Bleb Membrane Research Articles

Inverse bleb membrane protrusions pump fluid within the early mouse embryo.

Inverse bleb membrane protrusions pump fluid within the early mouse embryo.

Differences in Lipid Order and Dynamics in Plasma Membranes Assessed by Nonlinear Optical Microscopy.

When amphiphilic polar dyes were added to the cells, they intercalated predominantly in the outer leaf of the plasma membrane, making them active for second harmonic generation (SHG). The fluorescence of the dye enabled simultaneous 3D imaging of SHG and two-photon excited fluorescence (TPF). Because SHG intensity is sensitive to the alignment of the dyes, which reflects lipid ordering in the plasma membrane, we assessed the difference in lipid ordering by comparing the SHG intensity normalized to the TPF intensity. Together with an enzyme release assay that detects pore formation in the plasma membrane, our SHG assay revealed how polycations affect lipid ordering at low concentrations, where membrane damage has not yet been examined. By scaling the results of the assays with the charge concentration of the two polycations, polyethylenimine (PEI) and poly-l-lysine (PLL), we found that PEI reduced the lipid order more than PLL, and PLL formed more pores than PEI. A comparison of the SHG and TPF images of the wounded cells revealed that one of the lipid dynamics (flip-flop) was significantly enhanced in the bleb membrane. Moreover, the SHG assay indicated that the biocompatible polymer, poly(N-(2-hydroxypropyl)methacrylamide), did not affect the lipid order. Thus, our technique allows the assessment of the plasma membrane structure at the molecular level.

Myosin II-interacting guanine nucleotide exchange factor promotes bleb retraction via stimulating cortex reassembly at the bleb membrane.

Blebs are involved in various biological processes such as cell migration, cytokinesis, and apoptosis. While the expansion of blebs is largely an intracellular pressure-driven process, the retraction of blebs is believed to be driven by RhoA activation that leads to the reassembly of the actomyosin cortex at the bleb membrane. However, it is still poorly understood how RhoA is activated at the bleb membrane. Here, we provide evidence demonstrating that myosin II–interacting guanine nucleotide exchange factor (MYOGEF) is implicated in bleb retraction via stimulating RhoA activation and the reassembly of an actomyosin network at the bleb membrane during bleb retraction. Interaction of MYOGEF with ezrin, a well-known regulator of bleb retraction, is required for MYOGEF localization to retracting blebs. Notably, knockout of MYOGEF or ezrin not only disrupts RhoA activation at the bleb membrane, but also interferes with nonmuscle myosin II localization and activation, as well as actin polymerization in retracting blebs. Importantly, MYOGEF knockout slows down bleb retraction. We propose that ezrin interacts with MYOGEF and recruits it to retracting blebs, where MYOGEF activates RhoA and promotes the reassembly of the cortical actomyosin network at the bleb membrane, thus contributing to the regulation of bleb retraction.

Membrane shrinkage and cortex remodelling are predicted to work in harmony to retract blebs.

Numerous cell types undergo an oscillatory form of dynamics known as blebbing, whereby pressure-driven spherical protrusions of membrane (known as blebs) expand and contract over the cell's surface. Depending on the cell line, blebs play important roles in many different phenomena including mitosis and locomotion. The expansion phase of cellular blebbing has been mathematically modelled in detail. However, the active processes occurring during the retraction phase are not so well characterized. It is thought that blebs retract because a cortex reforms inside, and adheres to, the bleb membrane. This cortex is retracted into the cell and the attached bleb membrane follows. Using a computational model of a cell's membrane, cortex and interconnecting adhesions, we demonstrate that cortex retraction alone cannot account for bleb retraction and suggest that the mechanism works in tandem with membrane shrinking. Further, an emergent hysteresis loop is observed in the intracellular pressure, which suggests a potential mechanism through which a secondary bleb can be initiated as a primary bleb contracts.

Integrin-mediated Membrane Blebbing Is Dependent on Sodium-Proton Exchanger 1 and Sodium-Calcium Exchanger 1 Activity

Integrin signaling and membrane blebbing modulate cell adhesion, spreading, and migration. However, the relationship between integrin signaling and membrane blebbing is unclear. Here, we show that an integrin-ligand interaction induces both membrane blebbing and changes in membrane permeability. Sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are membrane proteins located on the bleb membrane. Inhibition of NHE1 disrupts membrane blebbing and decreases changes in membrane permeability. However, inhibition of NCX1 enhances cell blebbing; cells become swollen because of NHE1 induced intracellular sodium accumulation. Our study found that NHE1 induced sodium influx is a driving force for membrane bleb growth, while sodium efflux (and calcium influx) induced by NCX1 in a reverse mode results in membrane bleb retraction. Together, these findings reveal a novel function for NHE1 and NCX1 in membrane blebbing and permeability, and establish a link between membrane blebbing and integrin signaling.

Stretch-Activated Potassium Channels in Hypotonically Induced Blebs of Atrial Myocytes

Stress in the lipids of the cell membrane may be responsible for activating stretch-activated channels (SACs) in nonspecialized sensory cells such as cardiac myocytes, where they are likely to play a role in cardiac mechanoelectric feedback. We examined the influence of the mechanical microenvironment on the gating of stretch-activated potassium channels (SAKCs) in rat atrial myocytes. The goal was to examine the role of the cytoskeleton in the gating process. We recorded from blebs that have minimal cytoskeleton and cells treated with cytochalasin B (cyto-B) to disrupt filamentous actin. Histochemical and electron microscopic techniques confirmed that the bleb membrane was largely free of F-actin. Channel currents showed mechanosensitivity and potassium selectivity and were activated by low pH and arachidonic acid, similar to properties of TREK-1. Some patches showed a time-dependent decrease in current that may be adaptation or inactivation, and since this decrease appeared in control cells and blebs, it is probably not the result of adaptation in the cytoskeleton. Cyto-B treatment and blebbing caused an increase in background channel activity, suggesting a transfer of stress from actin to bilayer and then to the channel. The slope sensitivity of gating before and after cyto-B treatment was similar to that of blebs, implying the characteristic change of dimensions associated with channel gating was the same in the three mechanical environments. The mechanosensitivity of SAKCs appears to be the result of interaction with membrane lipids and not of direct involvement of the cytoskeleton.

Life and Times of a Cellular Bleb

Blebs are spherical cellular protrusions that occur in many physiological situations. Two distinct phases make up the life of a bleb, each of which have their own biology and physics: expansion, which lasts ∼30 s, and retraction, which lasts ∼2 min. We investigate these phases using optical microscopy and simple theoretical concepts, seeking information on blebbing itself, and on cytomechanics in general. We show that bleb nucleation depends on pressure, membrane-cortex adhesion energy, and membrane tension, and test this experimentally. Bleb growth occurs through a combination of bulk flow of lipids and delamination from the cell cortex via the formation and propagation of tears. In extreme cases, this can give rise to a traveling wave around the cell periphery, known as “circus movement.” When growth stalls, an actin cortex reforms under the bleb membrane, and retraction starts, driven by myosin-II. Using flicker spectroscopy, we find that retracting blebs are fivefold more rigid than expanding blebs, an increase entirely explained by the properties of the newly formed cortical actin mesh. Finally, using artificially nucleated blebs as pressure sensors, we show that cells rounded up in mitosis possess a substantial intracellular pressure.

Identification of the cardiac ryanodine receptor channel in membrane blebs of sarcoplasmic reticulum

Blebs of the sarcoplasmic reticulum (SR) membrane of heart muscle cells were generated after saponin perforation of the plasma membrane followed by complete hypercontraction of the cell. Although characteristic proteins of the plasma membrane, namely the β1-adrenoreceptor and Gαi, were stained by monoclonal antibodies in the hypercontracted cells, these proteins could not be detected in the adjacent blebs. Monoclonal antibodies to the cardiac ryanodine receptor (RyR2), calsequestrin and SERCA2 bound at different amounts to surface components of the blebs and to components of the hypercontracted cells. From the immunofluorescence signals we conclude that the blebs are mainly constituted of corbular and junctional SR membrane, and only to a lesser extent of network SR membrane. Deconvolution microscopy revealed that the membrane location of RyR2, calsequestrin and SERCA2 in the bleb is comparable to native SR membrane. At the bleb membrane giga-ohm seals could be obtained and patches could be excised in a way that single-channel currents could be measured, although these are not completely identified.

A voltage-sensitive cation channel present in clusters in lobster skeletal muscle membrane.

The single channel properties of a voltage-sensitive cation channel are described in a study of ion channel activity in enzymatically induced blebs of lobster skeletal muscle membrane. This cation channel, one of several that are spontaneously active in excised patches from bleb membrane, can be distinguished from other channels on the basis of its large single channel conductance (293 pS), voltage-sensitive gating properties, the presence of a subconductance state of the fully open channel, and a weak selectivity for K > Na. At hyperpolarizing voltages, this channel displays flickering or bursting behavior, and a single state of the fully open channel is observed. At depolarizing voltages, the mean channel open time increases and a second longer-lived open state is observed. The voltage dependence of the mean channel open time and the linear i-V relation of this channel predict that the macroscopic current carried through this cation channel would be outwardly rectifying. Channels of this type are infrequently observed in this preparation, but when present in the patch are often present in multiple copies. We describe a statistical test for examining the clustering of ion channels in excised patches of membrane. The result of this test shows that the cation channels appear in clusters in the blebs.

Na current in membrane blebs: implications for channel mobility and patch clamp recording

When suction was applied to loose patch clamp pipettes while recording from enzymatically dissociated muscle fibers, large membrane blebs formed within the pipettes. We initiated a study of these suction-induced blebs because ion channels in the blebs would complicate or possibly invalidate loose patch voltage clamp measurements of membrane current density. The low lateral mobility (Stühmer and Almers, 1982) and steep gradients of Na channels at the end-plate and tendon (Caldwell et al., 1986) imply tight binding of Na channels to cytoskeletal elements and led us to expect few, if any, Na channels in the blebs. Bleb formation produced an increase in membrane capacitance, as expected from the increase in membrane area. Bleb formation also increased the Na current, indicating that the blebs contained Na channels. Assuming that the increased capacitance and Na current were due to lipid and Na channels moving from membrane outside the pipette, ejection of the bleb from the pipette was expected to bring the capacitance and Na current back to their original values. Capacitance did return to its original value, but Na current was lower than expected. The decrease in Na current is explained by Na channels moving from the patch membrane into the bleb. Normalization of bleb and patch Na current to their respective capacitances revealed that bleb membrane had a Na channel density almost 50% that of normal surface membrane. Thus, bleb membrane is neither devoid of proteins nor truly representative of the normal surface membrane from which it arose. It is enriched in membrane lipids and is relatively protein poor. Two conclusions can be drawn.(ABSTRACT TRUNCATED AT 250 WORDS)

Irreversible injury in anoxic hepatocytes precipitated by an abrupt increase in plasma membrane permeability.

Using low-light digitized video microscopy, the onset, progression, and reversibility of anoxic injury were assessed in single hepatocytes isolated from fasted rats. Cell-surface bleb formation occurred in three stages over 1-3 h after anoxia. Stage I was characterized by formation of numerous small blebs. In stage II, small blebs enlarged by coalescence and fusion to form a few large terminal blebs. Near the end of stage II, cells began to swell rapidly, ending with the apparent breakdown of one of the terminal blebs. Breakdown of the bleb membrane initiated stage III of injury and was coincident with a rapid increase of nonspecific permeability to organic cationic and anionic molecules. On reoxygenation, stages I and II were fully reversible, and plasma membrane blebs were resorbed completely within 6 min of reoxygenation without loss of viability. Stage III, however, was not reversible, and no morphological changes occurred on reoxygenation. The results indicate that onset of cell death owing to anoxia is a rapid event initiated by a sudden increase of nonspecific plasma membrane permeability caused by rupture of a terminal bleb. Anoxic injury is reversible until this event occurs.

Rotation of a single swollen thylakoid vesicle in a rotating electric field. Electrical properties of the photosynthetic membrane and their modification by ionophores, lipophilic ions and pH

Rotation of single swollen thylakoid vesicles (‘blebs’) was induced by means of a rotating electric field of strength 104 V · cm −1, inducing a membrane voltage of 72 mV peak. Within the range of medium conductives described (40–300 μS · cm −1), measurement of the field frequency (2–100 kHz) giving maximum rotation rate is equivalent to measuring the electrical time constant of the bleb membrane. Hence the membrane capacity (specific capacitance) was determined, and the value found at pH 8.1 (0.93 ± 0.07 μF · cm −2) is in agreement with values deduced from measurements using other techniques. However, the capacity was also found to decreased with pH: a minimum value of 0.77 ± 0.01 μF · cm −2 was measured at pH 4.4. The present study was extended to measurements of the effects of the lipid-soluble anion of dipicrylamine on the membrane capacity. At pH 7.2 and dipicrylamine concentration of 1.0 μM, a minimum estimate of the apparent membrane capacity was found to be 2.0 ± 0.2 μF · cm −2, with 2.6 ± 0.2 μF · cm −2 being observed at 5.0 μM concentration. In addition, it was found possible to measure the membrane resistivity (specific resistance) in the presence of either gramicidin (1.0 to 10 nM) or valinomycin (1.0 to 10 μM). In the case of gramicidin, it was possible to derive a maximum estimate of the mean channel conductance, and this agrees very well with the values for individual, single channels that may be deduced from artificial bilayer work. Unless the gramicidin channels in blebs are in fact substantially more conductive than in artificial bilayers, this indicates that a high percentage of the added gramicidin forms channels which are open for most of the time. In the case of valinomycin, a much greater amount had to be added to produce the same reduction of membrane resistivity as seen with a given concentration of gramicidin. However, calculations indicate that the majority of this effect is due to the difference in partioning behaviour of the two ionophores.

Electrophotoluminescence and the electrical properties of the photosynthetic membrane. I. Initial kinetics and the charging capacitance of the membrane

Preilluminated chloroplast membranes, and particularly hypotonically swollen vesicles (blebs), give rise to a strong characteristic luminescence (electrophotoluminescence, EPL; Ellenson and Sauer, 1976, Photochem. Photobiol., 23:113-123; Arnold and Azzi, 1971, Photochem. Photobiol., 14:233-240) during the application of a strong external electric field. A detailed kinetic study of EPL was carried out and the initial kinetics from the field onset are reported here. The fast rise time (less than 0.2 mus) of the applied external electric field together with a high instrumental time resolution allowed the observation of a characteristic delay (lag time) between the field onset and the appearance of the induced emission. The lag time decreased with increase in the applied field strength and/or the conductivity of the suspension and is interpreted to be a consequence of (a) the necessity to reach a threshold electrical potential difference in the bleb membrane, below which no emission can be triggered, and (b) the finite time required to attain such a transmembranal field during the charging process of the membrane. A quantitative analysis, connecting the lag time, the controllable experimental parameters, and the membrane electrical characteristics is presented. Its verification was carried out in both size-selected and heterogeneous bleb populations. In the latter, experiments were consistent with the assumption that the lag time reflects the charging of the largest blebs. The results indicate (a) the possibility of directly measuring the specific membrane capacitance, yielding an estimate of Cm = 1.2 +/- 0.3 microF/cm2 (the precision being particle size-homogeneity dependent); (b) A minimal transmembranal potential difference (of approximately 240 mV) is necessary to induce electrophotoluminescence; and (c) the lag duration depends on the time elapsed between the preillumination and the external field application. Correlated with the study of ionophore effects on the lag time, this suggests additivity of the light- and field-induced transmembrane potentials in attaining the threshold for emission.

Ultrastructural studies on apical blebs of striated ducts in the rat submandibular gland.

Apical blebs of rat submandibular gland striated ducts were studied by electron microscopy. The glands were stimulated by starvation of the animals for 24 h whereafter they were fed 15 min before death and tissue removal. After stimulation most striated duct cells developed apical blebs, with various shapes and usually without granules and vesicles. A semi-separating filamentous structure separates the cell from the content of the bleb. Apparently only the vesicular and granular content can pass through this structure and enter the bleb. Following the appearance of small ruptures in the bleb membrane, the bleb is discarded into the lumen of the duct where it disintegrates. Typical wall-like protrusions on the luminal surface of the duct cell, are probably, rudiments of the ruptured bleb membrane. It is suggested that the apical blebs are manifestations of apocrine secretion.

Enhanced molecular diffusibility in muscle membrane blebs: release of lateral constraints.

Measurements of lateral molecular diffusion on blebs formed on the surfaces of isolated muscle cells and myoblasts are reported. These blebbed membranes retain integral proteins but apparently separate from the detectable cytoskeleton. On blebs, acetylcholine receptors, concanavalin A receptors, and stearoyldextran extrinsic model receptor molecules are free to diffuse with a diffusion coefficient (D) approximately 3 x 10(-9) cm2/s, which is close to the value predicted for hydrodynamic drag in the lipid membrane. In contrast, diffusion of these typical receptors in intact cell membranes is constrained to D approximately less than 10(-10) cm2/s with substantial fractions virtually nondiffusible (D less than 10(-12) cm2/s). Lipid analog diffusion is also slightly enhanced in blebs as expected of evanescent lipid protein interaction. This strong enhancement of membrane protein diffusion is attributed to release from unidentified natural constraints that is induced in some way by detachment of the bleb membrane.