scholarly journals The mechano-sensitive ion channel Piezo mediates Rho activation and actin stress fibre formation in Drosophila nephrocytes

2021 ◽  
Author(s):  
Sybille Koehler ◽  
Barry Denholm
Keyword(s):  
Shear Stress ◽  
Function Analysis ◽  
Mechanical Force ◽  
Loss Of Function ◽  
Actin Stress Fibre ◽  
Biomechanical Force ◽  
Channel Opening ◽  
Intracellular Ca ◽  
A Cell ◽  
Sense And Respond

Mechanotransduction is an important process of sensing physical forces in the environment of organisms, tissues and cells and transducing them into a biochemical response. Due to their position on the glomerular capillaries, podocytes are exposed to near-constant biomechanical force, which can fluctuate widely. These include shear stress and hydrostatic pressure. A pathological increase in these forces can induce morphological change to podocytes, their detachment from the glomerular basement membrane and subsequent loss into the primary urine. The ability to sense and respond to variations in mechanical force would be beneficial to a cell exposed to these conditions. It is likely podocytes have such mechanisms, however their identity are unknown. Here we investigated the hypothesis that the mechanotransducer Piezo is involved in a mechanotransduction pathway in Drosophila nephrocytes, the podocyte homologue in the fly. We find Piezo is expressed in nephrocytes and localizes to the nephrocyte diaphragm. The Piezo agonist YODA, which stimulates channel opening in the absence of mechanical force, leads to a significant increase in intracellular Ca++ upon shear stress in the nephrocyte. This leads to activation of Rho1, delineating a putative Piezo mechanotransductive pathway in these cells. Loss of function analysis revealed minor defects in nephrocyte filtration function. In contrast, we show that elevated Piezo levels resulted in constantly oscillating Ca++ signals even in the absence of shear stress, increased active Rho1 and accumulation of actin stress fibers, culminating in a severe nephrocyte filtration phenotype, suggesting that pathway hyperactivity is detrimental. We asked if this phenotype could be reversed by blocking Piezo activity pharmacologically using the tarantula toxin GsMTx4. Treatment with GsMTx4 brought levels of activated Rho1 into the normal range. This work delineates a mechanotransductive pathway in nephrocytes involving Piezo, Ca++, Rho1 and the actin-cytoskeleton, and suggest this is part of a mechanism by which nephrocytes sense and adapt to changes in mechanical force.

scholarly journals Maturation of the Na,K-ATPase in the Endoplasmic Reticulum in Health and Disease

2021 ◽  
Author(s):  
Vitalii Kryvenko ◽  
Olga Vagin ◽  
Laura A. Dada ◽  
Jacob I. Sznajder ◽  
István Vadász
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Signaling ◽  
Loss Of Function ◽  
Α Subunit ◽  
Rate Limiting Step ◽  
Atpase Subunits ◽  
A Cell ◽  
Health And Disease ◽  
And Function ◽  
Rate Limiting

Abstract The Na,K-ATPase establishes the electrochemical gradient of cells by driving an active exchange of Na+ and K+ ions while consuming ATP. The minimal functional transporter consists of a catalytic α-subunit and a β-subunit with chaperon activity. The Na,K-ATPase also functions as a cell adhesion molecule and participates in various intracellular signaling pathways. The maturation and trafficking of the Na,K-ATPase include co- and post-translational processing of the enzyme in the endoplasmic reticulum (ER) and the Golgi apparatus and subsequent delivery to the plasma membrane (PM). The ER folding of the enzyme is considered as the rate-limiting step in the membrane delivery of the protein. It has been demonstrated that only assembled Na,K-ATPase α:β-complexes may exit the organelle, whereas unassembled, misfolded or unfolded subunits are retained in the ER and are subsequently degraded. Loss of function of the Na,K-ATPase has been associated with lung, heart, kidney and neurological disorders. Recently, it has been shown that ER dysfunction, in particular, alterations in the homeostasis of the organelle, as well as impaired ER-resident chaperone activity may impede folding of Na,K-ATPase subunits, thus decreasing the abundance and function of the enzyme at the PM. Here, we summarize our current understanding on maturation and subsequent processing of the Na,K-ATPase in the ER under physiological and pathophysiological conditions. Graphic Abstract

scholarly journals Mechanical force effect on the two-state equilibrium of the hyaluronan-binding domain of CD44 in cell rolling

2015 ◽  
Vol 112 (22) ◽  
pp. 6991-6996 ◽  
Author(s):  
Takashi Suzuki ◽  
Miho Suzuki ◽  
Shinji Ogino ◽  
Ryo Umemoto ◽  
Noritaka Nishida ◽  
...  
Keyword(s):  
Shear Stress ◽  
Conformational Change ◽  
Mechanical Force ◽  
Binding Domain ◽  
Terminal Region ◽  
Hematopoietic Progenitor Cell ◽  
High Shear ◽  
Cell Rolling ◽  
High Affinity ◽  
C Terminus

CD44 is the receptor for hyaluronan (HA) and mediates cell rolling under fluid shear stress. The HA-binding domain (HABD) of CD44 interconverts between a low-affinity, ordered (O) state and a high-affinity, partially disordered (PD) state, by the conformational change of the C-terminal region, which is connected to the plasma membrane. To examine the role of tensile force on CD44-mediated rolling, we used a cell-free rolling system, in which recombinant HABDs were attached to beads through a C-terminal or N-terminal tag. We found that the rolling behavior was stabilized only at high shear stress, when the HABD was attached through the C-terminal tag. In contrast, no difference was observed for the beads coated with HABD mutants that constitutively adopt either the O state or the PD state. Steered molecular dynamics simulations suggested that the force from the C terminus disrupts the interaction between the C-terminal region and the core of the domain, thus providing structural insights into how the mechanical force triggers the allosteric O-to-PD transition. Based on these results, we propose that the force applied from the C terminus enhances the HABD–HA interactions by inducing the conformational change to the high-affinity PD transition more rapidly, thereby enabling CD44 to mediate lymphocyte trafficking and hematopoietic progenitor cell homing under high-shear conditions.

scholarly journals Laminar shear stress upregulates endothelial Ca2+-activated K+ channels KCa2.3 and KCa3.1 via a Ca2+/calmodulin-dependent protein kinase kinase/Akt/p300 cascade

2013 ◽  
Vol 305 (4) ◽  
pp. H484-H493 ◽  
Author(s):  
Jun Takai ◽  
Alexandra Santu ◽  
Haifeng Zheng ◽  
Sang Don Koh ◽  
Masanori Ohta ◽  
...  
Keyword(s):  
Shear Stress ◽  
Transcriptional Activation ◽  
Static Condition ◽  
Fold Increase ◽  
Laminar Shear Stress ◽  
Human Coronary Artery ◽  
Intracellular Ca ◽  
Kinase Cascade ◽  
Dependent Protein ◽  
Laminar Shear

In endothelial cells (ECs), Ca2+-activated K+ channels KCa2.3 and KCa3.1 play a crucial role in the regulation of arterial tone via producing NO and endothelium-derived hyperpolarizing factors. Since a rise in intracellular Ca2+ levels and activation of p300 histone acetyltransferase are early EC responses to laminar shear stress (LS) for the transcriptional activation of genes, we examined the role of Ca2+/calmodulin-dependent kinase kinase (CaMKK), the most upstream element of a Ca2+/calmodulin-kinase cascade, and p300 in LS-dependent regulation of KCa2.3 and KCa3.1 in ECs. Exposure to LS (15 dyn/cm2) for 24 h markedly increased KCa2.3 and KCa3.1 mRNA expression in cultured human coronary artery ECs (3.2 ± 0.4 and 45 ± 10 fold increase, respectively; P < 0.05 vs. static condition; n = 8–30), whereas oscillatory shear (OS; ± 5 dyn/cm2 × 1 Hz) moderately increased KCa3.1 but did not affect KCa2.3. Expression of KCa2.1 and KCa2.2 was suppressed under both LS and OS conditions, whereas KCa1.1 was slightly elevated in LS and unchanged in OS. Inhibition of CaMKK attenuated LS-induced increases in the expression and channel activity of KCa2.3 and KCa3.1, and in phosphorylation of Akt (Ser473) and p300 (Ser1834). Inhibition of Akt abolished the upregulation of these channels by diminishing p300 phosphorylation. Consistently, disruption of the interaction of p300 with transcription factors eliminated the induction of these channels. Thus a CaMKK/Akt/p300 cascade plays an important role in LS-dependent induction of KCa2.3 and KCa3.1 expression, thereby regulating EC function and adaptation to hemodynamic changes.

Tracings of Behavior of Myoblasts Cultured Under Couette Type of Shear Flow Between Parallel Disks

2021 ◽  
Author(s):  
Shigehiro Hashimoto ◽  
Hiroki Yonezawa
Keyword(s):  
Shear Stress ◽  
Shear Flow ◽  
Rotating Disk ◽  
Time Lapse ◽  
Mechanical Stimuli ◽  
Parallel Disks ◽  
Before And After ◽  
A Cell

Abstract A cell deforms and migrates on the scaffold under mechanical stimuli in vivo. In this study, a cell with division during shear stress stimulation has been observed in vitro. Before and after division, both migration and deformation of each cell were analyzed. To make a Couette-type shear flow, the medium was sandwiched between parallel disks (the lower stationary culture-disc and the upper rotating disk) with a constant gap. The wall shear stress (1.5 Pa &lt; τ &lt; 2 Pa) on the surface of the lower culture plate was controlled by the rotational speed of the upper disc. Myoblasts (C2C12: mouse myoblast cell line) were used in the test. After cultivation without flow for 24 hours for adhesion of the cells to the lower disk, constant τ was applied to the cells in the incubator for 7 days. The behavior of each cell during shear was tracked by time-lapse images observed by an inverted phase contrast microscope placed in the incubator. Experimental results show that each cell tends to divide after higher activities: deformation and migration. The tendency is remarkable at the shear stress of 1.5 Pa.

scholarly journals Acoustic Streaming and Its Applications

Fluids ◽  
2018 ◽  
Vol 3 (4) ◽  
pp. 108 ◽  
Author(s):  
Junru Wu
Keyword(s):  
Shear Stress ◽  
Acoustic Wave ◽  
Flow Velocity ◽  
Velocity Gradient ◽  
Oscillatory Flow ◽  
Acoustic Streaming ◽  
Small Scale ◽  
Solid Boundary ◽  
Streaming Velocity ◽  
A Cell

Broadly speaking, acoustic streaming is generated by a nonlinear acoustic wave with a finite amplitude propagating in a viscid fluid. The fluid volume elements of molecules, d V , are forced to oscillate at the same frequency as the incident acoustic wave. Due to the nature of the nonlinearity of the acoustic wave, the second-order effect of the wave propagation produces a time-independent flow velocity (DC flow) in addition to a regular oscillatory motion (AC motion). Consequently, the fluid moves in a certain direction, which depends on the geometry of the system and its boundary conditions, as well as the parameters of the incident acoustic wave. The small scale acoustic streaming in a fluid is called “microstreaming”. When it is associated with acoustic cavitation, which refers to activities of microbubbles in a general sense, it is often called “cavitation microstreaming”. For biomedical applications, microstreaming usually takes place in a boundary layer at proximity of a solid boundary, which could be the membrane of a cell or walls of a container. To satisfy the non-slip boundary condition, the flow motion at a solid boundary should be zero. The magnitude of the DC acoustic streaming velocity, as well as the oscillatory flow velocity near the boundary, drop drastically; consequently, the acoustic streaming velocity generates a DC velocity gradient and the oscillatory flow velocity gradient produces an AC velocity gradient; they both will produce shear stress. The former is a DC shear stress and the latter is AC shear stress. It was observed the DC shear stress plays the dominant role, which may enhance the permeability of molecules passing through the cell membrane. This phenomenon is called “sonoporation”. Sonoporation has shown a great potential for the targeted delivery of DNA, drugs, and macromolecules into a cell. Acoustic streaming has also been used in fluid mixing, boundary cooling, and many other applications. The goal of this work is to give a brief review of the basic mathematical theory for acoustic microstreaming related to the aforementioned applications. The emphasis will be on its applications in biotechnology.

scholarly journals HMGB1 Promotes Systemic Lupus Erythematosus by Enhancing Macrophage Inflammatory Response

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Mudan Lu ◽  
Shanshan Yu ◽  
Wei Xu ◽  
Bo Gao ◽  
Sidong Xiong
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Inflammatory Response ◽  
Proinflammatory Cytokines ◽  
Lupus Erythematosus ◽  
Function Analysis ◽  
Critical Role ◽  
Loss Of Function ◽  
Systemic Lupus ◽  
Glycation End Products ◽  
Hmgb1 Expression

Background/Purpose. HMGB1, which may act as a proinflammatory mediator, has been proposed to contribute to the pathogenesis of multiple chronic inflammatory and autoimmune diseases including systemic lupus erythematosus (SLE); however, the precise mechanism of HMGB1 in the pathogenic process of SLE remains obscure.Method. The expression of HMGB1 was measured by ELISA and western blot. The ELISA was also applied to detect proinflammatory cytokines levels. Furthermore, nephritic pathology was evaluated by H&E staining of renal tissues.Results. In this study, we found that HMGB1 levels were significantly increased and correlated with SLE disease activity in both clinical patients and murine model. Furthermore, gain- and loss-of-function analysis showed that HMGB1 exacerbated the severity of SLE. Of note, the HMGB1 levels were found to be associated with the levels of proinflammatory cytokines such as TNF-αand IL-6 in SLE patients. Further study demonstrated that increased HMGB1 expression deteriorated the severity of SLE via enhancing macrophage inflammatory response. Moreover, we found that receptor of advanced glycation end products played a critical role in HMGB1-mediated macrophage inflammatory response.Conclusion. These findings suggested that HMGB1 might be a risk factor for SLE, and manipulation of HMGB1 signaling might provide a therapeutic strategy for SLE.

scholarly journals SENCR stabilizes vascular endothelial cell adherens junctions through interaction with CKAP4

2018 ◽  
Vol 116 (2) ◽  
pp. 546-555 ◽  
Author(s):  
Qing Lyu ◽  
Suowen Xu ◽  
Yuyan Lyu ◽  
Mihyun Choi ◽  
Christine K. Christie ◽  
...  
Keyword(s):  
Shear Stress ◽  
Endothelial Cell ◽  
Noncoding Rna ◽  
Rna Binding ◽  
Adherens Junctions ◽  
Vascular Endothelial Cell ◽  
Membrane Integrity ◽  
Laminar Shear Stress ◽  
Loss Of Function ◽  
Laminar Shear

SENCR is a human-specific, vascular cell-enriched long-noncoding RNA (lncRNA) that regulates vascular smooth muscle cell and endothelial cell (EC) phenotypes. The underlying mechanisms of action of SENCR in these and other cell types is unknown. Here, levels of SENCR RNA are shown to be elevated in several differentiated human EC lineages subjected to laminar shear stress. Increases in SENCR RNA are also observed in the laminar shear stress region of the adult aorta of humanized SENCR-expressing mice, but not in disturbed shear stress regions. SENCR loss-of-function studies disclose perturbations in EC membrane integrity resulting in increased EC permeability. Biotinylated RNA pull-down and mass spectrometry establish an abundant SENCR-binding protein, cytoskeletal-associated protein 4 (CKAP4); this ribonucleoprotein complex was further confirmed in an RNA immunoprecipitation experiment using an antibody to CKAP4. Structure–function studies demonstrate a noncanonical RNA-binding domain in CKAP4 that binds SENCR. Upon SENCR knockdown, increasing levels of CKAP4 protein are detected in the EC surface fraction. Furthermore, an interaction between CKAP4 and CDH5 is enhanced in SENCR-depleted EC. This heightened association appears to destabilize the CDH5/CTNND1 complex and augment CDH5 internalization, resulting in impaired adherens junctions. These findings support SENCR as a flow-responsive lncRNA that promotes EC adherens junction integrity through physical association with CKAP4, thereby stabilizing cell membrane-bound CDH5.

scholarly journals Mutations that Affect Channel Opening of Innexin Hemichannels in the C. elegans Gonad

2020 ◽  
Author(s):  
Todd Starich ◽  
David Greenstein
Keyword(s):  
Cell Proliferation ◽  
Gap Junctions ◽  
Germ Cell ◽  
Structural Model ◽  
Loss Of Function ◽  
C Elegans ◽  
Somatic Gonad ◽  
Channel Opening ◽  
Germline Proliferation ◽  
Germ Cell Proliferation

In C. elegans, gap junctions couple cells of the somatic gonad with the germline to support germ cell proliferation and gametogenesis. We previously characterized a strong loss-of-function mutation (T239I) affecting the second extracellular loop (EL2) of the somatic INX-8 hemichannel subunit. These mutant hemichannels form non-functional gap junctions with germline-expressed innexins. Here we describe the characterization of mutations that restore germ cell proliferation in the T239I EL2 mutant background. We recovered seven intragenic mutations located in diverse domains of INX-8 but not the EL domains. These second-site mutations compensate for the original channel defect to varying degrees, from nearly complete wild-type rescue, to partial rescue of germline proliferation. One suppressor mutation (E350K) supports the innexin cryo-EM structural model that the channel pore opening is surrounded by a cytoplasmic dome. Two suppressor mutations (S9L and I36N) may form leaky hemichannels that support germline proliferation but cause the demise of somatic sheath cells. Phenotypic analyses of three other suppressors reveal an equivalency in the rescue of germline proliferation and comparable delays in gametogenesis but a graded rescue of fertility. These latter mutations may be useful to probe interactions with the biochemical pathways that produce the molecules transiting through soma-germline gap junctions.

scholarly journals Asterosap, an Egg Jelly Peptide, Elevate Intracellular Ca2+ and Activate the Motility of Spermatozoa

2013 ◽  
Vol 19 (1) ◽  
pp. 79-88 ◽  
Author(s):  
MS Islam ◽  
T Akhter ◽  
M Matsumoto
Keyword(s):  
Plasma Membrane ◽  
Cyclic Gmp ◽  
Guanylyl Cyclase ◽  
Acrosome Reaction ◽  
Nickel Chloride ◽  
Selective Inhibitor ◽  
Ion Flux ◽  
Intracellular Ca ◽  
A Cell ◽  
Egg Jelly

Components from the outer envelopes of the egg that influence the flagellar beating and acrosome reaction of spermatozoa are regulated by ion flux across the plasma membrane. Asterosap, a sperm-activating peptide from the starfish egg jelly layer, causes a transient increase in intracellular cyclic GMP (cGMP) through the activation of the asterosap receptor, a guanylyl cyclase (GC), and causes an increase in intracellular Ca2+. Here we describe the pathway of asterosap-induced Ca2+ elevation using different Ca2+ channel antagonists. Fluo-4 AM, a cell permeable Ca2+ sensitive dye was used to determine the channel caused by the asterosap-induced Ca2+ elevation in spermatozoa. Different L-type Ca2+ channel antagonists, a non specific Ca2+ channel antagonist (nickel chloride), and a store-operated Ca2+ channel (SOC) antagonist do not show any significant response on asterosap-induced Ca2+ elevation, whereas KB-R7943, a selective inhibitor against Na+/Ca2+ exchanger (NCX) inhibited effectively. We also analyzed the flagellar movement of spermatozoa in artificial seawater (ASW) containing the asterosap at 100 nM ml?1. We found that spermatozoa swam vigorously with more symmetrical flagellar movement in asterosap than in ASW and KB-R7943 significantly inhibited the flagellar movement.DOI: http://dx.doi.org/10.3329/pa.v19i1.17358 Progress. Agric. 19(1): 79 - 88, 2008 

scholarly journals When Coupled to Natural Transformation in Acinetobacter sp. Strain ADP1, PCR Mutagenesis Is Made Less Random by Mismatch Repair

2005 ◽  
Vol 71 (11) ◽  
pp. 7610-7612 ◽  
Author(s):  
Alison Buchan ◽  
L. Nicholas Ornston
Keyword(s):  
Structure Function ◽  
Mismatch Repair ◽  
Protein Function ◽  
Natural Transformation ◽  
Function Analysis ◽  
Repair System ◽  
Loss Of Function ◽  
Nucleotide Substitutions ◽  
Full Spectrum ◽  
Mismatch Repair System

ABSTRACT Random PCR mutagenesis is a powerful tool for structure-function analysis of targeted proteins, especially when coupled with DNA integration through natural transformation followed by selection for loss of function. The technique has been applied successfully to structure-function analysis of transcriptional regulators, enzymes, and transporters in Acinetobacter sp. strain ADP1. However, the mismatch repair system prevents the full spectrum of nucleotide substitutions that may be selected at the level of protein function from being recovered. This barrier may be overcome by introducing PCR-mutagenized genes into strains in which the corresponding genes have been deleted.

Sign in / Sign up

Export Citation Format

Share Document