Effects of cholesterol on nano-mechanical properties of the living cell plasma membrane

Khatibzadeh, Nima; Gupta, Sharad; Farrell, Brenda; Brownell, William E.; Anvari, Bahman

doi:10.1039/c2sm25263e

Cited by 81 publications

(89 citation statements)

References 57 publications

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“…Histological experiments using a photomicrogram would detect the changes in F-actin in GTPγS-treated OHCs. Indeed, Khatibzadeh recently reported that F-actin depolymerization reduces the effect of cholesterol depletion on membrane mechanics, suggesting the possibility that cortical lattice may directly influence outer hair cell membrane mechanics [22]. The change in cytoskeletal-membrane interactions mechanics with change in cholesterol concentration is consistent with the change in prestin function observed in the present study.…”

Section: Discussionsupporting

confidence: 82%

Effects of cholesterol alterations are mediated via G-protein-related pathways in outer hair cells

Nagaki

Kakehata

Kitani

et al. 2013

Pflugers Arch - Eur J Physiol

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Cholesterol is an essential component of cell membranes, and determines their rigidity and fluidity. Alterations in membrane cholesterol by MβCD or water-soluble cholesterol affect the stiffness, capacitance, motility, and cell length of outer hair cells (OHCs). This suggests that reconstruction of the cytoskeleton may be induced by cholesterol alterations. In this study, we investigated intracellular signaling pathways involving G proteins to determine whether they modulate the changes in voltage-dependent capacitance caused by cholesterol alterations. Membrane capacitance of isolated guinea pig OHCs were assessed using a two-sine voltage stimulus protocol superimposed onto a voltage ramp (200 ms duration) from −150 to +140 mV. One group of OHCs was treated with 100 μM guanosine 5′-O-(3-thiotriphosphate) tetralithium salt (GTPγS), the GTP analog, administrated into individual cells via patch pipettes. Another group of OHCs was internally perfused with 600 μM guanosine 5′-(β-thio) diphosphate trilithium salt (GDPβS), the GDP analog. A third group was perfused with internal solution only as a control. Application of 1 mM MβCD shifted non-linear capacitance curves to the depolarized direction of the control group with reduction of the peak capacitance (C mpeak ). After the 10-min application of MβCD, shifts of voltage at C mpeak (V cmpeak ) and reduction of C mpeak were 73.32±11.09 mV and 9.09±2.10 pF, respectively (n=4). On the other hand, in the GTPγS-treated group, the shift of V cmpeak and reduction of C mpeak were attenuated remarkably. The shift of V cmpeak and reduction of C mpeak in the 10-min application of MβCD were 9.73± 10.92 mV and 3.08±1.91 pF, respectively (n=7). MβCD decreased the cell length by 16.53±4.27 % in the control group and by 6.45±6.22 % in the GTPγS group. In addition, we investigated the effects of GDPβS on cholesteroltreated OHCs. One millimolar cholesterol was externally applied after the 4-min application of 1 mM MβCD because the shift of V-C m function caused by cholesterol alone was small. Application of cholesterol shifted V-C m curves of the control group to the hyperpolarized direction with increase of the C mpeak . After the 10-min application of cholesterol, changes of V cmpeak and C mpeak were −9.19±6.68 mV and 2.14±0.44 pF, respectively (n=4). On the other hand, in the GDPβS-treated OHCs, the shift of V cmpeak and increase of C mpeak were attenuated markedly. The shift of V cmpeak and increase of C mpeak after 10 min were 5.13±10.46 mV and −0.55±1.39 pF, respectively (n=6). This study demonstrated that internally perfused GTPγS inhibited the MβCD effects and GDPβS inhibited the cholesterol effects, raising the possibility that G proteins may be involved in outer hair cell homeostasis as well as the possibility that cholesterol response may be G protein mediated. More study is required to clarify the detailed role of G proteins in the relation between cholesterol and the OHC cytoskeleton.

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Section: Discussionsupporting

confidence: 82%

Effects of cholesterol alterations are mediated via G-protein-related pathways in outer hair cells

Nagaki

Kakehata

Kitani

et al. 2013

Pflugers Arch - Eur J Physiol

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“…Motivated by the pivotal role of F-actin in the mechanical behavior of filopodia and other cellular protrusions, special focus has been on revealing the presence of F-actin within extracted membrane tubes (1, 13-16). However, apart from a single study (9), fluorescent visualization of the F-actin was achieved by staining and fixation of the cells, and literature contains conflicting results regarding the presence or absence of actin in membrane tubes pulled from living cells (8,11,17).Filopodia in living cells have the ability to rotate and bend by a so-far unknown mechanism (13,(18)(19)(20). For instance, filopodia have been reported to exhibit sharp kinks in neuronal cells (21-23) and macrophages (6).…”

mentioning

confidence: 99%

Helical buckling of actin inside filopodia generates traction

Leijnse

Oddershede

Bendix³

2014

Proc. Natl. Acad. Sci. U.S.A.

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Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell-cell communication. Filopodia contain crosslinked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle.membrane-cytoskeleton interactions | membrane nanotubes | filopodial retrograde flow | helical buckling | filopodia rotation T ubular membrane remodeling driven by the actin cytoskeleton plays a major role in both pathogenesis and in a healthy immune response. For instance, invadopodia, podosomes, and filopodia are crucial for invasion and migration of cells (1). Filopodia are thin (100 to 300 nm), tube-like, actin-rich structures that function as "antennae" or "tentacles" that cells use to probe and interact with their microenvironment (2-4). Such structures have been studied in vitro using model systems where the point-like 3D contacts with the extracellular matrix (ECM) have been mimicked by using optically trapped dielectric particles, functionalized with relevant ligands. These model systems allow for mechanical and visual control over filopodial dynamics and have significantly advanced the understanding of cellular mechanosensing (5).Extraction of membrane tubes, using optical trapping, has been used to investigate mechanical properties of the membrane-cytoskeleton system (6-10), or membrane cholesterol content (11), and has revealed important insight into the mechanism that peripheral proteins use to shape membranes (12). Motivated by the pivotal role of F-actin in the mechanical behavior of filopodia and other cellular protrusions, special focus has been on revealing the presence of F-actin within extracted membrane tubes (1, 13-16). However, apart from a single study (9), fluorescent visualization of the F-actin was achieved by staining and fixation of the cells, and literature contains conflicting results regarding the presence or absence of actin in membrane tubes pulled from living cells (8,11,17).Filopodia in living cells have the ability to rotate and bend by a so-far unknown mechanism (13,(18)(19)(20). For instance, filopodia ...

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“…The capacity of CD44 to bind to a ligand can depend on intracellular interactions with 1 Cells contain cytoskeletal networks that not only regulate cell morphology but also define the cellular mechanical properties of the cell and participate in the transduction of signals from various surface receptors to the cell nucleus. 22,[30][31][32] Thus, these results indicate that DTIC disrupts the local linkage of CD44 to the cytoskeleton, which may influence the interactions of the CD44 ligand-receptor.…”

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confidence: 95%

Effect of dacarbazine on CD44 in live melanoma cells as measured by atomic force microscopy-based nanoscopy

Huang

Zhang

et al. 2017

IJN

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CD44 ligand–receptor interactions are known to be involved in regulating cell migration and tumor cell metastasis. High expression levels of CD44 correlate with a poor prognosis of melanoma patients. In order to understand not only the mechanistic basis for dacarbazine (DTIC)-based melanoma treatment but also the reason for the poor prognosis of melanoma patients treated with DTIC, dynamic force spectroscopy was used to structurally map single native CD44-coupled receptors on the surface of melanoma cells. The effect of DTIC treatment was quantified by the dynamic binding strength and the ligand-binding free-energy landscape. The results demonstrated no obvious effect of DTIC on the unbinding force between CD44 ligand and its receptor, even when the CD44 nanodomains were reduced significantly. However, DTIC did perturb the kinetic and thermodynamic interactions of the CD44 ligand–receptor, with a resultant greater dissociation rate, lower affinity, lower binding free energy, and a narrower energy valley for the free-energy landscape. For cells treated with 25 and 75 μg/mL DTIC for 24 hours, the dissociation constant for CD44 increased 9- and 70-fold, respectively. The CD44 ligand binding free energy decreased from 9.94 for untreated cells to 8.65 and 7.39 kcal/mol for DTIC-treated cells, which indicated that the CD44 ligand–receptor complexes on DTIC-treated melanoma cells were less stable than on untreated cells. However, affinity remained in the micromolar range, rather than the millimolar range associated with nonaffinity ligands. Hence, the CD44 receptor could still be activated, resulting in intracellular signaling that could trigger a cellular response. These results demonstrate DTIC perturbs, but not completely inhibits, the binding of CD44 ligand to membrane receptors, suggesting a basis for the poor prognosis associated with DTIC treatment of melanoma. Overall, atomic force microscopy-based nanoscopic methods offer thermodynamic and kinetic insight into the effect of DTIC on the CD44 ligand-binding process.

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Effects of cholesterol on nano-mechanical properties of the living cell plasma membrane

Cited by 81 publications

References 57 publications

Effects of cholesterol alterations are mediated via G-protein-related pathways in outer hair cells

Effects of cholesterol alterations are mediated via G-protein-related pathways in outer hair cells

Helical buckling of actin inside filopodia generates traction

Effect of dacarbazine on CD44 in live melanoma cells as measured by atomic force microscopy-based nanoscopy

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