Bleb Expansion Research Articles

Three-Dimensional Printing Enabled Droplet Microfluidic Device for Real-Time Monitoring of Single-Cell Viability and Blebbing Activity.

Droplet-based microfluidics with the characteristics of high throughput, low sample consumption, increasing reaction speed, and homogeneous volume control have been demonstrated as a useful platform for biomedical research and applications. The traditional fabrication methods of droplet microfluidics largely rely on expensive instruments, sophisticated operations, and even the requirement of an ultraclean room. In this manuscript, we present a 3D printing-based droplet microfluidic system with a specifically designed microstructure for droplet generation aimed at developing a more accessible and cost-effective method. The performance of droplet generation and the encapsulation capacity of the setup were examined. The device was further applied to measure the variation in cell viability over time and monitor the cell's blebbing activity to investigate its potential ability and feasibility for single-cell analysis. The result demonstrated that the produced droplets remained stable enough to enable the long-time detection of cell viability. Additionally, cell membrane protrusions featuring the life cycle of bleb initiation, expansion, and retraction can be well-observed. Three-dimensional printing-based droplet microfluidics benefit from the ease of manufacture, which is expected to simplify the fabrication of microfluidics and expand the application of the droplet approach in biomedical fields.

Actin Turnover Required for Adhesion-Independent Bleb Migration

Cell migration is critical for many vital processes, such as wound healing, as well as harmful processes, such as cancer metastasis. Experiments have highlighted the diversity in migration strategies employed by cells in physiologically relevant environments. In 3D fibrous matrices and confinement between two surfaces, some cells migrate using round membrane protrusions, called blebs. In bleb-based migration, the role of substrate adhesion is thought to be minimal, and it remains unclear if a cell can migrate without any adhesion complexes. We present a 2D computational fluid-structure model of a cell using cycles of bleb expansion and retraction in a channel with several geometries. The cell model consists of a plasma membrane, an underlying actin cortex, and viscous cytoplasm. Cellular structures are immersed in viscous fluid which permeates them, and the fluid equations are solved using the method of regularized Stokeslets. Simulations show that the cell cannot effectively migrate when the actin cortex is modeled as a purely elastic material. We find that cells do migrate in rigid channels if actin turnover is included with a viscoelastic description for the cortex. Our study highlights the non-trivial relationship between cell rheology and its external environment during migration with cytoplasmic streaming.

3D Computational Modeling of Bleb Initiation Dynamics

Blebbing occurs in cells under high cortical tension when the membrane locally detaches from the actin cortex, resulting in pressure-driven flow of the cytosol and membrane expansion. Some cells use blebs as leading edge protrusions during cell migration, particularly in 3D environments such as a collagen matrix. Blebs can be initiated through either a localized loss of membrane-cortex adhesion or ablation of the cortex in a region. Bleb morphologies resulting from different initiation mechanisms have not been studied in detail, either experimentally or with theoretical models. Additionally, material properties of the cytoplasm, such as elasticity, have been shown to be important for limiting bleb size. A 3D dynamic computational model of the cell is presented that includes mechanics and the interactions of the cytoplasm, the actin cortex, the cell membrane, and the cytoskeleton. The model is used to quantify bleb expansion dynamics and shapes that result from simulations using different initiation mechanisms. The cytoplasm is modeled as a both viscous fluid and as a poroelastic material. Results from model simulations with a viscous fluid cytoplasm model show much broader blebs that expand faster when they are initiated via cortical ablation than when they are initiated by removing only membrane-cortex adhesion. Simulation results using the poroelastic model of the cytoplasm provide qualitatively similar bleb morphologies regardless of the initiation mechanism. Parameter studies on bleb expansion time, cytoplasmic stiffness, and permeability reveal different scaling properties, namely a smaller power-law exponent, in 3D simulations compared to 2D ones.

A Clockwork Bleb: cytoskeleton, calcium, and cytoplasmic fluidity.

When the plasma membrane (PM) detaches from the underlying actin cortex, the PM expands according to intracellular pressure and a spherical membrane protrusion called a bleb is formed. This bleb retracts when the actin cortex is reassembled underneath the PM. Whereas this phenomenon seems simple at first glance, there are many interesting, unresolved cell biological questions in each process. For example, what is the membrane source to enlarge the surface area of the PM during rapid bleb expansion? What signals induce actin reassembly for bleb retraction, and how is cytoplasmic fluidity regulated to allow rapid membrane deformation during bleb expansion? Furthermore, emerging evidence indicates that cancer cells use blebs for invasion, but little is known about how molecules that are involved in bleb formation, expansion, and retraction are coordinated for directional amoeboid migration. In this review, we discuss the molecular mechanisms involved in the regulation of blebs, which have been revealed by various experimental systems.

Cellular Blebs and Membrane Invaginations Are Coupled through Membrane Tension Buffering

Bleb-type cellular protrusions play key roles in a range of biological processes. It was recently found that bleb growth is facilitated by a local supply of membrane from tubular invaginations, but the interplay between the expanding bleb and the membrane tubes remains poorly understood. On the one hand, the membrane area stored in tubes may serve as a reservoir for bleb expansion. On the other hand, the sequestering of excess membrane in stabilized invaginations may effectively increase the cell membrane tension, which suppresses spontaneous protrusions. Here, we investigate this duality through physical modeling and in vivo experiments. In agreement with observations, our model describes the transition into a tube-flattening mode of bleb expansion while also predicting that the blebbing rate is impaired by elevating the concentration of the curved membrane proteins that form the tubes. We show both theoretically and experimentally that the stabilizing effect of tubes could be counterbalanced by the cortical myosin contractility. Our results largely suggest that proteins able to induce membrane tubulation, such as those containing N-BAR domains, can buffer the effective membrane tension—a master regulator of all cell deformations.

F/A-18 Aviator Successfully Returned to Flight After an In-Flight Spontaneous Pneumothorax

BACKGROUND: Spontaneous pneumothorax (PTX) is a diagnostic challenge in aviators given the common occurrence of musculoskeletal pain after flight and notorious underreporting of symptoms of other diseases in this group.CASE REPORT: A 24-yr-old active duty F/A-18 Weapon Systems Officer performed an anti-G straining maneuver (AGSM) in response to a 6.5-g warm-up turn during a training flight at 16,000 ft (4876.8 m) above sea level. He immediately developed right-sided thoracic back pain. The flight was terminated, he landed, and the pain improved. Over the next 5 d, he noticed the insidious development of pleuritic chest pain and dyspnea. His symptoms prompted presentation to an aviation medicine clinic where a large right sided PTX was identified. After transfer to a local emergency department, a large bore chest tube was placed. A CT scan showed bilateral apical blebs requiring right and subsequently left video assisted thoracoscopy (VATS) with chemical/mechanical pleurodesis and apical wedge resection. Pulmonary function testing (PFT) showed a mild restriction defect 2-1/2 mo after surgery. The patient also completed cardiopulmonary exercise testing (CPET), performing better than his predicted reference range. After a high resolution CT showed no remaining signs of bleb or cyst disease and another month of healing he was returned to flight.DISCUSSION: PTX should be considered in aviators with perithoracic pain after flight as several aspects of flight in high performance aircraft may increase the risk for PTX. These include positive pressure breathing through a facemask, repeated use of the AGSM, and the possibility of bleb expansion at altitude.DeYoung H, Ahmed Y, Buckley J. F/A-18 aviator successfully returned to flight after an in-flight spontaneous pneumothorax. Aerosp Med Hum Perform. 2018; 89(11):1008-1012.

Bleb Expansion in Migrating Cells Depends on Supply of Membrane from Cell Surface Invaginations

Cell migration is essential for morphogenesis, organ formation, and homeostasis, with relevance for clinical conditions. The migration of primordial germ cells (PGCs) is a useful model for studying this process in the context of the developing embryo. Zebrafish PGC migration depends on the formation of cellular protrusions in form of blebs, a type of protrusion found in various cell types. Here we report on the mechanisms allowing the inflation of the membrane during bleb formation. We show that the rapid expansion of the protrusion depends on membrane invaginations that are localized preferentially at the cell front. The formation of these invaginations requires the function of Cdc42, and their unfolding allows bleb inflation and dynamic cell-shape changes performed by migrating cells. Inhibiting the formation and release of the invaginations strongly interfered with bleb formation, cell motility, and the ability of the cells to reach their target.

Involvement of β- and γ-actin isoforms in actin cytoskeleton organization and migration abilities of bleb-forming human colon cancer cells.

Amoeboid movement is characteristic for rounded cells, which do not form strong adhesion contacts with the ECM and use blebs as migratory protrusions. It is well known that actin is the main component of mature forms of these structures, but the exact role fulfilled by non-muscle actin isoforms β- and γ- in bleb formation and migration of these cells is still not fully understood. The aim of this study was to establish the role of β- and γ-actin in migration of bleb-forming cancer cells using isoform-specific antibodies and expression of fluorescently tagged actin isoforms. We observed, after staining with monoclonal antibodies, that both actins are present in these cells in the form of a cortical ring as well as in the area of blebs. Additionally, using simultaneous expression of differentially tagged β- and γ-actin in cells, we observed that the actin isoforms are present together in a single bleb. They were involved during bleb expansion as well as retraction. Also present in the area of these protrusions formed by both isoforms were the bleb markers–ezrin and myosin II. The overexpression of β- or γ-actin led to actin cytoskeletal rearrangement followed by the growth of migration and invasion abilities of examined human colon cancer cells, LS174T line. In summary these data prove that both actin isoforms have an impact on motility of bleb-forming cancer cells. Moreover, we conclude that monoclonal antibodies directed against actin isoforms in combination with the tagged actins are good tools to study their role in important biological processes.

Cell Surface Mechanochemistry and the Determinants of Bleb Formation, Healing, and Travel Velocity

Blebs are pressure-driven cell protrusions implicated in cellular functions such as cell division, apoptosis, and cell motility, including motility of protease-inhibited cancer cells. Because of their mechanical nature, blebs inform us about general cell-surface mechanics, including membrane dynamics, pressure propagation throughout the cytoplasm, and the architecture and dynamics of the actin cortex. Mathematical models including detailed fluid dynamics have previously been used to understand bleb expansion. Here, we develop mathematical models in two and three dimensions on longer timescales that recapitulate the full bleb life cycle, including both expansion and healing by cortex reformation, in terms of experimentally accessible biophysical parameters such as myosin contractility, osmotic pressure, and turnover of actin and ezrin. The model provides conditions under which blebbing occurs, and naturally gives rise to traveling blebs. The model predicts conditions under which blebs travel or remain stationary, as well as the bleb traveling velocity, a quantity that has remained elusive in previous models. As previous studies have used blebs as reporters of membrane tension and pressure dynamics within the cell, we have used our system to investigate various pressure equilibration models and dynamic, nonuniform membrane tension to account for the shape of a traveling bleb. We also find that traveling blebs tend to expand in all directions unless otherwise constrained. One possible constraint could be provided by spatial heterogeneity in, for example, adhesion density.

Intracellular Pressure Dynamics in Blebbing Cells

Blebs are pressure-driven protrusions that play an important role in cell migration, particularly in three-dimensional environments. A bleb is initiated when the cytoskeleton detaches from the cell membrane, resulting in the pressure-driven flow of cytosol toward the area of detachment and local expansion of the cell membrane. Recent experiments involving blebbing cells have led to conflicting hypotheses regarding the timescale of intracellular pressure propagation. The interpretation of one set of experiments supports a poroelastic model of the cytoplasm that leads to slow pressure equilibration when compared to the timescale of bleb expansion. A different study concludes that pressure equilibrates faster than the timescale of bleb expansion. To address this discrepancy, a dynamic computational model of the cell was developed that includes mechanics of and the interactions among the cytoplasm, the actin cortex, the cell membrane, and the cytoskeleton. The model results quantify the relationship among cytoplasmic rheology, pressure, and bleb expansion dynamics, and provide a more detailed picture of intracellular pressure dynamics. This study shows the elastic response of the cytoplasm relieves pressure and limits bleb size, and that both permeability and elasticity of the cytoplasm determine bleb expansion time. Our model with a poroelastic cytoplasm shows that pressure disturbances from bleb initiation propagate faster than the timescale of bleb expansion and that pressure equilibrates slower than the timescale of bleb expansion. The multiple timescales in intracellular pressure dynamics explain the apparent discrepancy in the interpretation of experimental results.

Utility of Operative Glaucoma Tube Shunt Viscoelastic Bolus Flush.

ABSTRACTObjective: To assess the utility of viscoelastic injection to induce bleb expansion and decrease intraocular pressure (IOP) in eyes with encapsulated glaucoma tube shunt blebs.Design: Case series.Subjects and participants: Forty-three glaucomatous eyes, including 13 eyes with congenital, 13 uveitic, 5 neovascular, 5 open angle, 4 narrow angle and 3 traumatic glaucomas.Methods, interventions or testing: All patients underwent viscoelastic flush procedure. A pre-bent 27 or 30-gauge cannula was passed through a 25-gauge paracentesis, advanced over the iris across the anterior chamber, and insinuated into the tube shunt lumen. Once the cannula was firmly lodged in position, 0.45 to 0.85 ml of viscoelastic was injected to hyperinflate the bleb.Main outcome measures: Paired t-tests were performed comparing preoperative IOP and number of medications used preoperatively vs levels measured at 1, 6, 12, 18 and 24 months.Results: Intraocular pressure was reduced from a mean preoperative level of 26.0 ± 1.2 (sem) mm Hg to 15.8 ± 1.0 at 1 month, remaining stable thereafter at each 6-month interval with 15.1 ± 1.1 mm Hg at 24 months (p < 0.0001). Medication use did not vary significantly from baseline. Pressure remained < 21 mm Hg after 2 years in 85% of eyes cannulated within 1 year of primary tube shunt implantation (n = 23), and in 62% of eyes cannulated more than 1 year after tube shunt placement (n = 20).Conclusion: Tube shunt expansion with bolus viscoelastic flush successfully restored encapsulated bleb function, providing a substantial (~10 mm Hg) IOP decrease into the mid-normal pressure range. This persisted in the majority of treated eyes for the entire study period.How to cite this article: Groth SL, Greider KL, Sponsel WE. Utility of Operative Glaucoma Tube Shunt Viscoelastic Bolus Flush. J Curr Glaucoma Pract 2015;9(3):73-76.

Extraction of Blebs in Human Embryonic Stem Cell Videos.

Blebbing is an important biological indicator in determining the health of human embryonic stem cells (hESC). Especially, areas of a bleb sequence in a video are often used to distinguish two cell blebbing behaviors in hESC: dynamic and apoptotic blebbings. This paper analyzes various segmentation methods for bleb extraction in hESC videos and introduces a bio-inspired score function to improve the performance in bleb extraction. Full bleb formation consists of bleb expansion and retraction. Blebs change their size and image properties dynamically in both processes and between frames. Therefore, adaptive parameters are needed for each segmentation method. A score function derived from the change of bleb area and orientation between consecutive frames is proposed which provides adaptive parameters for bleb extraction in videos. In comparison to manual analysis, the proposed method provides an automated fast and accurate approach for bleb sequence extraction.

Insights into Cytoplasmic Rheology Gained from Modeling Cellular Blebbing

Blebbing occurs when the cytoskeleton detaches from the cell membrane, resulting in the pressure-driven flow of cytosol towards the area of detachment and local expansion of the cell membrane. Recent experiments involving blebbing cells have led to conflicting hypotheses regarding the timescale of intracellular pressure propagation. The interpretation of one set of experiments supports a poroelastic cytoplasmic model which leads to slow pressure equilibration when compared to the timescale of bleb expansion. A different study concludes that pressure equilibrates faster than the timescale of bleb expansion. To address this, a dynamic computational model of the cell was developed that includes mechanics of and the interactions between the intracellular fluid, the actin cortex, the cell membrane, and the cytoskeleton. The model results quantify the relative importance of cytoskeletal elasticity and drag in bleb expansion dynamics. This study also shows that recent multi-bleb experimental results can be explained by the combination of cytoskeletal poroelasticity with either dynamic membrane-cortex adhesion or cortical reformation.

Bleb Formation in a Hela Cell Induced by Temperature Gradient

The ability to sense and respond to temperature changes is necessary for biological systems. However, the response of individual cells to local temperature gradient is poorly understood. We have observed actomyosin dynamics within a cell which responds to local thermo-stimulation produced by focusing infrared (IR) laser. We found that the thermo-stimulation provokes spherical expansions of the membrane, termed blebs, in mitotic HeLa cells along the temperature gradient. Similar blebs are observed during cell migration, cell spreading, cytokinesis, and apoptosis. It has been proposed that the blebs form due to an increase in intracellular hydrostatic pressure, which results in the breakage of actomyosin cortex. Blebs can be induced artificially by laser ablation or by local delivery of actin-depolymerizing drugs (Sedzinski et al., 2011; Paluch et al., 2005). Cortical tension is also important for bleb expansion (Tinevez et al., 2009). However, the specific mechanism of bleb formation remains unclear. In this study, we demonstrate that blebs are induced by temperature gradient. Different types of blebs were observed depending on the steepness of temperature gradient at the ambient temperature between 40 and 50°C. Mitotic cells which responded to the thermo-gradient formed asymmetrical actomyosin network. A similar, but uniform increase in the ambient temperature did not induce the directional bleb formation, suggesting that the temperature gradient within a cell is essential for the bleb formation. When the motor function of myosin II or the polymerization/depolymerization dynamics of actin had been suppressed, smaller blebs were produced. These results hint to a new perspective that individual cells respond to local thermo-stimulation through shifting the protein activity or concentration distribution.

Role of cortical tension in bleb growth

Blebs are spherical membrane protrusions often observed during cell migration, cell spreading, cytokinesis, and apoptosis, both in cultured cells and in vivo. Bleb expansion is thought to be driven by the contractile actomyosin cortex, which generates hydrostatic pressure in the cytoplasm and can thus drive herniations of the plasma membrane. However, the role of cortical tension in bleb formation has not been directly tested, and despite the importance of blebbing, little is known about the mechanisms of bleb growth. In order to explore the link between cortical tension and bleb expansion, we induced bleb formation on cells with different tensions. Blebs were nucleated in a controlled manner by laser ablation of the cortex, mimicking endogenous bleb nucleation. Cortical tension was modified by treatments affecting the level of myosin activity or proteins regulating actin turnover. We show that there is a critical tension below which blebs cannot expand. Above this threshold, the maximal size of a bleb strongly depends on tension, and this dependence can be fitted with a model of the cortex as an active elastic material. Together, our observations and model allow us to relate bleb shape parameters to the underlying cellular mechanics and provide insights as to how bleb formation can be biochemically regulated during cell motility.

Blebbing of Dictyostelium cells in response to chemoattractant

Stimulation of Dictyostelium cells with a high uniform concentration of the chemoattractant cyclic-AMP induces a series of morphological changes, including cell rounding and subsequent extension of pseudopodia in random directions. Here we report that cyclic-AMP also elicits blebs and analyse their mechanism of formation. The surface area and volume of cells remain constant during blebbing indicating that blebs form by the redistribution of cytoplasm and plasma membrane rather than the exocytosis of internal membrane coupled to a swelling of the cell. Blebbing occurs immediately after a rapid rise and fall in submembraneous F-actin, but the blebs themselves contain little F-actin as they expand. A mutant with a partially inactivated Arp2/3 complex has a greatly reduced rise in F-actin content, yet shows a large increase in blebbing. This suggests that bleb formation is not enhanced by the preceding actin dynamics, but is actually inhibited by them. In contrast, cells that lack myosin-II completely fail to bleb. We conclude that bleb expansion is likely to be driven by hydrostatic pressure produced by cortical contraction involving myosin-II. As blebs are induced by chemoattractant, we speculate that hydrostatic pressure is one of the forces driving pseudopod extension during movement up a gradient of cyclic-AMP.