotubes or C70(C(COOH)2)4-8. Compared with single-walled carbon nanotubes, C70(C(COOH)2)4-8 exerted more serious adverse effects on BY-2 cells.

Upregulation of Nestin Protects Podocytes from Apoptosis Induced by Puromycin Aminonucleoside

Background: Nestin is an intermediate filament protein widely used as a marker of stem cells or progenitor cells. Nestin is also highly expressed in the glomerular podocyte, a type of terminally differentiated epithelial cell. Little is known about the significance of nestin in podocytes. Methods: Puromycin aminonucleoside (PAN) was injected into the rats to produce a PAN nephrosis model. Transmission electronic microscopy and terminal dUTP nick end-labeling assay were used to examine the podocyte foot process (FP) effacement and apoptosis, respectively. A mouse podocyte cell line was cultured and incubated with PAN. Immunoblot was used to examine the level of nestin expression both in vivo and in vitro. Enhanced green fluorescence protein-tagged plasmids containing nestin shRNA were transfected into the cultured podocytes to silence nestin expression. F-actin arrangement within cultured podocytes was investigated by immunofluorescence, while the apoptosis rate was examined by both Hoechst stain and flow cytometry. Results: In the PAN-induced rat nephrosis model, podocyte nestin expression was increased in the absence of apparent podocyte apoptosis, even though the FP was significantly effaced. In the cultured mouse podocytes, PAN upregulated nestin expression in a time-dependent manner within 24 h of treatment. Notably, no significant apoptosis occurred, however knocking down nestin expression resulted in a remarkable derangement of actin cytoskeleton and an increase in apoptosis in the cultured podocytes 24 h after being incubated with PAN. Conclusions: Upregulation of nestin expression during PAN nephrosis could protect podocytes from apoptosis and that this process is mediated by maintaining the regular arrangement of actin cytoskeleton.

Hypothyroidism decreases proinsulin gene expression and the attachment of its mRNA and eEF1A protein to the actin cytoskeleton of INS-1E cells

The actions of thyroid hormone (TH) on pancreatic beta cells have not been thoroughly explored, with current knowledge being limited to the modulation of insulin secretion in response to glucose, and beta cell viability by regulation of pro-mitotic and pro-apoptotic factors. Therefore, the effects of TH on proinsulin gene expression are not known. This led us to measure: a) proinsulin mRNA expression, b) proinsulin transcripts and eEF1A protein binding to the actin cytoskeleton, c) actin cytoskeleton arrangement, and d) proinsulin mRNA poly(A) tail length modulation in INS-1E cells cultured in different media containing: i) normal fetal bovine serum - FBS (control); ii) normal FBS plus 1 µM or 10 nM T3, for 12 h, and iii) FBS depleted of TH for 24 h (Tx). A decrease in proinsulin mRNA content and attachment to the cytoskeleton were observed in hypothyroid (Tx) beta cells. The amount of eEF1A protein anchored to the cytoskeleton was also reduced in hypothyroidism, and it is worth mentioning that eEF1A is essential to attach transcripts to the cytoskeleton, which might modulate their stability and rate of translation. Proinsulin poly(A) tail length and cytoskeleton arrangement remained unchanged in hypothyroidism. T3 treatment of control cells for 12 h did not induce any changes in the parameters studied. The data indicate that TH is important for proinsulin mRNA expression and translation, since its total amount and attachment to the cytoskeleton are decreased in hypothyroid beta cells, providing evidence that effects of TH on carbohydrate metabolism also include the control of proinsulin gene expression.

A QUICK Screen for Lrrk2 Interaction Partners – Leucine-rich Repeat Kinase 2 is Involved in Actin Cytoskeleton Dynamics

Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinson's disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement, and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function.