Membrane-cytoskeleton Adhesion Research Articles

Roles of microglial membranes in Alzheimer's disease.

The majority of Alzheimer's disease (AD) risk genes are highly and selectively expressed by microglia in the brain. Several of these genes are related to lipid and cholesterol metabolism, lipid synthesis, lipid transport, endocytosis, exocytosis and phagocytosis. Therefore, studying the roles of cellular membrane biophysics in microglial function should improve our understanding of the AD pathology. In this chapter, we discuss how lipid rafts and membrane-cytoskeleton adhesion impact microglial-mediated oxidative stress and clearance of amyloid-β peptide (Aβ). We also discuss potential roles of lipid membrane-bound extracellular vesicles as carriers of pathological factors to promote inflammation and cytotoxicity.

Facilitation of Rapid Temporal Processing by Ion Channel Cooperativity Suggests Coordination through Membrane Electromechanics

The ability of neuronal populations to rapidly encode and process information is crucial for such basic computations as coincidence detection, grouping by synchrony and spike-timing-dependent plasticity. Theoretical analyses have linked these abilities to the fast-onset dynamics of action potentials (APs). A computational analysis that invokes cooperative activation of Na+ ion channels at the axon initial segment (AIS) predicts rapid AP onset. The near simultaneous gating of ion channels results in a channel-density dependent hyperpolarizing shift in the population activation curve and rapid AP initiation. The biophysical basis for intra-channel coupling is unknown. Membrane mechanics is known to modulate ion channel function and membrane tethers have been shown to generate electromechanical force at frequencies up to 10 kHz. We propose that membrane electromechanics contributes to cooperative gating of sodium channels at the AIS by providing a fast and direct mechanism for coordinating inter-channel activity. Ion channels are clustered by the actin based cytoskeleton in the AIS. Since cooperative activation is dependent on the density of Na+ channels, their clustering enhances cooperative gating. We find that membrane-cytoskeleton adhesion is stronger in the AIS than it is in the neuronal soma using optical tweezers. This is consistent with recent experiments showing the removal of a cytoskeletal scaffold protein disrupts sodium channel function and results in impaired network excitability. We also use optical tweezers to measure membrane electromechanical force generation in voltage clamped cells. The use of concomitant variation between electromechanical force and channel function to determine the membrane's role in ion channel gating will be discussed.

Membrane Tension in Rapidly Moving Cells Is Determined by Cytoskeletal Forces

Membrane tension plays an essential role in cell motility. The load imposed by the tensed membrane restrains actin polymerization, promotes rear retraction, and influences membrane transport. Moreover, membrane tension is crucial for large-scale coordination of cell boundary dynamics. Despite its importance, little is known about how membrane tension is set and regulated in cells. The prevailing hypothesis is that membrane tension is largely controlled by membrane-cytoskeleton adhesion and/or changes in membrane area. In this work, we measure the apparent membrane tension in rapidly moving fish epithelial keratocytes under normal and perturbed conditions with a tether-pulling assay. We find that enlargement of the cell surface area by fusion with giant unilamellar vesicles (GUVs) has only minor effects on membrane tension and on cell movement. However, modulation of the cytoskeletal forces has a substantial influence on tension: reduction of the actin-pushing forces along the cell's leading edge leads to a significant decrease in membrane tension, whereas increase of the strength of adhesion and/or decrease of myosin-induced contraction leads to higher tension. We find that the membrane tension in rapidly moving keratocytes is primarily determined by a mechanical force balance between the cell membrane and cytoskeletal forces. Our results highlight the role of membrane tension as a global mechanical regulator of cell behavior.

Amyloid-β Peptide on Sialyl-LewisX-Selectin-Mediated Membrane Tether Mechanics at the Cerebral Endothelial Cell Surface

Increased deposition of amyloid-β peptide (Aβ) at the cerebral endothelial cell (CEC) surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB) in Alzheimer's disease (AD). In this study, quantitative immunofluorescence microscopy (QIM) and atomic force microscopy (AFM) with cantilevers biofunctionalized by sialyl-Lewisx (sLex) were employed to investigate Aβ-altered mechanics of membrane tethers formed by bonding between sLex and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Aβ to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs) and human primary CECs. AFM data also showed the ability for Aβ to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Aβ lowered the overall force of membrane tether formation (Fmtf), and produced a bimodal population of Fmtf, suggesting subcellular mechanical alterations in membrane tethering. The lower Fmtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Aβ and latrunculin A decreased membrane stiffness, suggesting a lower membrane-cytoskeleton adhesion, a factor resulting in lower Fmtf. In addition, these cerebral endothelial alterations induced by Aβ were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Aβ to enhance p-selectin expression at the CEC surface and induce cytoskeleton reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB.

Cellular Membrane Tether (Nanotube) Retraction, Mobility, and Coalescence

During leukocyte rolling on the endothelium, membrane tethers (nanotubes)are extracted simultaneously from both leukocytes and endothelial cells because of the force imposed by the blood flow. Tether extraction has been shown to stabilize leukocyte rolling by increasing the lifetime of the adhesive selectin-ligand bonds that mediate leukocyte rolling. Over the past two decades, tether extraction has been studied extensively, both experimentally and theoretically. In contrast, much less is known about tether retraction. Tether retraction may occur in several occasions. For example, upon the breakage of the selectin-ligand bonds during leukocyte rolling, extracted tethers may retract back to the cells. In addition, during simultaneous tether extraction (two tethers, one from a leukocyte and the other from an endothelial cell, linked in series by receptor-ligand bonds), one tether will retract when the pulling force falls below the larger threshold force. In this work, with the micro-cantilever technique where latex beads affixed on silicon cantilevers were used as the force transducer, we extracted tethers either perpendicular or tangential to the neutrophil surface. Little movement of the tether-cell junction was observed during tangential tether extraction and no coalescence was observed during multiple tether extraction. Following adhesion rupture, spontaneous tether retraction was visualized by membrane staining, which revealed two phases: one was fast and exponential, whereas the other was slow and linear. Both phases can be reproduced with a mechanical model, showing that the first phase was dominated by elastic deformation recovery and the latter slow phase was driven by the far-field membrane tension on the cell body and the membrane-cytoskeleton adhesion. These results show for the first time how neutrophil tethers shorten upon instantaneous force removal and illustrate further how membrane tethers contribute to neutrophil rolling stability during the inflammatory response.

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

In this study, we investigated the effects of membrane cholesterol content on the mechanical properties of cell membranes by using optical tweezers. We pulled membrane tethers from human embryonic kidney cells using single and multi-speed protocols, and obtained time-resolved tether forces. We quantified various mechanical characteristics including the tether equilibrium force, bending modulus, effective membrane viscosity, and plasma membrane-cytoskeleton adhesion energy, and correlated them to the membrane cholesterol level. Decreases in cholesterol concentration were associated with increases in the tether equilibrium force, tether stiffness, and adhesion energy. Tether diameter and effective viscosity increased with increasing cholesterol levels. Disruption of cytoskeletal F-actin significantly changed the tether diameters in both non-cholesterol and cholesterol-manipulated cells, while the effective membrane viscosity was unaffected by F-actin disruption. The findings are relevant to inner ear function where cochlear amplification is altered by changes in membrane cholesterol content.

How cholesterol regulates endothelial biomechanics

As endothelial cells form the barrier between blood flow and surrounding tissue, many of their functions depend on mechanical integrity, in particular those of the plasma membrane. As component and organizer of the plasma membrane, cholesterol is a regulator of cellular mechanical properties. Disruption of cholesterol balance leads to impairment of endothelial functions and eventually to disease. The mechanical properties of the membrane are strongly affected by the cytoskeleton. As Phosphatidylinositol-4,5-bisphosphate (PIP2) is a key mediator between the membrane and cytoskeleton, it also affects cellular biomechanical properties. Typically, PIP2 is concentrated in cholesterol-rich microdomains, such as caveolae and lipid rafts, which are particularly abundant in the endothelial plasma membrane. We investigated the connection between cholesterol and PIP2 by extracting membrane tethers from bovine aortic endothelial cells (BAEC) at different cholesterol levels and PIP2 conditions. Our results suggest that in BAEC the role of PIP2, as a mediator of membrane-cytoskeleton adhesion, is regulated by cholesterol. Our findings confirm the specific role of cholesterol in endothelial cells and may have implications for cholesterol-dependent vascular pathologies.

Single-Molecule Adhesion Forces and Attachment Lifetimes of Myosin-I Phosphoinositide Interactions

Phosphoinositides regulate the activities and localization of many cytoskeletal proteins involved in crucial biological processes, including membrane-cytoskeleton adhesion. Yet little is known about the mechanics of protein-phosphoinositide interactions, or about the membrane-attachment mechanics of any peripheral membrane proteins. Myosin-Ic (myo1c) is a molecular motor that links membranes to the cytoskeleton via phosphoinositide binding, so it is particularly important to understand the mechanics of its membrane attachment. We used optical tweezers to measure the strength and attachment lifetime of single myo1c molecules as they bind beads coated with a bilayer of 2% phosphatidylinositol 4,5-bisphosphate and 98% phosphatidylcholine. Adhesion forces measured under ramp-load ranged between 5.5 and 16 pN at loading rates between 250 and 1800 pN/s. Dissociation rates increased linearly with constant force (0.3–2.5 pN), with rates exceeding 360 s −1 at 2.5 pN. Attachment lifetimes calculated from adhesion force measurements were loading-rate-dependent, suggesting nonadiabatic behavior during pulling. The adhesion forces of myo1c with phosphoinositides are greater than the motors stall forces and are within twofold of the force required to extract a lipid molecule from the membrane. However, attachment durations are short-lived, suggesting that phosphoinositides alone do not provide the mechanical stability required to anchor myo1c to membranes during multiple ATPase cycles.

Myosin motor function: the ins and outs of actin-based membrane protrusions

Cells build plasma membrane protrusions supported by parallel bundles of F-actin to enable a wide variety of biological functions, ranging from motility to host defense. Filopodia, microvilli and stereocilia are three such protrusions that have been the focus of intense biological and biophysical investigation in recent years. While it is evident that actin dynamics play a significant role in the formation of these organelles, members of the myosin superfamily have also been implicated as key players in the maintenance of protrusion architecture and function. Based on a simple analysis of the physical forces that control protrusion formation and morphology, as well as our review of available data, we propose that myosins play two general roles within these structures: (1) as cargo transporters to move critical regulatory components toward distal tips and (2) as mediators of membrane-cytoskeleton adhesion.

Myosin-I Dependent Membrane-Cytoskeleton Adhesion as a Regulator of Cell Surface Morphology

All cellular functions involving deformation of the plasma membrane (e.g. endocytosis, exocytosis, the formation of surface protrusions) are regulated by the apparent membrane tension (Tm), a composite of the surface tension (σ) and the adhesion provided by molecules linking membrane to the actin cytoskeleton (γ). Using an optical trap based tether force assay, we recently demonstrated that class I myosins, a family of membrane-binding actin-based motor proteins, control membrane tension by mediating membrane-cytoskeleton adhesion. More specifically, these studies revealed that the membrane-cytoskeleton adhesion provided by myosin-I increases apparent membrane tension as indicated by the force required to pull a single membrane tether from the cell surface. Interestingly, the physical links to the cytoskeleton provided by myosin-I also allow the cell to form multiple adjacent tethers. However, when multiple tethers are pulled, tether lifetimes appear to decrease as apparent membrane tension increases. Here we present a thermodynamic model, which accounts for the impact of apparent membrane tension on the lifetime of multiple tethers. We argue that, in the context of our experimental geometry, the global increase in apparent membrane tension that results from greater membrane-cytoskeleton adhesion, works locally to accelerate the rupture of bonds between the membrane and cytoskeleton, which would otherwise prevent individual membrane tethers from coalescing. We also elaborate on this concept to develop a separate model, which shows that tuning the level of membrane-cytoskeleton adhesion may enable cells to vary the density of surface protrusions (i.e. # of structures per unit area membrane). As such, myosin-I dependent membrane-cytoskeleton adhesion emerges as the key regulator of cell surface morphology.

Myosin-1a

Myosin-1a is one of eight monomeric, membrane binding class I myosins expressed in vertebrates.1 As the most abundant actin-based motor protein found in the enterocyte microvillus, myosin-1a has long been known to interact with the apical membrane via a highly basic C-terminal tail domain.2 Several recent studies shed light on possible functional consequences of this protein/lipid interaction. In vitro and in vivo studies of microvillar function have revealed that myosin-1a can move apical membrane along core actin bundles, leading to the release of small vesicles from microvillar tips.3,4 Additional studies indicate that myosin-1a and other class I myosins contribute to membrane-cytoskeleton adhesion, which enables the apical membrane to resist deformation.5 These findings clearly position myosin-1a as an important player in apical membrane movement and structural stability. How this motor is able to fulfill these two seemingly distinct functions is currently unclear, but will serve as the focus of our discussion below.

Computational analysis of the tether-pulling experiment to probe plasma membrane-cytoskeleton interaction in cells.

Tethers are thin membrane tubes that can be formed when relatively small and localized forces are applied to cellular membranes and lipid bilayers. Tether pulling experiments have been used to better understand the fine membrane properties. These include the interaction between the plasma membrane and the underlying cytoskeleton, which is an important factor affecting membrane mechanics. We use a computational method aimed at the interpretation and design of tether pulling experiments in cells with a strong membrane-cytoskeleton attachment. In our model, we take into account the detailed information in the topology of bonds connecting the plasma membrane and the cytoskeleton. We compute the force-dependent piecewise membrane deflection and bending as well as modes of stored energy in three major regions of the system: body of the tether, membrane-cytoskeleton attachment zone, and the transition zone between the two. We apply our method to three cells: cochlear outer hair cells (OHCs), human embryonic kidney (HEK) cells, and Chinese hamster ovary (CHO) cells. OHCs have a special system of pillars connecting the membrane and the cytoskeleton, and HEK and CHO cells have the membrane-cytoskeleton adhesion arrangement via bonds (e.g., PIP2), which is common to many other cells. We also present a validation of our model by using experimental data on CHO and HEK cells. The proposed method can be an effective tool in the analyses of experiments to probe the properties of cellular membranes.

The effect of cellular cholesterol on membrane-cytoskeleton adhesion

Whereas recent studies suggest that cholesterol plays important role in the regulation of membrane proteins, its effect on the interaction of the cell membrane with the underlying cytoskeleton is not well understood. Here, we investigated this by measuring the forces needed to extract nanotubes (tethers) from the plasma membrane, using atomic force microscopy. The magnitude of these forces provided a direct measure of cell stiffness, cell membrane effective surface viscosity and association with the underlying cytoskeleton. Furthermore, we measured the lateral diffusion constant of a lipid analog DiIC12, using fluorescence recovery after photobleaching, which offers additional information on the organization of the membrane. We found that cholesterol depletion significantly increased the adhesion energy between the membrane and the cytoskeleton and decreased the membrane diffusion constant. An increase in cellular cholesterol to a level higher than that in control cells led to a decrease in the adhesion energy and the membrane surface viscosity. Disassembly of the actin network abrogated all the observed effects, suggesting that cholesterol affects the mechanical properties of a cell through the underlying cytoskeleton. The results of these quantitative studies may help to better understand the biomechanical processes accompanying the development of atherosclerosis.

CONTINUOUS MEMBRANE-CYTOSKELETON ADHESION REQUIRES CONTINUOUS ACCOMMODATION TO LIPID AND CYTOSKELETON DYNAMICS

The plasma membrane of most animal cells conforms to the cytoskeleton and only occasionally separates to form blebs. Previous studies indicated that many weak interactions between cytoskeleton and the lipid bilayer kept the surfaces together to counteract the normal outward pressure of cytoplasm. Either the loss of adhesion strength or the formation of gaps in the cytoskeleton enables the pressure to form blebs. Membrane-associated cytoskeleton proteins, such as spectrin and filamin, can control the movement and aggregation of membrane proteins and lipids, e.g., phosphoinositol phospholipids (PIPs), as well as blebbing. At the same time, lipids (particularly PIPs) and membrane proteins affect cytoskeleton and signaling dynamics. We consider here the roles of the major phosphatidylinositol-4,5-diphosphate (PIP2) binding protein, MARCKS, and PIP2 levels in controlling cytoskeleton dynamics. Further understanding of dynamics will provide important clues about how membrane-cytoskeleton adhesion rapidly adjusts to cytoskeleton and membrane dynamics.

Phospholipase C activation by anesthetics decreases membrane-cytoskeleton adhesion.

Many different amphiphilic compounds cause an increase in the fluid-phase endocytosis rates of cells in parallel with a decrease in membrane-cytoskeleton adhesion. These compounds, however, do not share a common chemical structure, which leaves the mechanism and even site of action unknown. One possible mechanism of action is through an alteration of inositol lipid metabolism by modifying the cytoplasmic surface of the plasma membrane bilayer. By comparing permeable amphiphilic amines used as local anesthetics with their impermeable analogs, we find that access to the cytoplasmic surface is necessary to increase endocytosis rate and decrease membrane-cytoskeleton adhesion. In parallel, we find that the level of phosphatidylinositol 4,5-bisphosphate (PIP(2)) in the plasma membrane is decreased and cytoplasmic Ca(2+) is increased only by permeable amines. The time course of both the decrease in plasma membrane PIP(2) and the rise in Ca(2+) parallels the decrease in cytoskeleton-membrane adhesion. Inositol labeling shows that phosphatidylinositol-4-phosphate levels are increased by the permeable anesthetics, indicating that lipid turnover is increased. Consistent with previous observations, phospholipase C (PLC) inhibitors block anesthetic effects on the PIP(2) and cytoplasmic Ca(2+) levels, as well as the drop in adhesion. Therefore, we suggest that PLC activity is increased by amine anesthetics at the cytoplasmic surface of the plasma membrane, which results in a decrease in membrane-cytoskeleton adhesion.

Cell control by membrane-cytoskeleton adhesion.

The rates of mechanochemical processes, such as endocytosis, membrane extension and membrane resealing after cell wounding, are known to be controlled biochemically, through interaction with regulatory proteins. Here, I propose that these rates are also controlled physically, through an apparently continuous adhesion between plasma membrane lipids and cytoskeletal proteins.

Membrane Tether Formation from Blebbing Cells

Membrane tension has been proposed to be important in regulating cell functions such as endocytosis and cell motility. The apparent membrane tension has been calculated from tether forces measured with laser tweezers. Both membrane-cytoskeleton adhesion and membrane tension contribute to the tether force. Separation of the plasma membrane from the cytoskeleton occurs in membrane blebs, which could remove the membrane-cytoskeleton adhesion term. In renal epithelial cells, tether forces are significantly lower on blebs than on membranes that are supported by cytoskeleton. Furthermore, the tether forces are equal on apical and basolateral blebs. In contrast, tether forces from membranes supported by the cytoskeleton are greater in apical than in basolateral regions, which is consistent with the greater apparent cytoskeletal density in the apical region. We suggest that the tether force on blebs primarily contains only the membrane tension term and that the membrane tension may be uniform over the cell surface. Additional support for this hypothesis comes from observations of melanoma cells that spontaneously bleb. In melanoma cells, tether forces on blebs are proportional to the radius of the bleb, and as large blebs form, there are spikes in the tether force in other cell regions. We suggest that an internal osmotic pressure inflates the blebs, and the pressure calculated from the Law of Laplace is similar to independent measurements of intracellular pressures. When the membrane tension term is subtracted from the apparent membrane tension over the cytoskeleton, the membrane-cytoskeleton adhesion term can be estimated. In both cell systems, membrane-cytoskeleton adhesion was the major factor in generating the tether force.