Direct Measurement of the Cortical Tension during the Growth of Membrane Blebs

Mechanics is at the heart of many cellular processes and its importance has received considerable attention during the last two decades. In particular, the tension of cell membranes, and more specifically of the cell cortex, is a key parameter that determines the mechanical behavior of the cell periphery. However, the measurement of tension remains challenging due to its dynamic nature. Here we show that a noninvasive interferometric technique can reveal time-resolved effective tension measurements by a high-accuracy determination of edge fluctuations in expanding cell blebs of filamin-deficient melanoma cells. The introduced technique shows that the bleb tension is ∼10–100 pN/μm and increases during bleb growth. Our results directly confirm that the subsequent stop of bleb growth is induced by an increase of measured tension, possibly mediated by the repolymerized actin cytoskeleton.     

RAB19 Directs Cortical Remodeling and Membrane Growth for Primary Ciliogenesis

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Direct Measurement of Potential Difference across the Human Red Blood Cell Membrane

The electrical potential difference across the human red cell membrane has been measured directly. A biological amplifier with neutralized input capacity was used. Human red cells in modified Ringer solution were impaled individually with 3 M KCl-filled glass microelectrodes. Movements of the microelectrodes were effected by Leitz micromanipulators. Results showed a potential difference of -8.0 ± 0.21 (SEM) mv, the inside being negative with respect to the outside. This value is approximately that calculated by using the Nernst equation considering the intracellular and extracellular chloride concentrations.

As a control, similar measurements were made on nylon microcapsules containing hemoglobin. The measured potential of -0.52 ± 0.02 (SEM) mv, which agreed very well with the value calculated on the basis of Donnan equilibrium, was much smaller in magnitude as compared to the results for the red cell, and there was evidence of fixed charges on the microcapsule membrane. There was no evidence of this in the case of the red cell.

     

Proteomics Characterization of Cell Membrane Blebs in Human Retinal Pigment Epithelium Cells

Age-related macular degeneration (AMD) is the leading cause of legal blindness among the elderly population in the industrialized world, affecting about 14 million people in the United States alone. Smoking is a major environmental risk factor for AMD, and hydroquinone is a major component in cigarette smoke. Hydroquinone induces the formation of cell membrane blebs in human retinal pigment epithelium (RPE). Blebs may accumulate and eventually contribute first to sub-RPE deposits and then drusen formation, which is a prominent histopathologic feature in eyes with AMD. As an attempt to better understand the mechanisms involved in early AMD, we sought to investigate the proteomic profile of RPE blebs. Isolated blebs were subjected to SDS-PAGE fractionation, and in-gel trypsin-digested peptides were analyzed by LC-MS/MS that lead to the identification of a total of 314 proteins. Identified proteins were predominantly involved in oxidative phosphorylation, cell junction, focal adhesion, cytoskeleton regulation, and immunogenic processes. Importantly basigin and matrix metalloproteinase-14, key proteins involved in extracellular matrix remodeling, were identified in RPE blebs and shown to be more prevalent in AMD patients. Altogether our findings suggest, for the first time, the potential involvement of RPE blebs in eye disease and shed light on the implication of cell-derived microvesicles in human pathology.Age-related macular degeneration (AMD)¹ is one of the most common pathologies in the retina, consisting in a chronic degenerative disorder that constitutes the leading cause of blindness in the elderly, probably affecting 14 million people in the United States. AMD is a multifactorial disease in nature in which age is the predominant risk factor, although there are also environmental factors involved. In this regard, smoking is thought to be a major environmental risk factor as supported by extensive epidemiological evidence (1–5). AMD develops in two different stages: early AMD (also referred to as dry AMD) and the late stage of AMD known as wet AMD by virtue of the extensive neovascularization taking place in the retina choroid. Although there is a fair understanding of the mechanisms involved in wet AMD, little is known about dry AMD and its transition into the most severe stage of this disorder, i.e. wet AMD (6).Early AMD targets the retinal pigment epithelium (RPE) and the Bruch membrane (BrM) in the retina. The RPE constitutes a cell monolayer that is crucial to maintain a normal photoreceptor function. In fact, RPE participates in the cycling of the visual molecules, provides nutrients to rods and cones, and is responsible for withdrawing waste debris from the outer segments of photoreceptors (7). The early stage of AMD is characterized by initial deregulation of the normal extracellular matrix (ECM) turnover leading to thickening of the BrM, sub-RPE deposit accumulation, and drusen formation (8). As mentioned earlier, cumulative evidence suggests that smoking may constitute a major risk factor for early AMD. In fact, we and others have provided evidence that hydroquinone (HQ), a major component of cigarette smoke, has the ability to deregulate the ECM (9–12). Aside from cigarette smoke, HQ is a compound of environmental relevance because of its broad presence in plastics, foodstuff, and air pollution (13, 14).Mild injuries inflected to the retina elicit a cellular response in the RPE consisting in pinching off small areas of the plasma membrane, which renders small microvesicles called blebs (15). The reason(s) behind membrane blebbing remains unknown, although it has been postulated to be an attempt to discard damaged cellular constituents by the RPE cell (8). Under prolonged injury, blebs may accumulate between the RPE and the basal lamina underneath this cell monolayer. Based on this concept, a plausible role for blebs in the pathogenesis of dry AMD has been suggested as a likely contributor to build-up of the sub-RPE deposits, which are characteristic of the early stages of this disorder (8). To date, however, RPE bleb composition and potential functions remain largely unexplored.However, membrane bleb or microvesicle production stimulated by a variety of stress has been extensively described in many different cell types (16–23). To gain a better understanding of the functional relevance of blebs in general and the pathogenic mechanism(s) involved in early AMD in particular, we sought to investigate the identity of proteins carried by human RPE blebs. Previously microvesicles from lymphocytes have been subjected to analysis leading to the identification of a number of proteins (24). In our study, we show the proteomics characterization of stress-induced blebs in RPE cells from human retina. We report identification of several proteins, some of them potentially involved in matrix metalloproteinase (MMP) activation, membrane lipid raft formation, and immunogenic processes. Interestingly RPE blebs were found to carry basigin (including highly glycosylated species) and MMP-14, which are key proteins regulating the ECM turnover and remodeling. A previous proteomics study also has revealed the presence of basigin in the blebs from malignant lymphocytes (24). In the present study, we intended to gain some insight into the functional characterization of blebs to unravel some of the biological consequences of cell membrane blebbing in disease.     

pH Effect of the Sphingomyelin Membrane Interfacial Tension

The effect of pH on the interfacial tension of a sphingomyelin membrane in aqueous solution has been studied. Three models describing H⁺ and OH⁻ ion adsorption on the bilayer lipid surface are presented. In models I and II, the membrane surface is continuous, with uniformly distributed functional groups as centers of H⁺ and OH⁻ ion adsorption. In model III, the membrane surface is composed of lipid molecules, with and without adsorbed H⁺ and OH⁻ ions. The contribution of each individual lipid molecule to the overall interfacial tension of the bilayer was assumed to be additive in models I and II. In model III, the Gibbs isotherm was used to describe adsorption of H⁺ and OH⁻ ions at the bilayer surface. Theoretical equations are derived to describe the interfacial tension as a function of pH for all three models. Maximum interfacial tension was observed experimentally at the isoelectric point.     

The Role of Membrane Lateral Tension in Calcium-Induced Membrane Fusion

Calcium-induced fusion of liposomes was studied with a view to understand the role of membrane tension in this process. Lipid mixing due to fusion was monitored by following fluorescence of rhodamine-phosphatidyl-ethanolamine incorporated into liposomal membrane at a self-quenching concentration. The extent of lipid mixing was found to depend on the rate of calcium addition: at slow rates it was significantly lower than when calcium was injected instantly. The vesicle inner volume was then made accessible to external calcium by adding calcium ionophore A23187. No effect on fusion was observed at high rates of calcium addition while at slow rates lipid mixing was eliminated. Fusion of labeled vesicles with a planar phospholipid membrane (BLM) was studied using fluorescence microscopy. Above a threshold concentration specific for each ion, Ca²⁺, Mg²⁺, Cd²⁺ and La³⁺ induce fusion of both charged and neutral membranes. The threshold calcium concentration required for fusion was found to be dependent on the vesicle charge, but not on the BLM charge. Pretreatment of vesicles with ionophore and calcium inhibited vesicle fusion with BLM. This effect was reversible: chelation of calcium prior to the application of vesicle to BLM completely restored their ability to fuse. These results support the hypothesis that tension in the outer monolayer of lipid vesicle is a primary reason for membrane destabilization promoting membrane fusion. How this may be a common mechanism for both purely lipidic and protein-mediated membrane fusion is discussed. Received: 27 September 1999/Revised: 22 March 2000     

Modulation of Cortical Oscillations by Low-Frequency Direct Cortical Stimulation Is State-Dependent

Cortical oscillations play a fundamental role in organizing large-scale functional brain networks. Noninvasive brain stimulation with temporally patterned waveforms such as repetitive transcranial magnetic stimulation (rTMS) and transcranial alternating current stimulation (tACS) have been proposed to modulate these oscillations. Thus, these stimulation modalities represent promising new approaches for the treatment of psychiatric illnesses in which these oscillations are impaired. However, the mechanism by which periodic brain stimulation alters endogenous oscillation dynamics is debated and appears to depend on brain state. Here, we demonstrate with a static model and a neural oscillator model that recurrent excitation in the thalamo-cortical circuit, together with recruitment of cortico-cortical connections, can explain the enhancement of oscillations by brain stimulation as a function of brain state. We then performed concurrent invasive recording and stimulation of the human cortical surface to elucidate the response of cortical oscillations to periodic stimulation and support the findings from the computational models. We found that (1) stimulation enhanced the targeted oscillation power, (2) this enhancement outlasted stimulation, and (3) the effect of stimulation depended on behavioral state. Together, our results show successful target engagement of oscillations by periodic brain stimulation and highlight the role of nonlinear interaction between endogenous network oscillations and stimulation. These mechanistic insights will contribute to the design of adaptive, more targeted stimulation paradigms.     

Lamellipodia and Membrane Blebs Drive Efficient Electrotactic Migration of Rat Walker Carcinosarcoma Cells WC 256

The endogenous electric field (EF) may provide an important signal for directional cell migration during wound healing, embryonic development and cancer metastasis but the mechanism of cell electrotaxis is poorly understood. Additionally, there is no research addressing the question on the difference in electrotactic motility of cells representing various strategies of cell movement—specifically blebbing vs. lamellipodial migration. In the current study we constructed a unique experimental model which allowed for the investigation of electrotactic movement of cells of the same origin but representing different modes of cell migration: weakly adherent, spontaneously blebbing (BC) and lamellipodia forming (LC) WC256 cells. We report that both BC and LC sublines show robust cathodal migration in a physiological EF (1–3 V/cm). The directionality of cell movement was completely reversible upon reversing the field polarity. However, the full reversal of cell direction after the change of EF polarity was much faster in the case of BC (10 minutes) than LC cells (30 minutes). We also investigated the distinct requirements for Rac, Cdc42 and Rho pathways and intracellular Ca²⁺ in electrotaxis of WC256 sublines forming different types of cell protrusions. It was found that Rac1 is required for directional movement of LC to a much greater extent than for BC, but Cdc42 and RhoA are more crucial for BC than for LC cells. The inhibition of ROCK did not affect electrotaxis of LC in contrast to BC cells. The results also showed that intracellular Ca²⁺ is essential only for the electrotactic reaction of BC cells. Moreover, inhibition of MLCK and myosin II did not affect the electrotaxis of LC in contrast to BC cells. In conclusion, our results revealed that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of WC 256 carcinosarcoma cells, however directional migration is mediated by different signalling pathways.     

Regulation of the Epithelial Na+ Channel by Membrane Tension

The sensitivity of αβγ rat epithelial Na⁺ channel (rENaC) to osmotically or mechanically induced changes of membrane tension was investigated in the Xenopus oocyte expression system, using both dual electrode voltage clamp and cell-attached patch clamp methodologies. ENaC whole-cell currents were insensitive to mechanical cell swelling caused by direct injection of 90 or 180 nl of 100-mM KCl. Similarly, ENaC whole-cell currents were insensitive to osmotic cell swelling caused by a 33% decrease of bathing solution osmolarity. The lack of an effect of cell swelling on ENaC was independent of the status of the actin cytoskeleton, as ENaC remained insensitive to osmotic and mechanical cell swelling in oocytes pretreated with cytochalasin B for 2–5 h. This apparent insensitivity of ENaC to increased cell volume and changes of membrane tension was also observed at the single channel level in membrane patches subjected to negative or positive pressures of 5 or 10 in. of water. However, and contrary to the lack of an effect of cell swelling, ENaC currents were inhibited by cell shrinking. A 45-min incubation in a 260-mosmol solution (a 25% increase of solution osmolarity) caused a decrease of ENaC currents (at −100 mV) from −3.42 ± 0.34 to −2.02 ± 0.23 μA (n = 6). This decrease of current with cell shrinking was completely blocked by pretreatment of oocytes with cytochalasin B, indicating that these changes of current are not likely related to a direct effect of cell shrinking. We conclude that αβγ rENaC is not directly mechanosensitive when expressed in a system that can produce a channel with identical properties to those found in native epithelia.     

Theoretical Analysis of Membrane Tension in Moving Cells

Lateral tension in cell plasma membranes plays an essential role in regulation of a number of membrane-related intracellular processes and cell motion. Understanding the physical factors generating the lateral tension and quantitative determination of the tension distribution along the cell membrane is an emerging topic of cell biophysics. Although experimental data are accumulating on membrane tension values in several cell types, the tension distribution along the membranes of moving cells remains largely unexplored. Here we suggest and analyze a theoretical model predicting the tension distribution along the membrane of a cell crawling on a flat substrate. We consider the tension to be generated by the force of actin network polymerization against the membrane at the cell leading edge. The three major factors determining the tension distribution are the membrane interaction with anchors connecting the actin network to the lipid bilayer, the membrane interaction with cell adhesions, and the force developing at the rear boundary due to the detachment of the remaining cell adhesion from the substrate in the course of cell crawling. Our model recovers the experimentally measured values of the tension in fish keratocytes and their dependence on the number of adhesions. The model predicts, quantitatively, the tension distribution between the leading and rear membrane edges as a function of the area fractions of the anchors and the adhesions.     

Theoretical Analysis of Membrane Tension in Moving Cells

Lateral tension in cell plasma membranes plays an essential role in regulation of a number of membrane-related intracellular processes and cell motion. Understanding the physical factors generating the lateral tension and quantitative determination of the tension distribution along the cell membrane is an emerging topic of cell biophysics. Although experimental data are accumulating on membrane tension values in several cell types, the tension distribution along the membranes of moving cells remains largely unexplored. Here we suggest and analyze a theoretical model predicting the tension distribution along the membrane of a cell crawling on a flat substrate. We consider the tension to be generated by the force of actin network polymerization against the membrane at the cell leading edge. The three major factors determining the tension distribution are the membrane interaction with anchors connecting the actin network to the lipid bilayer, the membrane interaction with cell adhesions, and the force developing at the rear boundary due to the detachment of the remaining cell adhesion from the substrate in the course of cell crawling. Our model recovers the experimentally measured values of the tension in fish keratocytes and their dependence on the number of adhesions. The model predicts, quantitatively, the tension distribution between the leading and rear membrane edges as a function of the area fractions of the anchors and the adhesions.     

Binding of Plasma Membrane Lipids Recruits the Yeast Integral Membrane Protein Ist2 to the Cortical ER

Recruitment of cytosolic proteins to individual membranes is governed by a combination of protein–protein and protein–membrane interactions. Many proteins recognize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] at the cytosolic surface of the plasma membrane (PM). Here, we show that a protein–lipid interaction can also serve as a dominant signal for the sorting of integral membrane proteins. Interaction with phosphatidly-inositolphosphates (PIPs) at the PM is involved in the targeting of the polytopic yeast protein Ist2 to PM-associated domains of the cortical endoplasmic reticulum (ER). Moreover, binding of PI(4,5)P₂ at the PM functions as a dominant mechanism that targets other integral membrane proteins to PM-associated domains of the cortical ER. This sorting to a subdomain of the ER abolishes proteasomal degradation and trafficking along the classical secretory (sec) pathway. In combination with the localization of IST2 mRNA to the bud tip and other redundant signals in Ist2, binding of PIPs leads to efficient accumulation of Ist2 at domains of the cortical ER from where the protein may reach the PM independently of the function of the sec-pathway.     

Simultaneous Regulation of Cytokinetic Furrow and Nucleus Positions by Cortical Tension Contributes to Proper DNA Segregation during Late Mitosis

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Membrane Tension Gates ERK-Mediated Regulation of Pluripotent Cell Fate

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Three-Dimensional Balance of Cortical Tension and Axial Contractility Enables Fast Amoeboid Migration

Fast amoeboid migration requires cells to apply mechanical forces on their surroundings via transient adhesions. However, the role these forces play in controlling cell migration speed remains largely unknown. We used three-dimensional force microscopy to measure the three-dimensional forces exerted by chemotaxing Dictyostelium cells, and examined wild-type cells as well as mutants with defects in contractility, internal F-actin crosslinking, and cortical integrity. We showed that cells pull on their substrate adhesions using two distinct, yet interconnected mechanisms: axial actomyosin contractility and cortical tension. We found that the migration speed increases when axial contractility overcomes cortical tension to produce the cell shape changes needed for locomotion. We demonstrated that the three-dimensional pulling forces generated by both mechanisms are internally balanced by an increase in cytoplasmic pressure that allows cells to push on their substrate without adhering to it, and which may be relevant for amoeboid migration in complex three-dimensional environments.     

The Effect of Cortical Elasticity and Active Tension on Cell Adhesion Mechanics

We consider a cell as an elastic, contractile shell surrounding a liquid incompressible cytoplasm and with nonspecific adhesion. We perform numerical simulations of this model to study the mechanics of cell-cell separation. By variation of parameters, we are able to recover well-known limits of the Johnson-Kendall-Roberts theory, the Derjaguin-Muller-Toporov model, adhesive vesicles with surface tension (Brochard-Wyart and de Gennes derivation), and thin elastic shells. We further locate biological cells on this parameter space by comparison to existing experiments on S180 cells. Using this model, we show that mechanical parameters can be obtained that are consistent with both dual pipette aspiration and micropipette aspiration, a problem not successfully tackled so far. We estimate a cortex elastic modulus of E_c ≈ 15 kPa, an effective cortex thickness of t_c ≈ 0.3 μm, and an active tension of γ ≈ 0.4 nN/μm. With these parameters, a Johnson-Kendall-Roberts-like scaling of the separation force is recovered. Finally, the change of contact radius with applied force in a pull-off experiment was investigated. For small forces, a scaling similar to both the Brochard-Wyart and de Gennes derivation and the Derjaguin-Muller-Toporov model is found.     

Plasma Membrane Tethering of the Cortical ER Necessitates Its Finely Reticulated Architecture

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Highlights? The VAP proteins sustain the ER-plasma membrane attachment in fission yeast ? Footprint of the ER on the cortex is functionally insulated from the cytosol ? Division-site placement requires a patchwork of cortical sites accessible to cytosol