scholarly journals Adherent cell remodeling on micropatterns is modulated by Piezo1 channels

2020 ◽  
Author(s):  
Deekshitha Jetta ◽  
Mohammad Reza Bahrani Fard ◽  
Frederick Sachs ◽  
Katie Munechika ◽  
Susan Z. Hua
Keyword(s):  
Nuclear Envelope ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Cation Channel ◽  
Hek293 Cells ◽  
Adherent Cell ◽  
Environmental Cues ◽  
Adherent Cells ◽  
Cell Extension ◽  
Cell Remodeling

AbstractAdherent cells utilize local environmental cues to make decisions on their growth and movement. We have previously shown that HEK293 cells grown on the fibronectin stripe patterns were elongated. Here we show that Piezo1 function is involved in cell spreading. Inhibiting the Rho-ROCK pathway also reversibly inhibited cell extension indicating that myosin contractility is involved. Piezo1 expressing HEK cells plated on fibronectin stripes elongated, while a knockout of Piezo1 eliminated elongation. Inhibiting Piezo1 conductance using GsMTx4 or Gd3+ blocked cell spreading, but the cells grew thin tail-like extensions along the patterns. Images of GFP-tagged Piezo1 showed plaques of Piezo1 moving to the extrusion edges, co-localized with focal adhesions. Surprisingly, in non-spreading cells Piezo1 was located primarily on the nuclear envelope. The growth of thin extrusion tails did not occur in Piezo1 knockout cells suggesting that Piezo1 may have functions besides acting as a cation channel.

scholarly journals Adherent cell remodeling on micropatterns is modulated by Piezo1 channels

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Deekshitha Jetta ◽  
Mohammad Reza Bahrani Fard ◽  
Frederick Sachs ◽  
Katie Munechika ◽  
Susan Z. Hua
Keyword(s):  
Nuclear Envelope ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Cation Channel ◽  
Hek293 Cells ◽  
Adherent Cell ◽  
Environmental Cues ◽  
Adherent Cells ◽  
Cell Extension ◽  
Cell Remodeling

AbstractAdherent cells utilize local environmental cues to make decisions on their growth and movement. We have previously shown that HEK293 cells grown on the fibronectin stripe patterns were elongated. Here we show that Piezo1 function is involved in cell spreading. Piezo1 expressing HEK cells plated on fibronectin stripes elongated, while a knockout of Piezo1 eliminated elongation. Inhibiting Piezo1 conductance using GsMTx4 or Gd3+ blocked cell spreading, but the cells grew thin tail-like extensions along the patterns. Images of GFP-tagged Piezo1 showed plaques of Piezo1 moving to the extrusion edges, co-localized with focal adhesions. Surprisingly, in non-spreading cells Piezo1 was located primarily on the nuclear envelope. Inhibiting the Rho-ROCK pathway also reversibly inhibited cell extension indicating that myosin contractility is involved. The growth of thin extrusion tails did not occur in Piezo1 knockout cells suggesting that Piezo1 may have functions besides acting as a cation channel.

scholarly journals Focal adhesions are sites of integrin extension

2010 ◽  
Vol 188 (6) ◽  
pp. 891-903 ◽  
Author(s):  
Janet A. Askari ◽  
Christopher J. Tynan ◽  
Stephen E.D. Webb ◽  
Marisa L. Martin-Fernandez ◽  
Christoph Ballestrem ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Focal Adhesions ◽  
Resonance Energy ◽  
Cell Spreading ◽  
Integrin Subunit ◽  
Adherent Cells ◽  
Extended Form ◽  
Integrin Α5β1 ◽  
Mediate Cell

Integrins undergo global conformational changes that specify their activation state. Current models portray the inactive receptor in a bent conformation that upon activation converts to a fully extended form in which the integrin subunit leg regions are separated to enable ligand binding and subsequent signaling. To test the applicability of this model in adherent cells, we used a fluorescent resonance energy transfer (FRET)–based approach, in combination with engineered integrin mutants and monoclonal antibody reporters, to image integrin α5β1 conformation. We find that restricting leg separation causes the integrin to adopt a bent conformation that is unable to respond to agonists and mediate cell spreading. By measuring FRET between labeled α5β1 and the cell membrane, we find extended receptors are enriched in focal adhesions compared with adjacent regions of the plasma membrane. These results demonstrate definitely that major quaternary rearrangements of β1-integrin subunits occur in adherent cells and that conversion from a bent to extended form takes place at focal adhesions.

scholarly journals Cell Adhesion Promotes Ebola Virus Envelope Glycoprotein-Mediated Binding and Infection

2008 ◽  
Vol 82 (14) ◽  
pp. 7238-7242 ◽  
Author(s):  
Derek Dube ◽  
Kathryn L. Schornberg ◽  
Tzanko S. Stantchev ◽  
Matthew I. Bonaparte ◽  
Sue E. Delos ◽  
...  
Keyword(s):  
Envelope Glycoprotein ◽  
Ebola Virus ◽  
Cell Spreading ◽  
Cell Types ◽  
Pseudotyped Virus ◽  
Adherent Cell ◽  
Adherent Cells ◽  
Receptor Binding Domain ◽  
Virus Envelope ◽  
Nonadherent Cells

ABSTRACT Ebola virus infects a wide variety of adherent cell types, while nonadherent cells are found to be refractory. To explore this correlation, we compared the ability of pairs of related adherent and nonadherent cells to bind a recombinant Ebola virus receptor binding domain (EboV RBD) and to be infected with Ebola virus glycoprotein (GP)-pseudotyped particles. Both human 293F and THP-1 cells can be propagated as adherent or nonadherent cultures, and in both cases adherent cells were found to be significantly more susceptible to both EboV RBD binding and GP-pseudotyped virus infection than their nonadherent counterparts. Furthermore, with 293F cells the acquisition of EboV RBD binding paralleled cell spreading and did not require new mRNA or protein synthesis.

Inactivation of Surface-adherent Listeria monocytogenes Hypochlorite and Heat

1991 ◽  
Vol 54 (1) ◽  
pp. 4-6 ◽  
Author(s):  
SHIN-HO LEE ◽  
JOSEPH F. FRANK
Keyword(s):  
Stainless Steel ◽  
Listeria Monocytogenes ◽  
Cell Population ◽  
Incubation Time ◽  
Cell Populations ◽  
Adherent Cell ◽  
Adherent Cells ◽  
D Cells

Inactivation by hypochlorite of Listeria monocytogenes cells adherent to stainless steel was determined. Adherent cell populations were prepared by incubating stainless steel slides with a 24 h culture of L. monocytogenes for 4 h at 21°C. Adherent microcolonies were prepared by growing L. monocytogenes on stainless steel slides submerged in a 1:15 dilution of tryptic soy broth at 21°C. The slides were then rinsed and transferred to fresh sterile broth every 2 d with a total incubation time of 8 d. Although the 4 h and 8 d adherent populations were at similar levels, 8 d adherent cells were over 100 times more resistant than the 4 h adherent cell population when exposed to 200 ppm hypochlorite for 30 s. When stainless steel slides containing adherent cells were heated at 72°C both adherent cell populations were inactivated after 1 min. Detectable numbers of L. monocytogenes remained on stainless steel slides after treatment at 65°C for 3 min when adherent 8 d cells were tested but not when adherent 4 h cells were used.

Myocilin binding to Hep II domain of fibronectin inhibits cell spreading and incorporation of paxillin into focal adhesions

2005 ◽  
Vol 303 (2) ◽  
pp. 218-228 ◽  
Author(s):  
Donna M. Peters ◽  
Kathleen Herbert ◽  
Brenda Biddick ◽  
Jennifer A. Peterson
Keyword(s):  
Focal Adhesions ◽  
Cell Spreading

Syndesmos, a protein that interacts with the cytoplasmic domain of syndecan-4, mediates cell spreading and actin cytoskeletal organization

2000 ◽  
Vol 113 (2) ◽  
pp. 315-324 ◽  
Author(s):  
P.C. Baciu ◽  
S. Saoncella ◽  
S.H. Lee ◽  
F. Denhez ◽  
D. Leuthardt ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Cytoplasmic Domain ◽  
Cellular Protein ◽  
Actin Stress Fiber ◽  
Independent Manner ◽  
Intracellular Proteins ◽  
Central Regions ◽  
A Cell

Syndecan-4 is a cell surface heparan sulfate proteoglycan which, in cooperation with integrins, transduces signals for the assembly of focal adhesions and actin stress fibers in cells plated on fibronectin. The regulation of these cellular events is proposed to occur, in part, through the interaction of the cytoplasmic domains of these transmembrane receptors with intracellular proteins. To identify potential intracellular proteins that interact with the cytoplasmic domain of syndecan-4, we carried out a yeast two-hybrid screen in which the cytoplasmic domain of syndecan-4 was used as bait. As a result of this screen, we have identified a novel cellular protein that interacts with the cytoplasmic domain of syndecan-4 but not with those of the other three syndecan family members. The interaction involves both the membrane proximal and variable central regions of the cytoplasmic domain. We have named this cDNA and encoded protein syndesmos. Syndesmos is ubiquitously expressed and can be myristylated. Consistent with its myristylation and syndecan-4 association, syndesmos colocalizes with syndecan-4 in the ventral plasma membranes of cells plated on fibronectin. When overexpressed in NIH 3T3 cells, syndesmos enhances cell spreading, actin stress fiber and focal contact formation in a serum-independent manner.

scholarly journals Establishment of an adherent cell layer from human umbilical cord blood

2000 ◽  
Vol 23 (3) ◽  
pp. 519-522 ◽  
Author(s):  
Zeni Z.C. Alfonso ◽  
Eduardo D. Forneck ◽  
Waldir F. Allebrandt ◽  
Nance B. Nardi
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
Adherent Cell ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Adherent Cells ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

In addition to bone marrow and peripheral blood, stem cells also occur in human umbilical cord blood (HUCB), and there is an increasing interest in the use of this material as an alternative source for bone marrow transplantation and gene therapy. In vitro hematopoiesis has been maintained for up to 16 weeks in HUCB cultures, but the establishment of an adherent, stromal layer has consistently failed. Adherent cell precursors among mononuclear cells from HUCB were sought for in long-term cultures. Mononuclear cells obtained from cord blood after full term, normal deliveries were cultivated at different concentrations in Iscove's modified Dulbecco's medium (IMDM) with weekly feeding. An adherent layer was detected in 16 of 30 cultures, 12 of which were plated at cell concentrations higher than 2 x 10(6) cells/ml. In contrast to bone marrow cultures, in which the stroma is detected early, in most (10/16) positive cultures from HUCB the adherent layer was identified only after the fourth week of culture. The cells never reached confluence and detached from the plate approximately four weeks after detection. May-Grünwald-Giemsa staining of positive cultures revealed fibroblast- or endothelial-like adherent cells in an arrangement different from that of bone marrow stroma in 13 samples. In two of these, the adherent cells were organized into characteristic, delimited cords of cells. Unlike bone marrow cultures, fat cells were never observed in the adherent layers. A rapid development of large myeloid cells in the first week of culture was characteristic of negative cultures and these cells were maintained for up to 12 weeks. HUCB contains adherent cell precursors which occur in lower numbers than in bone marrow and may be at a different (possibly less mature) stage of differentiation.

scholarly journals Microinjection of antibodies against talin inhibits the spreading and migration of fibroblasts

1992 ◽  
Vol 102 (4) ◽  
pp. 753-762
Author(s):  
G.H. Nuckolls ◽  
L.H. Romer ◽  
K. Burridge
Keyword(s):  
Extracellular Matrix ◽  
Tissue Culture ◽  
Actin Filaments ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
And Migration ◽  
Extracellular Matrix Receptors

Talin is believed to be one of the key proteins involved in linking actin filaments to extracellular matrix receptors in focal adhesions. Our strategy for studying the function of talin has been to inactivate talin in living fibroblasts in tissue culture through the microinjection of affinity-purified, polyclonal anti-talin antibodies. The effect of the injected anti-talin antibodies on cell spreading was found to depend on how recently the cells had been plated. Cells that were in the process of spreading on a fibronectin substratum, and which had newly developed focal adhesions, were induced to round up and to disassemble many of the adhesions. However, if fibroblasts were allowed to spread completely before they were microinjected with the anti-talin antibody, focal adhesions remained intact and the flat morphology of the cells was unaffected. The percentage of cells that were able to maintain a spread morphology despite the injection of anti-talin antibodies increased during the first few hours after plating on fibronectin substrata. Fibroblasts that were allowed to spread completely before microinjection with the anti-talin antibody retained both intact focal adhesions and a flat, well-spread morphology, but failed to migrate effectively. Our experiments do not directly address the role of talin in mature focal adhesions, but they indicate that talin is essential for the spreading and migration of fibroblasts on fibronectin as well as for the development and initial maintenance of focal adhesions on this substratum.

scholarly journals Lipid droplets disrupt mechanosensing in human hepatocytes

2020 ◽  
Vol 319 (1) ◽  
pp. G11-G22
Author(s):  
LiKang Chin ◽  
Neil D. Theise ◽  
Abigail E. Loneker ◽  
Paul A. Janmey ◽  
Rebecca G. Wells
Keyword(s):  
Mechanical Stress ◽  
Nuclear Localization ◽  
Lipid Droplets ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Human Hepatocytes ◽  
Stress Fibers ◽  
Primary Hepatocytes ◽  
The Impact ◽  
Lipid Loading

This work examines the impact of lipid loading on mechanosensing by human hepatocytes. In cirrhotic livers, the presence of large (although not small) lipid droplets increased nuclear localization of the mechanotransducer YAP. In primary hepatocytes in culture, lipid droplets led to decreased stiffness-induced cell spreading and disrupted focal adhesions and stress fibers; the presence of large lipid droplets resulted in increased YAP nuclear localization. Collectively, the data suggest that lipid droplets induce intracellular mechanical stress.

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