Signaling clusters in the cell membrane

Large-scale molecular assemblies, or signaling clusters, at the cell membrane are emerging as important regulators of cell signaling. Here, we review new findings and describe shared characteristics common to signaling clusters from a diverse set of cellular systems. The well-known T cell receptor cluster serves as our paradigmatic model. Specifically, each cluster initiates recruitment of hundreds of molecules to the membrane, interacts with the actin cytoskeleton, and contains a significant fraction of the entire signaling process. Probed by recent advancements in patterning and microscopy techniques, the signaling clusters display functional outcomes that are not readily predictable from the individual components.     

Cortactin phosphorylation as a switch for actin cytoskeletal network and cell dynamics control

Cortactin is an important molecular scaffold for actin assembly and organization. Novel mechanistic functions of cortactin have emerged with more interacting partners identified, revealing its multifaceted roles in regulating actin cytoskeletal networks that are necessary for endocytosis, cell migration and invasion, adhesion, synaptic organization and cell morphogenesis. These processes are mediated by its multi-domains binding to F-actin and Arp2/3 complex and various SH3 targets. Furthermore, its role in actin remodeling is subjected to regulation by tyrosine and serine/threonine kinases. Elucidating the mechanisms underlying cortactin phosphorylation and its functional consequences would provide new insights to various aspects of cell dynamics control.     

miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration

During keratinocyte differentiation and stratification, cells undergo extensive remodeling of their actin cytoskeleton, which is important to control cell mobility and to coordinate and stabilize adhesive structures necessary for functional epithelia. Limited knowledge exists on how the actin cytoskeleton is remodeled in epithelial stratification and whether cell shape is a key determinant to trigger terminal differentiation. In this paper, using human keratinocytes and mouse epidermis as models, we implicate miR-24 in actin adhesion dynamics and demonstrate that miR-24 directly controls actin cable formation and cell mobility. miR-24 overexpression in proliferating cells was sufficient to trigger keratinocyte differentiation both in vitro and in vivo and directly repressed cytoskeletal modulators (PAK4, Tks5, and ArhGAP19). Silencing of these targets recapitulated the effects of miR-24 overexpression. Our results uncover a new regulatory pathway involving a differentiation-promoting microribonucleic acid that regulates actin adhesion dynamics in human and mouse epidermis.     

Engagement of the NG2 proteoglycan triggers cell spreading via rac and p130cas

Cells that express the NG2 proteoglycan will spread on surfaces coated with monoclonal antibodies against this membrane-spanning protein. On surfaces coated with the N143 monoclonal antibody, this cell spreading occurs by extension of lamellipodia, suggesting that activation of the small GTPase rac is involved in the observed morphological change. Support for this hypothesis comes from the finding of increased levels of GTP-bound rac in cells spreading on N143-coated surfaces. Furthermore, lamellipodia extension is blocked by transfection of cells with the dominant negative rac construct N17rac, but not by transfection with N17cdc42. Formation of lamellipodia on the N143-coated surfaces is also inhibited by transfection of the dominant negative CasdeltaSD construct. This result implicates p130cas as an additional functional player in NG2-mediated cell spreading.     

Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells

When cultivated on substrates that prevent cell adhesion (the polymer polyhydroxyethylmethacrylate, bovine serum albumin, and Teflon), human endothelial cells (EC) rapidly lost viability with a half-life of approximately 10 h. Dying EC showed the morphological and biochemical characteristics of apoptosis. The apoptotic process of suspended EC was delayed by the protein synthesis inhibitor cycloheximide. To obtain information as to the mechanism involved in the apoptosis of suspended EC, we investigated whether adhesion to matrix proteins or integrin occupancy in EC retaining a round shape may affect EC suicide. EC bound to low coating concentration of either fibronectin or vitronectin, retaining a round shape and failing to organize actin microfilaments, underwent to rapid cell death; by contrast, cells on high substrate concentrations became flattened, showed actin microfilament organization, and retained viability. Addition of saturating amounts of soluble vitronectin to suspended round-shaped EC did not reduce the process of apoptosis. Finally, when suspended EC bound Gly-Arg-Gly-Asp- Ser-coated microbeads (approximately 10 microbeads/cell), yet retaining a round shape, the apoptotic process was not affected. Oncogene- transformed EC in suspension were less susceptible to cell death and apoptosis than normal EC. Overall, these data indicate that cell attachment to matrix or integrin binding per se is not sufficient for maintaining cell viability, and that cells need to undergo some minimal degree of shape change to survive. Modulation of interaction with the extracellular matrix can, therefore, be an important target for the control of angiogenesis.     

Initial dynamics of cell spreading are governed by dissipation in the actin cortex

The initial stages of spreading of a suspended cell onto a substrate under the effect of (specific or nonspecific) adhesion exhibit a universal behavior, which is cell-type independent. We show that this behavior is governed only by cell-scale phenomena. This can be understood if the main retarding force that opposes cell adhesion is of mechanical origin, that is, dissipation occurring during the spreading. By comparing several naive models that generate different patterns of dissipation, we show by numerical simulation that only dissipation due to the deformation of the actin cortex is compatible with the experimental observations. This viscous-like dissipation corresponds to the energetic cost of rearranging the cytoskeleton, and is the trace of all dissipative events occurring in the cell cortex during the early spreading, such as the binding and unbinding of cross-linkers and molecular friction.     

Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane‐adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel‐fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large‐scale lipid phase separation and protein segregation in intact, protein‐crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible.     

Study of actin filament ends in the human red cell membrane

There is conflicting evidence concerning the state of the actin protofilaments in the membrane cytoskeleton of the human red cell. To resolve this uncertainty, we have analysed their characteristics with respect to nucleation of G-actin polymerization. The effects of cytochalasin E on the rate of elongation of the protofilaments have been measured in a medium containing 0.1 M-sodium chloride and 5 mM-magnesium chloride, using pyrene-labelled G-actin. At an initial monomer concentration far above the critical concentration for the negative ("pointed") end of F-actin, high concentrations of cytochalasin reduce the elongation rate of free F-actin by about 70%. The residual rate is presumed to correspond to the elongation rate at the negative ends. By contrast, the elongation rate on red cell ghosts or cytoskeletons falls to zero, allowing for the background of self-nucleated polymerization of the G-actin. The critical concentration of the actin in the red cell membrane has been measured after elongation of the filaments by added pyrenyl-G-actin in the same solvent. It was found to be 0.07 microM, compared with 0.11 microM under the same conditions for actin alone. This is consistent with prediction for the case of blocked negative ends on the red cell actin. The rate of elongation of actin filaments, free and in the red cell membrane cytoskeleton, has been measured as a function of the concentration of an added actin-capping protein, plasma gelsolin, with a high affinity for the positive ends. The elongation rate falls linearly with increasing gelsolin concentration until it approaches a minimum when the gelsolin has bound to all positive filament ends. The elongation rate at this point corresponds to the activity of the negative ends, and its ratio to the unperturbed polymerization rate (in the absence of capping proteins) is indistinguishable from zero in the case of ghosts, but about 1 : 4 in the case of F-actin. When ATP is replaced in the system by ADP, so that the critical concentrations at the two filament ends are equalized, the difference is equally well-marked: for F-actin, the rate at the equivalence point is about 40% of that in the absence of capping protein, whereas for ghosts the nucleated polymerization rate at the equivalence point is again zero, indicating that under these conditions the negative ends contribute little or not at all to the rate of elongation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role for actin filament turnover and a myosin II motor in cytoskeleton-driven disassembly of the epithelial apical junctional complex

Disassembly of the epithelial apical junctional complex (AJC), composed of the tight junction (TJ) and adherens junction (AJ), is important for normal tissue remodeling and pathogen-induced disruption of epithelial barriers. Using a calcium depletion model in T84 epithelial cells, we previously found that disassembly of the AJC results in endocytosis of AJ/TJ proteins. In the present study, we investigated the role of the actin cytoskeleton in disassembly and internalization of the AJC. Calcium depletion induced reorganization of apical F-actin into contractile rings. Internalized AJ/TJ proteins colocalized with these rings. Both depolymerization and stabilization of F-actin inhibited ring formation and disassembly of the AJC, suggesting a role for actin filament turnover. Actin reorganization was accompanied by activation (dephosphorylation) of cofilin-1 and its translocation to the F-actin rings. In addition, Arp3 and cortactin colocalized with these rings. F-actin reorganization and disassembly of the AJC were blocked by blebbistatin, an inhibitor of nonmuscle myosin II. Myosin IIA was expressed in T84 cells and colocalized with F-actin rings. We conclude that disassembly of the AJC in calcium-depleted cells is driven by reorganization of apical F-actin. Mechanisms of such reorganization involve cofilin-1-dependent depolymerization and Arp2/3-assisted repolymerization of actin filaments as well as myosin IIA-mediated contraction.     

Effect of membrane tension on the physical properties of DOPC lipid bilayer membrane

Molecular dynamics simulations of a dioleoylphosphocholine (DOPC) lipid bilayer were performed to explore its mechanosensitivity. Variations in the bilayer properties, such as area per lipid, volume, thickness, hydration depth (HD), hydration thickness (HT), lateral diffusion coefficient, and changes in lipid structural order were computed in the membrane tension range 0 to 15dyn/cm. We determined that an increase in membrane tension results in a decrease in the bilayer thickness and HD of ~5% and ~5.7% respectively, whereas area per lipid, volume, and HT/HD increased by 6.8%, 2.4%, and 5% respectively. The changes in lipid conformation and orientation were characterized using orientational (S(2)) and deuterium (S(CD)) order parameters. Upon increase of membrane tension both order parameters indicated an increase in lipid disorder by 10-20%, mostly in the tail end region of the hydrophobic chains. The effect of membrane tension on lipid lateral diffusion in the DOPC bilayer was analyzed on three different time scales corresponding to inertial motion, anomalous diffusion and normal diffusion. The results showed that lateral diffusion of lipid molecules is anomalous in nature due to the non-exponential distribution of waiting times. The anomalous and normal diffusion coefficients increased by 20% and 52% when the membrane tension changed from 0 to 15dyn/cm, respectively. In conclusion, our studies showed that membrane tension causes relatively significant changes in the area per lipid, volume, polarity, membrane thickness, and fluidity of the membrane suggesting multiple mechanisms by which mechanical perturbation of the membrane could trigger mechanosensitive response in cells.     

A Highlights from MBoC Selection: Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane

Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (∼25–30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ∼60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane.     

Microvilli and blebs as sources of reserve surface membrane during cell spreading

The increase in surface area that occurs as cells spread from the rounded to the flattened state has been examined in synchronized BHK 21 cells in the scanning electron microscope. Rounded cells, whether in mitosis or dissociated and freshly seeded in culture, are covered with a mixture of folds, blebs, and microvilli. As cells spread, these protuberances disappear, first in the flattening marginal region and progressively submarginally until the entire cell surface is virtually smooth. The estimated surface area of rounded post-mitotic daughter cells, taking microvilli into account, is close to that of fully spread cells 4 h after mitosis. Likewise, rounded early mitotic mother cells, which are also covered with microvilli, have approximately the same surface as fully spread cells just prior to mitosis. These findings suggest that cells possess a membrane reserve in their microvilli and other protuberances which can be utilized for spreading and initiating cell locomotion.     

Nanoscale dynamics of actin filaments in the red blood cell membrane skeleton

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15–18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature, and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ are not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200–300 nm apart interspersed with dimmer F-actin staining regions. Single molecule stochastic optical reconstruction microscopy imaging of Alexa 647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, nonrandom distribution of F-actin nodes. Treatment of RBCs with latrunculin A and cytochalasin D indicates that F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape, and membrane deformability.     

Modeling the kinetics of cell membrane spreading on substrates with ligand density gradient

An analytical model is developed for the effect of surface gradient in ligand density on the adhesion kinetics of a curved elastic membrane with mobile receptors. The displacement and speed of spreading at the edge of adhesion zone as well as the density profile of receptors along the membrane are predicted as a function of time. According to results, in the diffusion-controlled regime, the front edge displacement of adhesion zone and the rate of membrane spreading decreased with increasing the ligand density in a certain direction. Furthermore, the displacement of the edge of the adhesion zone did not scale with the square root of time, as observed on substrates with uniform ligand density.     

Arp2/3 complex inhibition radically alters lamellipodial actin architecture,suspended cell shape,and the cell spreading process

Recent studies have investigated the dendritic actin cytoskeleton of the cell edge''s lamellipodial (LP) region by experimentally decreasing the activity of the actin filament nucleator and branch former, the Arp2/3 complex. Here we extend these studies via pharmacological inhibition of the Arp2/3 complex in sea urchin coelomocytes, cells that possess an unusually broad LP region and display correspondingly exaggerated centripetal flow. Using light and electron microscopy, we demonstrate that Arp2/3 complex inhibition via the drug CK666 dramatically altered LP actin architecture, slowed centripetal flow, drove a lamellipodial-to-filopodial shape change in suspended cells, and induced a novel actin structural organization during cell spreading. A general feature of the CK666 phenotype in coelomocytes was transverse actin arcs, and arc generation was arrested by a formin inhibitor. We also demonstrate that CK666 treatment produces actin arcs in other cells with broad LP regions, namely fish keratocytes and Drosophila S2 cells. We hypothesize that the actin arcs made visible by Arp2/3 complex inhibition in coelomocytes may represent an exaggerated manifestation of the elongate mother filaments that could possibly serve as the scaffold for the production of the dendritic actin network.     

Low cholesterol triggers membrane microdomain-dependent CD44 shedding and suppresses tumor cell migration

CD44 is a cell surface adhesion molecule for hyaluronan and is implicated in tumor invasion and metastasis. Proteolytic cleavage of CD44 plays a critical role in the migration of tumor cells and is regulated by factors present in the tumor microenvironment, such as hyaluronan oligosaccharides and epidermal growth factor. However, molecular mechanisms underlying the proteolytic cleavage on membranes remain poorly understood. In this study, we demonstrated that cholesterol depletion with methyl-β-cyclodextrin, which disintegrates membrane lipid rafts, enhances CD44 shedding mediated by a disintegrin and metalloproteinase 10 (ADAM10) and that cholesterol depletion disorders CD44 localization to the lipid raft. We also evaluated the effect of long term cholesterol reduction using a statin agent and demonstrated that statin enhances CD44 shedding and suppresses tumor cell migration on a hyaluronan-coated substrate. Our results indicate that membrane lipid organization regulates CD44 shedding and propose a possible molecular mechanism by which cholesterol reduction might be effective for preventing and treating the progression of malignant tumors.