scholarly journals Disruption of membrane cholesterol organization impairs the concerted activity of PIEZO1 channel clusters

2019 ◽  
Author(s):  
P. Ridone ◽  
E. Pandzic ◽  
M. Vassalli ◽  
C. D. Cox ◽  
A. Macmillan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Unsaturated Fatty Acids ◽  
Applied Pressure ◽  
Super Resolution ◽  
Membrane Cholesterol ◽  
Mechanical Stimuli ◽  
Channel Inactivation ◽  
Electrophysiological Recordings ◽  
Concerted Activity ◽  
Mechanosensitive Ion Channel

ABSTRACTThe human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichment or depletion by methyl-β-Cyclodextrin (MBCD) and disruption of membrane cholesterol organization by dynasore affects PIEZO1-GFP’s response to mechanical force. Electrophysiological recordings in the cell-attached configuration revealed that MBCD caused a rightward shift in the PIEZO1-GFP pressure-response curve, increased channel latency in response to mechanical stimuli and markedly slowed channel inactivation. The same effects were seen in native PIEZO1 in N2A cells. STORM super-resolution imaging revealed that, at the nano-scale, PIEZO1-GFP channels in the membrane associate as clusters sensitive to membrane manipulation. Both cluster distribution and diffusion rates were affected by treatment with MBCD (5 mM). Supplementation of poly-unsaturated fatty acids appeared to sensitize the PIEZO1-GFP response to applied pressure. Together, our results indicate that PIEZO1 function is directly dependent on the membrane mechanical properties and lateral organization of membrane cholesterol domains which coordinates the concerted activity of PIEZO1 channels.SUMMARYThe essential mammalian mechanosensitive channel PIEZO1 organizes in the plasma membrane into nanometric clusters which depend on the integrity of cholesterol domains to rapidly detect applied force and especially inactivate syncronously, the most commonly altered feature of PIEZO1 in pathology.

scholarly journals Disruption of membrane cholesterol organization impairs the activity of PIEZO1 channel clusters

2020 ◽  
Vol 152 (8) ◽  
Author(s):  
Pietro Ridone ◽  
Elvis Pandzic ◽  
Massimo Vassalli ◽  
Charles D. Cox ◽  
Alexander Macmillan ◽  
...  
Keyword(s):  
Applied Pressure ◽  
Membrane Cholesterol ◽  
Mechanical Stimuli ◽  
Membrane Composition ◽  
Superresolution Imaging ◽  
Channel Inactivation ◽  
Electrophysiological Recordings ◽  
Mechanosensitive Ion Channel ◽  
Diffusion Rates ◽  
And Diffusion

The human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichment or depletion by methyl-β-cyclodextrin (MBCD) and disruption of membrane cholesterol organization by dynasore affects PIEZO1-GFP’s response to mechanical force. Electrophysiological recordings in the cell-attached configuration revealed that MBCD caused a rightward shift in the PIEZO1-GFP pressure–response curve, increased channel latency in response to mechanical stimuli, and markedly slowed channel inactivation. The same effects were seen in native PIEZO1 in N2A cells. STORM superresolution imaging revealed that, at the nanoscale, PIEZO1-GFP channels in the membrane associate as clusters sensitive to membrane manipulation. Both cluster distribution and diffusion rates were affected by treatment with MBCD (5 mM). Supplementation of polyunsaturated fatty acids appeared to sensitize the PIEZO1-GFP response to applied pressure. Together, our results indicate that PIEZO1 function is directly dependent on the membrane composition and lateral organization of membrane cholesterol domains, which coordinate the activity of clustered PIEZO1 channels.

scholarly journals Monoclonal antibody detection of plasma membrane cholesterol microdomains responsive to cholesterol trafficking

2001 ◽  
Vol 42 (9) ◽  
pp. 1492-1500 ◽  
Author(s):  
Howard S. Kruth ◽  
Ina Ifrim ◽  
Janet Chang ◽  
Lia Addadi ◽  
Daniele Perl-Treves ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Antibody Detection ◽  
Membrane Cholesterol ◽  
Cholesterol Trafficking ◽  
Plasma Membrane Cholesterol

Cell culturing in a three-dimensional matrix affects the localization and properties of plasma membrane cholesterol

2009 ◽  
Vol 33 (10) ◽  
pp. 1079-1086 ◽  
Author(s):  
Nadezhda Stefanova ◽  
Galya Staneva ◽  
Diana Petkova ◽  
Teodora Lupanova ◽  
Roumen Pankov ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Three Dimensional ◽  
Membrane Cholesterol ◽  
Dimensional Matrix ◽  
Cell Culturing ◽  
Plasma Membrane Cholesterol

scholarly journals TACAN is an essential component of the mechanosensitive ion channel responsible for pain sensing

2018 ◽  
Author(s):  
L. Beaulieu-Laroche ◽  
M. Christin ◽  
AM Donoghue ◽  
F. Agosti ◽  
N. Yousefpour ◽  
...  
Keyword(s):  
Ion Channels ◽  
Heterologous Expression ◽  
Ion Channel ◽  
Behavioral Responses ◽  
Mechanical Stimuli ◽  
Thermal Pain ◽  
Fundamental Process ◽  
Mechanosensitive Ion Channel ◽  
Pain Stimuli ◽  
Essential Subunit

SummaryMechanotransduction, the conversion of mechanical stimuli into electrical signals, is a fundamental process underlying several physiological functions such as touch and pain sensing, hearing and proprioception. This process is carried out by specialized mechanosensitive ion channels whose identities have been discovered for most functions except pain sensing. Here we report the identification of TACAN (Tmem120A), an essential subunit of the mechanosensitive ion channel responsible for sensing mechanical pain. TACAN is expressed in a subset of nociceptors, and its heterologous expression increases mechanically-evoked currents in cell lines. Purification and reconstitution of TACAN in synthetic lipids generates a functional ion channel. Finally, knocking down TACAN decreases the mechanosensitivity of nociceptors and reduces behavioral responses to mechanical but not to thermal pain stimuli, without affecting the sensitivity to touch stimuli. We propose that TACAN is a pore-forming subunit of the mechanosensitive ion channel responsible for sensing mechanical pain.

scholarly journals Mechanosensitive Ion Channel Piezo1 Regulates Myocyte Fusion during Skeletal Myogenesis

2020 ◽  
Author(s):  
Huascar Pedro Ortuste Quiroga ◽  
Shingo Yokoyama ◽  
Massimo Ganassi ◽  
Kodai Nakamura ◽  
Tomohiro Yamashita ◽  
...  
Keyword(s):  
Ion Channel ◽  
Molecular Mechanisms ◽  
Myoblast Fusion ◽  
Mechanical Stimuli ◽  
Muscle Satellite Cell ◽  
Myotube Formation ◽  
Mechanosensitive Ion Channel ◽  
And Function ◽  
Sirna Knockdown

AbstractMechanical stimuli such as stretch and resistance training are essential to regulate growth and function of skeletal muscle. However, the molecular mechanisms involved in sensing mechanical stress remain unclear. Here, the purpose of this study was to investigate the role of the mechanosensitive ion channel Piezo1 during myogenic progression. Muscle satellite cell-derived myoblasts and myotubes were modified with stretch, siRNA knockdown and agonist-induced activation of Piezo1. Direct manipulation of Piezo1 modulates terminal myogenic progression. Piezo1 knockdown suppressed myoblast fusion during myotube formation and maturation. This was accompanied by downregulation of the fusogenic protein Myomaker. Piezo1 knockdown also lowered Ca2+ influx in response to stretch. Conversely Piezo1 activation stimulated fusion and increased Ca2+ influx in response to stretch. These evidences indicate that Piezo1 is essential for myotube formation and maturation, which may have implications for msucular dystrophy prevention through its role as a mechanosensitive Ca2+ channel.

scholarly journals The nanoscale organization of the Wnt signaling integrator Dishevelled in the development-essential vegetal cortex domain of an egg and early embryo

2021 ◽  
Author(s):  
John H. Henson ◽  
Bakary Samasa ◽  
Charles B. Shuster ◽  
Athula H. Wikramanayake
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Super Resolution ◽  
Cell Types ◽  
Early Embryo ◽  
Ultrastructural Organization ◽  
C Terminus ◽  
Pathway Activation ◽  
Vegetal Cortex ◽  
Canonical Wnt

AbstractWnt/β-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the frizzled:LRP5/6 receptor complex with β-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica TEM to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D-SIM imaging of isolated egg cortices demonstrated the concentration gradient-like distribution of Dsh in the VCD, whereas higher resolution STED imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first division actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for canonical Wnt pathway activation at the sea urchin vegetal organization and may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.

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