scholarly journals Involvement of Actin Microfilament in Regulation of Pacemaking Activity Increased by Hypotonic Stress in Cultured ICCs of Murine Intestine

2015 ◽  
pp. 397-405 ◽  
Author(s):  
Z. Y. WANG ◽  
X. HUANG ◽  
D. H. LIU ◽  
H. L. LU ◽  
Y. C. KIM ◽  
...  
Keyword(s):  
Cytochalasin B ◽  
Imaging Techniques ◽  
Oscillation Amplitude ◽  
Mechanical Stimulus ◽  
Calcium Oscillations ◽  
Calcium Oscillation ◽  
Actin Microfilaments ◽  
Actin Microfilament ◽  
Hypotonic Stress ◽  
Pacemaker Currents

Distension is a regular mechanical stimulus in gastrointestinal (GI) tract. This study was designed to investigate the effect of hypotonic stress on pacemaking activity and determine whether actin microfilament is involved in its mechanism in cultured murine intestinal interstitial cells of Cajal (ICCs) by using whole-cell patch-clamp and calcium imaging techniques. Hypotonic stress induced sustained inward holding current from the baseline to –650±110 pA and significantly decreased amplitudes of pacemaker current. Hypotonic stress increased the intensity of basal fluorescence ratio (F/F0) from baseline to 1.09±0.03 and significantly increased Ca2+ oscillation amplitude. Cytochalasin-B (20 μM), a disruptor of actin microfilaments, significantly suppressed the amplitudes of pacemaker currents and calcium oscillations, respectively. Cytochalasin-B also blocked hypotonic stress-induced sustained inward holding current and hypotonic stress-induced increase of calcium oscillations. Phalloidin (20 μM), a stabilizer of actin microfilaments, significantly enhanced the amplitudes of pacemaker currents and calcium oscillations, respectively. Despite the presence of phalloidin, hypotonic stress was still able to induce an inward holding current and increased the basal fluorescence intensity. These results suggest that hypotonic stress induces sustained inward holding current via actin microfilaments and the process is mediated by alteration of intracellular basal calcium concentration and calcium oscillation in cultured intestinal ICCs.

scholarly journals Effects of Porcine Immature Oocyte Vitrification on Actin Microfilament Distribution and Chromatin Integrity During Early Embryo Development in vitro

2021 ◽  
Vol 9 ◽  
Author(s):  
Alma López ◽  
Yvonne Ducolomb ◽  
Eduardo Casas ◽  
Socorro Retana-Márquez ◽  
Miguel Betancourt ◽  
...  
Keyword(s):  
Embryo Development ◽  
Early Embryo ◽  
Actin Microfilaments ◽  
Cell Protection ◽  
Developmental Potential ◽  
Early Embryo Development ◽  
Immature Oocytes ◽  
Actin Microfilament ◽  
Development Rates ◽  
Chromatin Integrity

Vitrification is mainly used to cryopreserve female gametes. This technique allows maintaining cell viability, functionality, and developmental potential at low temperatures into liquid nitrogen at −196°C. For this, the addition of cryoprotectant agents, which are substances that provide cell protection during cooling and warming, is required. However, they have been reported to be toxic, reducing oocyte viability, maturation, fertilization, and embryo development, possibly by altering cell cytoskeleton structure and chromatin. Previous studies have evaluated the effects of vitrification in the germinal vesicle, metaphase II oocytes, zygotes, and blastocysts, but the knowledge of its impact on their further embryo development is limited. Other studies have evaluated the role of actin microfilaments and chromatin, based on the fertilization and embryo development rates obtained, but not the direct evaluation of these structures in embryos produced from vitrified immature oocytes. Therefore, this study was designed to evaluate how the vitrification of porcine immature oocytes affects early embryo development by the evaluation of actin microfilament distribution and chromatin integrity. Results demonstrate that the damage generated by the vitrification of immature oocytes affects viability, maturation, and the distribution of actin microfilaments and chromatin integrity, observed in early embryos. Therefore, it is suggested that vitrification could affect oocyte repair mechanisms in those structures, being one of the mechanisms that explain the low embryo development rates after vitrification.

scholarly journals Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells.

1990 ◽  
Vol 111 (1) ◽  
pp. 103-112 ◽  
Author(s):  
A Fernandez ◽  
D L Brautigan ◽  
M Mumby ◽  
N J Lamb
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Metabolic Labeling ◽  
Actin Microfilaments ◽  
Actin Microfilament ◽  
Nonmuscle Cells ◽  
Dependent Protein ◽  
Type 1 Phosphatase

Dynamic reorganization of the actin microfilament networks is dependent on the reversible phosphorylation of myosin light chain. To assess the potential role of protein phosphatases in this process in living nonmuscle cells, we have microinjected the purified type-1 and type-2A phosphatases into the cytoplasm of mammalian fibroblasts. Our studies reveal that elevating type-1 phosphatase levels led to the rapid (within 30 min) and fully reversible disassembly of the actin microfilament network as determined by immunofluorescence analysis. In contrast, microinjection of equivalent amounts of the purified type-2A phosphatase had no effect on actin microfilament organization. Metabolic labeling of cells after injection of purified phosphatases was used to analyze changes in protein phosphorylation. Concomitant with the disassembly of the actin microfilaments induced by type-1 phosphatase, there was an extensive dephosphorylation of myosin light chain. No such change was observed when cells were injected with type-2A phosphatase. In addition, after extraction of fibroblasts with Triton X-100, the type-1 phosphatase could be specifically localized by immunofluorescence to a fibrillar network of microfilaments. Furthermore, neutralizing type-1 phosphatase activity in vivo by microinjection of an affinity-purified antibody, prevented the reorganization of actin microfilaments that we had previously described following injection of cAMP-dependent protein kinase. These data support the notion that type 1 and type-2 phosphatases have distinct substrate specificity in living cells, and that type-1 phosphatase plays a predominant role in the dephosphorylation of myosin light chain and thus in the modulation of actin microfilament organization in vivo in intact nonmuscle cells.

Fast calcium wave propagation mediated by electrically conducted excitation and boosted by CICR

2008 ◽  
Vol 294 (4) ◽  
pp. C917-C930 ◽  
Author(s):  
J. M. A. M. Kusters ◽  
W. P. M. van Meerwijk ◽  
D. L. Ypey ◽  
A. P. R. Theuvenet ◽  
C. C. A. M. Gielen
Keyword(s):  
Intracellular Calcium ◽  
Chloride Channels ◽  
Calcium Release ◽  
Rat Kidney ◽  
Electrical Coupling ◽  
Calcium Oscillations ◽  
Calcium Wave ◽  
Calcium Oscillation ◽  
Pacemaker Cell ◽  
Pacemaker Cells

We have investigated synchronization and propagation of calcium oscillations, mediated by gap junctional excitation transmission. For that purpose we used an experimentally based model of normal rat kidney (NRK) cells, electrically coupled in a one-dimensional configuration (linear strand). Fibroblasts such as NRK cells can form an excitable syncytium and generate spontaneous inositol 1,4,5-trisphosphate (IP3)-mediated intracellular calcium waves, which may spread over a monolayer culture in a coordinated fashion. An intracellular calcium oscillation in a pacemaker cell causes a membrane depolarization from within that cell via calcium-activated chloride channels, leading to an L-type calcium channel-based action potential (AP) in that cell. This AP is then transmitted to the electrically connected neighbor cell, and the calcium inflow during that transmitted AP triggers a calcium wave in that neighbor cell by opening of IP3 receptor channels, causing calcium-induced calcium release (CICR). In this way the calcium wave of the pacemaker cell is rapidly propagated by the electrically transmitted AP. Propagation of APs in a strand of cells depends on the number of terminal pacemaker cells, the L-type calcium conductance of the cells, and the electrical coupling between the cells. Our results show that the coupling between IP3-mediated calcium oscillations and AP firing provides a robust mechanism for fast propagation of activity across a network of cells, which is representative for many other cell types such as gastrointestinal cells, urethral cells, and pacemaker cells in the heart.

Regulation of intracellular calcium oscillations in porcine tracheal smooth muscle cells

1997 ◽  
Vol 272 (3) ◽  
pp. C966-C975 ◽  
Author(s):  
Y. S. Prakash ◽  
M. S. Kannan ◽  
G. C. Sieck
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Confocal Microscopy ◽  
Ryanodine Receptor ◽  
Rise Time ◽  
Oscillation Amplitude ◽  
Calcium Oscillations ◽  
Tracheal Smooth Muscle ◽  
Release Mechanism ◽  
Ca2 Channels

Using real-time confocal microscopy, we examined the dynamic intracellular Ca2+ concentration ([Ca2+]i) response of porcine tracheal smooth muscle (TSM) cells to acetylcholine (ACh). Exposure to ACh caused regenerative, propagating [Ca2+]i oscillations. The amplitude and fall time of the [Ca2+]i oscillations were inversely correlated to basal [Ca2+]i, whereas the frequency and rise time were directly correlated to basal [Ca2+]i. ACh-induced [Ca2+]i oscillations were initiated in the absence of extracellular Ca2+ and after membrane depolarization with KCl, suggesting that 1) [Ca2+]i oscillations primarily arise by release from internal stores such as the sarcoplasmic reticulum (SR), and 2) Ca2+ influx is necessary for maintenance of oscillations. Exposure to both caffeine and ryanodine inhibited ongoing ACh-induced [Ca2+]i oscillations, suggesting a role for caffeine-sensitive ryanodine receptor (RyR) SR Ca2+ channels. Inhibition of SR Ca2+ reuptake by thapsigargin increased basal [Ca2+]i and decreased [Ca2+]i oscillation amplitude, suggesting that Ca2+ reuptake is also essential. The present results suggest that [Ca2+]i oscillations in porcine TSM cells involve repetitive Ca2+ release and reuptake from RyR channels, perhaps through a Ca2+ -induced Ca2+ release mechanism.

A disrupter of actin microfilaments impairs sulfonylurea-inhibitory gating of cardiac KATP channels

1996 ◽  
Vol 271 (6) ◽  
pp. H2710-H2716 ◽  
Author(s):  
P. A. Brady ◽  
A. E. Alekseev ◽  
L. A. Aleksandrova ◽  
L. A. Gomez ◽  
A. Terzic
Keyword(s):  
Katp Channel ◽  
Katp Channels ◽  
Hill Coefficient ◽  
Kinetic Properties ◽  
Actin Microfilaments ◽  
Actin Microfilament ◽  
Channel Inhibition ◽  
Dnase Treatment ◽  
Single Channel Currents ◽  
Sulfonylurea Drugs

The efficacy with which sulfonylurea drugs inhibit cardiac ATP-sensitive K+ (KATP) channels is reduced during metabolic compromise and cellular contracture. Disruption of the actin microfilament network, which occurs under similar conditions, reduces the sensitivity of the channel toward intracellular ATP. To investigate whether a disrupter of actin microfilaments could also affect the responsiveness of the KATP channel to sulfonylurea drugs, single-channel currents were measured in the inside-out configuration of excised patches from guinea pig ventricular myocytes. Treatment of the internal side of patches with deoxyribonuclease (DNase) I (100 micrograms/ml), which forms complexes with G actin and prevents actin filament formation, antagonized sulfonylurea-induced inhibition of KATP channels that was coupled with a loss of sensitivity to ATP. The apparent dissociation constant and Hill coefficient for the inhibitory effect of glyburide, a prototype sulfonylurea, on KATP-channel opening were, respectively, 0.13 microM and 0.95 before and 2.7 microM and 0.98 after DNase treatment. DNase did not alter intraburst kinetic properties of the channel. When DNase was denatured or coincubated with purified actin (200 micrograms/ml), it no longer decreased glyburide-induced channel inhibition. This suggests that sulfonylurea-inhibitory gating of cardiac KATP channels may also be regulated through a mechanism involving subsarcolemmal actin microfilament networks.

scholarly journals The Endothelial Cytoskeleton: Organization in Normal and Regenerating Endothelium*

1990 ◽  
Vol 18 (4a) ◽  
pp. 603-617 ◽  
Author(s):  
Avrum I. Gotlieb
Keyword(s):  
Shear Stress ◽  
Structural Integrity ◽  
Phorbol Esters ◽  
Actin Microfilaments ◽  
Actin Microfilament ◽  
Cytoskeleton Organization ◽  
Cell Locomotion ◽  
Microfilament Bundles ◽  
External Stimuli ◽  
Hemodynamic Shear Stress

The components of the endothelial cell cytoskeleton that have been shown to be important in maintaining endothelial structural integrity and in regulating endothelial repair include F-actin microfilament bundles, including stress fibers, and microtubules, and centrosomes. Endothelial cells contain peripheral and central actin microfilaments. The dense peripheral band (DPB) consists of peripheral actin microfilament bundles which are associated with vinculin adhesion plaques and are most prominent in low or no hemodynamic shear stress conditions. The central microfilaments are very prominent in areas of elevated hemodynamic shear stress. There is a redistribution of actin microfilaments characterized by a decrease of peripheral actin and an increase in central microfilaments under a variety of conditions, including exposure to thrombin, phorbol-esters, and hemodynamic shear stress. During reendothelialization, there is a sequential series of cytoskeletal changes. The DPB remains intact during the rapid lamellipodia mediated repair of very small wounds except at the base of the lamellipodia where it is splayed. The DPB is reduced or absent when cell locomotion occurs to repair a wound. In addition, when cell locomotion is required, the centrosome, in the presence of intact microtubules, redistributes to the front of the cell to establish cell polarity and acts as a modulator of the directionality of migration. This occurs prior to the loss of the DPB but does not occur in very small wounds that close without migration. Thus, the cytoskeleton is a dynamic intracellular system which regulates endothelial integrity and repair and is modulated by external stimuli that are present at the vessel wall-blood interface.

scholarly journals Roles of TRPV4 and piezo channels in stretch-evoked Ca2+ response in chondrocytes

2019 ◽  
Vol 245 (3) ◽  
pp. 180-189 ◽  
Author(s):  
Genlai Du ◽  
Li Li ◽  
Xinwang Zhang ◽  
Jianbing Liu ◽  
Jianqing Hao ◽  
...  
Keyword(s):  
Ion Channels ◽  
Transient Receptor Potential ◽  
Imaging System ◽  
Receptor Potential ◽  
Mechanical Stretch ◽  
Mechanical Stimulus ◽  
Calcium Oscillation ◽  
Transient Receptor Potential Vanilloid ◽  
Transient Receptor ◽  
Cartilage Disease

Chondrocyte mechanotransduction is not well understood, but recently, it has been proposed that mechanically activated ion channels such as transient receptor potential vanilloid 4 (TRPV4), Piezo1, and Piezo2 are of functional importance in chondrocyte mechanotransduction. The aim of this study was to distinguish the potential contributions of TRPV4, Piezo1, and Piezo2 in transducing different intensities of repetitive mechanical stimulus in chondrocytes. To study this, TRPV4-, Piezo1-, or Piezo2-specific siRNAs were transfected into cultured primary chondrocytes to knock down (KD) TRPV4, Piezo1, or Piezo2 expression, designated TRPV4-KD, Piezo1-KD, or Piezo2-KD cells. Then we used Flexcell® Tension System to apply cyclic tensile strains (CTS) of 3% to 18% at 0.5 Hz for 8 h to the knockdown and control siRNA-treated cells. Finally, using a Ca2+ imaging system, stretch-evoked intracellular Ca2+ ([Ca2+] i) influx in chondrocytes was examined to investigate the roles of TRPV4, Piezo1, and Piezo2 in Ca2+ signaling in response to different intensities of repetitive mechanical stretch stimulation. The characteristics of [Ca2+] i in chondrocytes evoked by stretch stimulation were stretch intensity dependent when comparing unstretched cells. In addition, stretch-evoked [Ca2+] i changes were significantly suppressed in TRPV4-KD, Piezo1-KD, or Piezo2-KD cells compared with control siRNA-treated cells, indicating that any channel essential for Ca2+ signaling induced by stretch stimulation in chondrocytes. Of note, they played different roles in calcium oscillation induced by different intensities of stretch stimulation. More specifically, TRPV4-mediated Ca2+ signaling played a central role in the response of chondrocytes to physiologic levels of strain (3% and 8% of strain), while Piezo2-mediated Ca2+ signaling played a central role in the response of chondrocytes to injurious levels of strain (18% of strain). These results provide a basis for further examination of mechanotransduction in cartilage and raise a possibility of therapeutically targeting Piezo2-mediated mechanotransduction for the treatment of cartilage disease induced by repetitive mechanical forces. Impact statement Chondrocytes in cartilage are constantly subjected to load-induced stimuli and regulate their metabolic activities in order to maintain cartilage homeostasis. Therefore, mechanotransduction is important in chondrocytes and is vital for their role in cartilage function. Our results indicate that chondrocytes might sense and distinguish the different intensities of repetitive mechanical stimulus by using different mechanosensitive ion channels. Specifically, TRPV4 is mainly responsible for sensing physiologic levels of repetitive CTS stimulus, while Piezo2 mainly contributes to chondrocyte sensing noxious levels of repetitive CTS loading. These results provide a basis for further examination of mechanotransduction in cartilage and raise the possibility of therapeutically targeting Piezo2-mediated mechanotransduction for the treatment of OA which is induced by injurious and repetitive mechanical stimulation.

Motility factor produced by malignant glioma cells: role in tumor invasion

1990 ◽  
Vol 73 (6) ◽  
pp. 881-888 ◽  
Author(s):  
Takanori Ohnishi ◽  
Norio Arita ◽  
Toru Hayakawa ◽  
Shuichi Izumoto ◽  
Takuyu Taki ◽  
...  
Keyword(s):  
Cell Migration ◽  
Malignant Glioma ◽  
Tumor Invasion ◽  
Cytochalasin B ◽  
Glioma Cells ◽  
Actin Microfilaments ◽  
Content Type ◽  
Motility Factor ◽  
Malignant Glioma Cells ◽  
Fine Print

✓ To better understand the cellular mechanism of tumor invasion, the production of a cell motility-stimulating factor by malignant glioma cells was studied in vitro. Serum-free conditioned media from cultures of rat C6 and human T98G cell lines contained a factor that stimulated the locomotion of the producer cells. This factor was termed the “glioma-derived motility factor.” The glioma-derived motility factor is a heat-labile protein with a molecular weight greater than 10 kD and has relative stability to acid. The factor showed not only chemotactic activity but also chemokinetic (stimulated random locomotion) activity in the two types of glioma cells studied. Although glioma-derived motility factors in conditioned media obtained from two different cell origins are likely to be the same, chemokinetic migration of T98G cells to their conditioned medium was much stronger than that of C6 cells to theirs. Coincubation of cells with cytochalasin B, which disrupts the assembly of cellular actin microfilaments, almost completely inhibited the cell migration stimulated by glioma-derived motility factor. Cytochalasin B also induced marked alterations in cell morphology, including cell retraction and arborization, while the drug did not affect cell attachment to culture dishes. These results indicate that glioma cells produce a motility factor which may play a role particularly when tumor cells are detached and migrate away from the original tumor mass, thus promoting tumor invasion. Also, glioma cell migration stimulated by the motility factor requires the normal organization of cytoskeletons such as actin microfilaments.

Actin microfilament and microtubule distribution patterns in the expanding root of Arabidopsis thaliana

2005 ◽  
Vol 83 (6) ◽  
pp. 579-590 ◽  
Author(s):  
David A Collings ◽  
Geoffrey O Wasteneys
Keyword(s):  
Arabidopsis Thaliana ◽  
Imaging Techniques ◽  
Cytoskeletal Proteins ◽  
Cell Cortex ◽  
Actin Microfilaments ◽  
Elongation Zone ◽  
Root Tips ◽  
Microtubule Organization ◽  
Microscopy Imaging ◽  
Distal Elongation Zone

Determination of the precise role(s) of actin microfilaments in the control of cell shape and elongation in the root tips of the model genetic system Arabidopsis thaliana (L.) Heynh is frustrated by inadequate microscopy imaging techniques. In this paper, we documented both microfilaments and microtubules in the root tips of Arabidopsis by double immunofluorescence labelling and computer-generated reconstruction of confocal image series. Our procedure, which complements the use of recently developed fluorescent reporter proteins, revealed hitherto undescribed aspects of the Arabidopsis microfilament cytoskeleton that may provide important clues about mechanisms behind cell elongation. We found that preservation of extensive arrays of transverse cortical microfilaments depends on unperturbed microtubule organization. Compared with ordinary epidermal cells, cells situated in the trichoblast or hair-forming cell files were comparatively devoid of endoplasmic microfilaments when in the distal elongation zone, well before hair formation begins. Computer-aided reconstructions also revealed that the nonexpanding end walls of cells in the distal elongation zone have radially oriented microtubules and randomly arranged microfilaments. In dividing cells, microfilaments became more prominent in the cell cortex, and subtle differences between microtubule and microfilament organization were seen within the phragmoplast. These observations will form the basis of understanding the roles of the cytoskeleton in controlling elongation in root tissues. In light of the many Arabidopsis mutants with altered root morphology, our methods offer a reliable approach to assess the function of cytoskeletal proteins and signalling systems in root morphogenesis.Key words: actin microfilaments, Arabidopsis thaliana, distal elongation zone, microtubules, phragmoplast, roots.

scholarly journals PIN2 Polarity Establishment in Arabidopsis in the Absence of an Intact Cytoskeleton

Biomolecules ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 222 ◽  
Author(s):  
Matouš Glanc ◽  
Matyáš Fendrych ◽  
Jiří Friml
Keyword(s):  
Subcellular Localization ◽  
Live Cell Imaging ◽  
Auxin Transport ◽  
Imaging Techniques ◽  
Actin Microfilaments ◽  
Coordinated Development ◽  
Arabidopsis Root ◽  
Polar Localization ◽  
Multicellular Organisms ◽  
Endomembrane Trafficking

Cell polarity is crucial for the coordinated development of all multicellular organisms. In plants, this is exemplified by the PIN-FORMED (PIN) efflux carriers of the phytohormone auxin: The polar subcellular localization of the PINs is instructive to the directional intercellular auxin transport, and thus to a plethora of auxin-regulated growth and developmental processes. Despite its importance, the regulation of PIN polar subcellular localization remains poorly understood. Here, we have employed advanced live-cell imaging techniques to study the roles of microtubules and actin microfilaments in the establishment of apical polar localization of PIN2 in the epidermis of the Arabidopsis root meristem. We report that apical PIN2 polarity requires neither intact actin microfilaments nor microtubules, suggesting that the primary spatial cue for polar PIN distribution is likely independent of cytoskeleton-guided endomembrane trafficking.

Sign in / Sign up

Export Citation Format

Share Document