Single ion channel recording in 3D culture of stem cells using patch-clamp technique.

Tri-dimensional (3D) cell aggregates or spheroids are considered to be closer to physiological conditions than traditional 2D cell culture. Mesenchymal stem cells (MSCs) assembling in spheroids have increased the survival of transplanted cells. The organization of stem cells in 3D culture affects cell microenvironment and their mechanical properties.

Store-Operated Ca Entry Contributes to Piezo1-Induced Ca Increase in Human Endometrial Stem Cells.

Endometrial mesenchymal stem cells (eMSCs) are a specific class of stromal cells which have the capability to migrate, develop and differentiate into different types of cells such as adipocytes, osteocytes or chondrocytes. It is this unique plasticity that makes the eMSCs significant for cellular therapy and regenerative medicine. Stem cells choose their way of development by analyzing the extracellular and intracellular signals generated by a mechanical force from the microenvironment.

The analysis of F-actin structure of mesenchymal stem cells by quantification of fractal dimension.

The actin cytoskeleton is indispensable for the motility and migration of all types of cells; therefore, it plays a crucial role in the ability of the tissues to repair. Mesenchymal stem cells are intensively used in regenerative medicine, but usually relatively low percent of transplanted cells reaches the injury. To overcome this evident limitation, researchers try to enhance the motility and migration rate of the cells.

Selective Chemical Activation of Piezo1 in Leukemia Cell Membrane: Single Channel Analysis.

Piezo1/2 are mechanosensitive calcium-permeable channels that can be activated by various modes of membrane deformation. The identification of the small molecule Yoda1, a synthetic Piezo1 agonist, revealed the possibility of chemical activation of the channel. Stimulating effects of Yoda1 on Piezo1 have been mainly documented using over-expressing cellular systems or channel proteins incorporated in artificial lipid bilayers.

Adenosine-5'-triphosphate suppresses proliferation and migration capacity of human endometrial stem cells.

Extracellular ATP through the activation of the P2X and P2Y purinergic receptors affects the migration, proliferation and differentiation of many types of cells, including stem cells. High plasticity, low immunogenicity and immunomodulation ability of mesenchymal stem cells derived from human endometrium (eMSCs) allow them to be considered a prominent tool for regenerative medicine. Here, we examined the role of ATP in the proliferation and migration of human eMSCs.

Downregulation of TRPV6 channel activity by cholesterol depletion in Jurkat T cell line.

Transient receptor potential vanilloid 6 (TRPV6) channels are key players in calcium metabolism of healthy and cancerous cells. Nevertheless, the mechanisms controlling abundance of these channels in plasma membrane of the cells to regulate Ca transport is still poorly understood. In this study, we provide the first evidence that TRPV6 calcium channels and Ca influx in Jurkat T cell line are modulated by cholesterol, a main lipid component of the plasma membrane.

Agonist-induced Piezo1 activation suppresses migration of transformed fibroblasts.

Increased migratory, invasive and metastatic potential is one of the main pathophysiological determinants of malignant cells. Mechanosensitive calcium-permeable ion channels are among the key membrane proteins that participate in processes of cellular motility. Local calcium influx via mechanosensitive channels was proposed to regulate calcium-dependent molecules involved in cell migration.

Cell Cycle-Dependent Expression of Bk Channels in Human Mesenchymal Endometrial Stem Cells.

The study of ion channels in stem cells provides important information about their role in stem cell fate. Previously we have identified the activity of calcium-activated potassium channels of big conductance (BK channels) in human endometrium-derived mesenchymal stem cells (eMSCs). BK channels could have significant impact into signaling processes by modulating membrane potential.

Extracellular protease trypsin activates amiloride-insensitive sodium channels in human leukemia cells.

Sodium influx is tightly regulated in the cells of blood origin. Amiloride-insensitive sodium channels were identified as one of the main sodium-transporting pathways in leukemia cells. To date, all known regulatory pathways of these channels are coupled with intracellular actin cytoskeleton dynamics.

Role of the Scaffold Protein MIM in the Actin-Dependent Regulation of Epithelial Sodium Channels (ENaC).

Epithelial Sodium Channels (ENaCs) are expressed in different organs and tissues, particularly in the cortical collecting duct (CCD) in the kidney, where they fine tune sodium reabsorption. Dynamic rearrangements of the cytoskeleton are one of the common mechanisms of ENaC activity regulation. In our previous studies, we showed that the actin-binding proteins cortactin and Arp2/3 complex are involved in the cytoskeleton-dependent regulation of ENaC and that their cooperative work decreases a channel's probability of remaining open; however, the specific mechanism of interaction between actin-binding proteins and ENaC is unclear.

Simvastatin induced actin cytoskeleton disassembly in normal and transformed fibroblasts without affecting lipid raft integrity.

Statins are the most commonly prescribed agents used to modulate cholesterol levels in course of hypercholesterolemia treatment because of their relative tolerability and LDL-C lowering effect. Recently, there are emerging interests in the perspectives of statin drugs as anticancer agents based on preclinical evidence of their antiproliferative, proapoptotic, and anti-invasive properties. Functional impact of statin application on transformed cells still remains obscure that requires systematic study on adequate cellular models to provide correct comparison with their non-transformed counterparts.

Local calcium signalling is mediated by mechanosensitive ion channels in mesenchymal stem cells.

Mechanical forces are implicated in key physiological processes in stem cells, including proliferation, differentiation and lineage switching. To date, there is an evident lack of understanding of how external mechanical cues are coupled with calcium signalling in stem cells. Mechanical reactions are of particular interest in adult mesenchymal stem cells because of their promising potential for use in tissue remodelling and clinical therapy.

[SPREADING OF NCTC CLONE 929 CELLS AFTER RESEEDING].

The period (1 h after reseeding) of behaviour of mouse NCTC clone 929 cells to the conditions of artificial cultivation was studied. The time-lapse imaging followed the processing of the cells with ImageJ program was applied. To characterize the parametres cell status we used the cell area (projection of the cell on substrate) and Rp/Ra ratio introduced earlier as a spreading coefficient (Kuz'minykh, Petrov, 2004).

TRPV5/V6 Channels Mediate Ca(2+) Influx in Jurkat T Cells Under the Control of Extracellular pH.

Regulation of cytoplasmic free calcium concentration [Ca(2+)]i is a key factor for the maintenance of cellular homeostasis in different cell types, including lymphocytes. During T lymphocyte activation as well as production of cytokines, sustained Ca(2+) influx is essential, however, it remains unclear how this influx is regulated. Previously, we reported the expression and functional activity of calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid type 5 and 6) in human leukemia Jurkat T cells.

Amiloride-insensitive sodium channels are directly regulated by actin cytoskeleton dynamics in human lymphoma cells.

Sodium influx mediated by ion channels of plasma membrane underlies fundamental physiological processes in cells of blood origin. However, little is known about the single channel activity and regulatory mechanisms of sodium-specific channels in native cells. In the present work, we used different modes of patch clamp technique to examine ion channels involved in Na-transporting pathway in U937 human lymphoma cells.

Obtaining spheroplasts of armored dinoflagellates and first single-channel recordings of their ion channels using patch-clamping.

Ion channels are tightly involved in various aspects of cell physiology, including cell signaling, proliferation, motility, endo- and exo-cytosis. They may be involved in toxin production and release by marine dinoflagellates, as well as harmful algal bloom proliferation. So far, the patch-clamp technique, which is the most powerful method to study the activity of ion channels, has not been applied to dinoflagellate cells, due to their complex cellulose-containing cell coverings.

Functional coupling of ion channels in cellular mechanotransduction.

The major players in the processes of cellular mechanotransduction are considered to be mechanosensitive (MS) or mechano-gated ion channels. Non-selective Ca(2+)-permeable channels, whose activity is directly controlled by membrane stretch (stretch-activated channels, SACs) are ubiquitously present in mammalian cells of different origin. Ca(2+) entry mediated by SACs presumably has a significant impact on various Ca(2+)-dependent intracellular and membrane processes.

Intracellular oxidation of hydroethidine: compartmentalization and cytotoxicity of oxidation products.

Hydroethidine (HE) is a blue fluorescent dye that is intracellularly converted into red-emitting products on two-electron oxidation. One of these products, namely 2-hydroxyethidium, is formed as the result of HE superoxide anion-specific oxidation, and so HE is widely used for the detection of superoxide in cells and tissues. In our experiments we exploited three cell lines of different origin: K562 (human leukemia cells), A431 (human epidermoid carcinoma cells), and SCE2304 (human mesenchymal stem cells derived from endometrium).

Angiotensin II has acute effects on TRPC6 channels in podocytes of freshly isolated glomeruli.

A key role for podocytes in the pathogenesis of proteinuric renal diseases has been established. Angiotensin II causes depolarization and increased intracellular calcium concentration in podocytes; members of the cation TRPC channels family, particularly TRPC6, are proposed as proteins responsible for calcium flux. Angiotensin II evokes calcium transient through TRPC channels and mutations in the gene encoding the TRPC6 channel result in the development of focal segmental glomerulosclerosis.

Arp2/3 complex inhibitors adversely affect actin cytoskeleton remodeling in the cultured murine kidney collecting duct M-1 cells.

Dynamic remodeling of the actin cytoskeleton plays an essential role in cell migration and various signaling processes in living cells. One of the critical factors that controls the nucleation of new actin filaments in eukaryotic cells is the actin-related protein 2/3 (Arp2/3) complex. Recently, two novel classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate F-actin have been discovered and described.

Expression of transient receptor potential vanilloid channels TRPV5 and TRPV6 in human blood lymphocytes and Jurkat leukemia T cells.

Regulation of Ca(2+) entry is a key process for lymphocyte activation, cytokine synthesis and proliferation. Several members of the transient receptor potential (TRP) channel family can contribute to changes in [Ca(2+)](in); however, the properties and expression levels of these channels in human lymphocytes continue to be elusive. Here, we established and compared the expression of the most Ca(2+)-selective members of the TRPs, Ca(2+) channels transient receptor potential vanilloid 5 and 6 (TRPV5 and TRPV6), in human blood lymphocytes (HBLs) and leukemia Jurkat T cells.

Regulation of TRPC6 Channels by Non-Steroidal Anti-Inflammatory Drugs.

Family focal segmental glomerulosclerosis (FSGS) is characterized by sclerosis and hyalinosis of particular loops of glomeruli and is one of the causes of the nephrotic syndrome. Certain mutations in the structure of TRPC6 channels are the genetic impetus for FSGS development resulting in podocytes functional abnormalities and various nephropathies. We have recently demonstrated that non-steroid anti-inflammatory drugs (NSAID) ibuprofen and diclofenac decrease the activity of endogenous TRPC like cal cium channels in the podocytes of the freshly isolated rat glomeruli.

Cholesterol depletion-induced inhibition of stretch-activated channels is mediated via actin rearrangement.

Cholesterol is a critical regulator of lipid bilayer dynamics and plasma membrane organization in eukaryotes. A variety of ion channels have been shown to be modulated by cellular cholesterol and partition into cholesterol-enriched membrane rafts. However, very little is known about functional role of membrane cholesterol in regulation of mechanically gated channels that are ubiquitously present in living cells.

Cortical actin binding protein cortactin mediates ENaC activity via Arp2/3 complex.

Epithelial Na(+) channel (ENaC) activity is regulated, in part, by the cortical cytoskeleton. Here we demonstrate that cortactin is highly expressed in the kidney cortex and polarized epithelial cells, and is localized to the cortical collecting duct. Coexpression of cortactin with ENaC decreases ENaC activity, as measured in patch-clamp experiments.