The effect of cellular cholesterol on membrane-cytoskeleton adhesion

Sun, Mingzhai; Northup, Nathan; Marga, Françoise; Huber, Tamás; Byfield, Fitzroy J.; Levitan, Irena; Forgács, Gabor

doi:10.1242/jcs.001370

Cited by 172 publications

(178 citation statements)

References 72 publications

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“…This observation was highly unexpected because previous studies showed that in membrane lipid bilayers an increase in membrane cholesterol increases the stiffness of the membrane 5,17 . Our further studies confirmed these observations using several independent approaches, including Atomic Force Microscopy 18,19 and Force Traction Microscopy …”

supporting

confidence: 80%

“…In contrast, optical tweezesrs are used to pull membrane nanotubes (tethers) to estimate cortical tension and membrane-cytoskeleton adhesion 37 . An alternative method to pull membrane tethers is to use the AFM, a method that has both significant similarities and differences as compared to optical tweezers 18,38 . The advantage of most of these methods is that they may provide detailed spatial information about the biomechanical properties of the individual cells.…”

Section: Discussionmentioning

confidence: 99%

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Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Kuhr

Byfield

et al. 2012

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Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 μm diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton. Video LinkThe video component of this article can be found at http://www.jove.com/video/3886/ Protocol Pulling Glass MicropipettesEquipment: Micropipette Puller, Microforge.Glass: Boroscillicate glass capillaries (~1.5 mm external diameter, ~1.4 mm internal diameter).1. Micropipettes are pulled using the same basic approach that is used to prepare glass microelectrodes for electrophysiology recordings.Briefly, a glass capillary is heated in th...

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supporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Kuhr

Byfield

et al. 2012

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“…Specifically, although the abundance and organization of the sphingolipid domains were influenced by cholesterol, nonrandom sphingolipid clustering was more drastically affected by cytoskeleton disruption than by cholesterol depletion. In fact, the altered sphingolipid organization observed after mβCD treatment was likely caused by cholesterol depletion-induced changes in cytoskeleton organization (31)(32)(33)35) and not by a loss of cohesive cholesterol-sphingolipid interactions. Because the cholesterol-sphingolipid interactions seem to play a minor role in sphingolipid domain formation, we conclude that the sphingolipid domains are not lipid rafts and are instead a distinctly different sphingolipid-enriched plasma membrane domain.…”

Section: Discussionmentioning

confidence: 93%

Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts

Frisz

Lou

Klitzing

et al. 2013

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

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Sphingolipids play important roles in plasma membrane structure and cell signaling. However, their lateral distribution in the plasma membrane is poorly understood. Here we quantitatively analyzed the sphingolipid organization on the entire dorsal surface of intact cells by mapping the distribution of 15 N-enriched ions from metabolically labeled 15 N-sphingolipids in the plasma membrane, using highresolution imaging mass spectrometry. Many types of control experiments (internal, positive, negative, and fixation temperature), along with parallel experiments involving the imaging of fluorescent sphingolipids-both in living cells and during fixation of living cellsexclude potential artifacts. Micrometer-scale sphingolipid patches consisting of numerous 15 N-sphingolipid microdomains with mean diameters of ∼200 nm are always present in the plasma membrane. Depletion of 30% of the cellular cholesterol did not eliminate the sphingolipid domains, but did reduce their abundance and longrange organization in the plasma membrane. In contrast, disruption of the cytoskeleton eliminated the sphingolipid domains. These results indicate that these sphingolipid assemblages are not lipid rafts and are instead a distinctly different type of sphingolipid-enriched plasma membrane domain that depends upon cortical actin.SIMS | stable isotope

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“…Furthermore, cytoskeletal assembly and disassembly appears to be closely associated with changes of membrane shape [34,35]. CHOL presumably plays a role in such shape changes, as the degree of cytoskeleton-membrane adhesion in plasma membranes was reported to depend on CHOL concentration in the membrane [36]. CHOL was also observed to be critically involved in the reversible wrinkling and folding of the membrane during the normal breathing cycle in the lung.…”

Section: Resultsmentioning

confidence: 99%

Cholesterol-Induced Buckling in Physisorbed Polymer-Tethered Lipid Monolayers

et al. 2013

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Abstract:The influence of cholesterol concentration on the formation of buckling structures is studied in a physisorbed polymer-tethered lipid monolayer system using epifluorescence microscopy (EPI) and atomic force microscopy (AFM). The monolayer system, built using the Langmuir-Blodgett (LB) technique, consists of 3 mol % poly(ethylene glycol) (PEG) lipopolymers and various concentrations of the phospholipid, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and cholesterol (CHOL). In the absence of CHOL, AFM micrographs show only occasional buckling structures, which is caused by the presence of the lipopolymers in the monolayer. In contrast, a gradual increase of CHOL concentration in the range of 0-40 mol % leads to fascinating film stress relaxation phenomena in the form of enhanced membrane buckling. Buckling structures are moderately deficient in CHOL, but do not cause any notable phospholipid-lipopolymer phase separation. Our experiments demonstrate that membrane buckling in physisorbed polymer-tethered membranes can be controlled through CHOL-mediated adjustment of membrane elastic properties. They further show that CHOL may have a notable impact on molecular confinement in the presence of crowding agents, such as lipopolymers. Our results are significant, because they offer an intriguing prospective on the role of CHOL on the material properties in complex membrane architecture. OPEN ACCESSPolymers 2013, 5 405

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The effect of cellular cholesterol on membrane-cytoskeleton adhesion

Cited by 172 publications

References 72 publications

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts

Cholesterol-Induced Buckling in Physisorbed Polymer-Tethered Lipid Monolayers

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