Ratiometric Imaging of the T-Cell Actin Cytoskeleton Reveals the Nature of Receptor-Induced Cytoskeletal Enrichment

The T-cell actin cytoskeleton mediates adaptive immune system responses to peptide antigens by physically directing the motion and clustering of T-cell receptors (TCRs) on the cell surface. When TCR movement is impeded by externally applied physical barriers, the actin network exhibits transient enrichment near the trapped receptors. The coordinated nature of the actin density fluctuations suggests that they are composed of filamentous actin, but it has not been possible to eliminate de novo polymerization at TCR-associated actin polymerizing factors as an alternative cause. Here, we use a dual-probe cytoskeleton labeling strategy to distinguish between stable and polymerizing pools of actin. Our results suggest that TCR-associated actin consists of a relatively high proportion of the stable cytoskeletal fraction and extends away from the cell membrane into the cell. This implies that actin enrichment at mechanically trapped TCRs results from three-dimensional bunching of the existing filamentous actin network.The T-cell actin cytoskeleton is critical for proper antigen recognition by the mammalian adaptive immune system. During T-cell receptor (TCR) triggering by antigen peptides presented on major histocompatibility proteins (pMHCs) on the surfaces of antigen-presenting cells (APCs), the T-cell actin cytoskeleton adopts a pattern of centrosymmetric retrograde flow (1–3). This simultaneously promotes further TCR triggering (4) and rearranges various T-cell membrane proteins and their APC counterparts into an organized cell-cell interface termed the immunological synapse (IS) (5–7). During this process, TCRs form microclusters that move to the center of the IS in an actin-dependent manner (8,9). When engineered physical barriers interrupt the centripetal motion of TCR clusters, actin flow slows near the pinned microclusters, and the cytoskeletal network transiently accumulates and dissipates at the sites (10,11). The amplitude and duration of the induced cytoskeletal fluctuations are much greater than would be expected for a random distribution of independent objects, indicating that the actin in the local environment is coordinated. Whether this coordination arises from a rearrangement in the existing F-actin network or represents de novo polymerization of the cytoskeleton, as predicted by the association of TCRs with actin polymerizing factors (12), remains unclear. Here, we use a dual-probe cytoskeleton labeling approach that has previously been applied to distinguish between stable and dynamic populations of actin by exploiting the different relative affinities of monomeric actin and actin-binding proteins toward each population (13). This strategy reveals that TCR-associated actin is composed primarily of the stable cytoskeletal fraction and that local enrichment results from three-dimensional bunching of the existing filamentous actin network.Primary T cells from mice transgenic for the AND TCR were triggered using synthetic APCs consisting of supported lipid bilayers functionalized with pMHC and the integrin ligand intercellular adhesion molecule 1. Nanopatterned metal grids on the bilayer substrate acted as diffusion barriers that prevented lateral transport of TCR-pMHC complexes (14,15). Transient enrichment of actin at TCR clusters trapped at these barriers was visualized using fluorescent fusions of actin itself (mKate2-β-actin) and the F-actin binding domain of utrophin (EGFP-UtrCH). Such a dual-probe strategy theoretically allows for discrimination between different pools of actin: dynamic populations characterized by high polymerization and/or short filament fragments tend to be relatively better labeled by direct actin fusions whereas stable populations composed of longer filaments can support higher labeling by fluorescent fusions of F-actin binding proteins. This visualization method has been validated in Xenopus oocytes, where it distinguishes actin populations during wound healing (13). It has not been explicitly applied to T cells; however, simultaneous labeling of the Jurkat cell cytoskeleton using EGFP-actin and Alexa 568-phalloidin reveals distinct populations of actin consistent with the results expected from Xenopus (13,16).Our results show that the T-cell periphery is relatively enriched in mKate2-β-actin (Fig. 1 C, box 1), while EGFP-UtrCH dominates toward the center of the IS (Fig. 1 C, box 2). We infer from this probe distribution that the cytoskeleton at the T-cell periphery is composed of short fragments and is a site of active polymerization, whereas at the center of the IS, actin filaments are longer and predominantly stable. This is consistent with previous models of the T-cell actin network (3,16). An effective way to highlight each of these cytoskeletal regions is to consider the relative ratios of the two probes at each location. In this case, a high UtrCH/actin ratio corresponds to stable actin, and a high actin/UtrCH ratio corresponds to dynamic actin (Fig. 1 D). When T cells are treated with cytochalasin D, an inhibitor of actin polymerization, the overall UtrCH/actin ratio of the cell decreases as would be expected from a general decrease in polymerized actin (see Movie S7 and Movie S8 in the Supporting Material). However, it should be noted that photobleaching can also shift the UtrCH/actin ratio over time. We limit quantitative analysis of the ratio to its spatial gradients at a single time point, but such analysis is possible in systems that permit rigorous calibration for probe expression and photobleaching.Open in a separate windowFigure 1Ratiometric imaging of the cytoskeleton in live T cells distinguishes between dynamic and stable actin populations. (A) mKate2-β-actin, (B) EGFP-UtrCH, and (C) merged images of a triggered T cell show different actin pools. The cutouts in panel C correspond to (1) a region high in dynamic actin featuring short, polymerizing filaments and/or actin monomers and (2) a region with a stable actin population featuring longer filaments to which UtrCH can bind. (D) The UtrCH/actin ratio image highlights pools of relatively high UtrCH (red) or actin (blue). (Scale bars: 5 μm.)Actin enrichment at trapped TCR clusters incorporates both mKate2-β-actin (Fig. 2, A and C) and EGFP-UtrCH (Fig. 2, B and C). The relative UtrCH/actin ratio at these sites (Fig. 2 D, box 2) is quite high relative to nearby background areas (Fig. 2 D, box 1), indicating that the actin is derived primarily from the stable actin population.Open in a separate windowFigure 2Receptor-induced cytoskeletal enrichment at sites of pinned TCRs corresponds to a primarily stable actin fraction. (A) mKate2-β-actin, (B) EGFP-UtrCH, and (C) merged images of a triggered T cell interacting with a nanopatterned supported lipid bilayer show actin enrichment corresponding to putative sites of pinned TCRs. (D) The UtrCH/actin ratio is high at sites displaying actin enrichment, indicating a primarily stable actin fraction in (1) these regions compared to (2) nearby background areas. (Scale bars: 5 μm.)The three-dimensional distribution of TCR-associated actin was analyzed in dual-labeled live T cells using a spinning disk confocal microscope. The recordings show actin extending away from the cell membrane in the vicinity of trapped TCRs, while the rest of the actin cytoskeleton remains relatively flat (Fig. 3 and see Fig. S1 in the Supporting Material). These protrusions of actin away from the membrane surface are predominantly composed of stable, filamentous actin, as indicated by their relatively high UtrCH/actin ratio (Fig. 3 B).Open in a separate windowFigure 3Three-dimensional ratiometric imaging shows that actin enrichment extends away from the cell membrane. Single planes from (A) merged mKate2-β-actin and EGFP-UtrCH and (B) UtrCH/actin ratio three-dimensional stacks show actin enrichment at the cell membrane. Cutouts represent Z projections passing through sites of (1) enrichment and (2) nearby background regions. The color distribution in panel B is analogous to that in Figs. 1D and ​and22D, and is omitted for clarity. (Scale bar: 5 μm in the x axis only. Scale box: 1 μm.)Our interpretation of these results is that the filamentous actin network is relatively dense at sites of pinned TCRs. This is the simplest explanation out of several possibilities, one of which is formin-mediated mKate2-β-actin-deficient actin nucleation (17). Filament bunching at pinned TCRs can arise from consistent biophysical properties without assuming heterogeneity between the biochemistry of these receptors and other actin-associated proteins such as those at the cell edge, where locally high probe ratios are absent.Although TCRs are intentionally trapped as part of this experimental strategy, it is likely APCs can naturally impede TCR ligand mobilities under certain circumstances, and this has been shown to impact T-cell signaling (18,19). Actin architecture near cell surface proteins has been extensively studied in focal adhesions of fibroblasts (20), but the lack of stress fibers in T cells makes it unlikely that the two structures are similar. Thus, receptor-induced cytoskeletal enrichment at TCR clusters adds to the catalog of actin behaviors in situ, which is conveniently probed by techniques such as ratiometric dual-probe imaging in live cells. These techniques can be coupled to various spatial analysis algorithms to further extend their utility.     

Binding of plasma membrane glycoproteins to the cytoskeleton during patching and capping is consistent with an entropy-enhancement model

Concentrations of concanavalin A that induced patching and capping of cell surface receptors on Dictyostelium discoideum also induce binding of the receptors to the cortical cytoskeleton, which was isolated by density-gradient centrifugation. The receptors were solubilized by deoxycholate, purified by affinity chromatography, and used to determine whether the receptors bound directly to the cytoskeletal protein, actin. As the concentration of actin was increased, many of the receptors became bound to purified filamentous rabbit muscle actin, even in the absence of concanavalin A. As in the ligation-induced binding of receptors to the cortical cytoskeleton in cells, concanavalin A induced much stronger binding of the purified receptors to filamentous actin. The results were consistent with a previously stated hypothesis that induction of receptor binding to the cytoskeleton during their patching and capping is driven by clustering the receptors, which reduces their translational entropy and by doing so enhances their avidity for the cytoskeleton.     

Rho GTPases: molecular switches that control the organization and dynamics of the actin cytoskeleton

The actin cytoskeleton plays a fundamental role in all eukaryotic cells it is a major determinant of cell morphology and polarity and the assembly and disassembly of filamentous actin structures provides a driving force for dynamic processes such as cell motility, phagocytosis, growth cone guidance and cytokinesis. The ability to reorganize actin filaments is a fundamental property of embryonic cells during development; the shape changes accompanying gastrulation and dorsal closure, for example, are dependent on the plasticity of the actin cytoskeleton, while the ability of cells or cell extensions, such as axons, to migrate within the developing embryo requires rapid and spatially organized changes to the actin cytoskeleton in response to the external environment. Work in mammalian cells over the last decade has demonstrated the central role played by the highly conserved Rho family of small GTPases in signal transduction pathways that link plasma membrane receptors to the organization of the actin cytoskeleton.     

The regulation and functional impact of actin assembly at cadherin cell–cell adhesions

Cadherin adhesion receptors are critical components for the maintenance of tissue architecture and organisation during development and in post-embryonic life. These receptors influence the actin cytoskeletal network by controlling its assembly at the junctions. Likewise, the actin cytoskeleton is required for cadherin integrity at cell–cell contacts. The junctional cytoskeleton is intrinsically dynamic and undergoes constant assembly and reorganisation to maintain a morphologically stable structure. This is governed by a host of molecular players that regulate actin assembly during nucleation and at post-nucleation stages. This review highlights the molecular machinery implicated in actin organisation at various stages of junctional assembly and its functional impact in simple epithelia and other model systems.     

Severing of stable microtubules by a mitotically activated protein in Xenopus egg extracts.

Eukaryotic cells disassemble and reorganize their cytoskeleton during the cell cycle and in response to environmental cues. Disassembly of the actin cytoskeleton is aided by proteins that sever filamentous actin, but microtubule-severing proteins thus far have not been identified. Here, we describe an activity in extracts from Xenopus eggs that rapidly severs stable microtubules along their length. Severing is elicited by a protein(s) whose activity is greatly stimulated during mitosis through a posttranslational mechanism. The microtubule-severing factor may be involved in disassembling the interphase microtubule network prior to constructing the mitotic spindle.     

Mediation of T-Cell Activation by Actin Meshworks

Although the actin cytoskeleton and T-cell receptor (TCR) signaling complexes are seemingly distinct molecular structures, they are tightly integrated in T cells. The signaling pathways initiated by TCRs binding to peptide MHC complexes are extensively influenced by the actin cytoskeletal activities of the motile phase before TCR signaling, the signalosome scaffolding function of the cytoskeleton, and the translocation of signaling clusters that precedes the termination of signaling at these complexes. As these three successive phases constitute essentially all the steps consequent to immune synapse formation, it has become clear that the substantial physical forces and signaling interactions generated by the actin cytoskeleton dominate the signaling life cycle of TCR signalosomes. We discuss the contributions of the actin cytoskeleton to TCR signaling phases and model some remaining questions about how specific cytoskeletal factors regulate TCR signaling outcomes.The activation of T cells is controlled primarily by T-cell receptors (TCRs) interacting with peptide-loaded major histocompatibility complexes (pMHCs) as T cells scan the surface of antigen presenting cells (APCs). Because T cells are continuously motile cells that transit through lymph nodes in their surveillance, it is clear that TCR triggering must occur within the context of physical forces that might rapidly separate TCRs from agonist pMHCs. Moreover, crawling T cells do not truly come to rest at the surfaces of APCs following TCR engagement. Instead, they continuously extend protrusions over APCs and move along the surface of their partner (Gunzer et al. 2000). In their initial encounters with antigen-bearing dendritic cells (DCs), T cells also often rapidly couple and uncouple on the order of minutes, rather than dwelling for extended periods of time on single DCs (Gunzer et al. 2000; Mempel et al. 2004). This dynamic coupling allows T cells to quickly sample a large proportion of the total APC membrane pool in search of their cognate antigen. Still, these transient contacts are productive—they induce calcium fluxes and the expression of markers of activated T cells—indicating that TCR signalosome outputs can be initiated in mere minutes and survive the dissolution of contacts, even under the mechanical stress of cytoskeletal remodeling.TCR signaling requires the dynamic recruitment of a macromolecular complex of kinases, scaffolding molecules, and other signaling effectors to a triggered TCR. Assembly of this macromolecular signaling complex must be very sensitive and occur rapidly, or there is a risk that the TCR will release the pMHC ligand, and the T cell will fail to register the antigen hit. Conversely, the signalosome assembly mechanism needs to discriminate against TCRs interacting transiently with a vast array of pMHCs presenting nonagonist peptides. Viewed in this manner, a scheme that rapidly dissociates TCRs from MHCs loaded with endogenous peptide, freeing them to rebind and test other MHCs, is desirable. It is notable that several TCR signaling factors carry binding sites for actin binding proteins or actin itself (Rozdzial et al. 1995; Zhang et al. 1999; Zeng et al. 2003; Phee et al. 2005; Gomez et al. 2006). Through these actin-associated factors, agonist-triggered TCRs rapidly assemble stabilized signaling platforms that survive mechanical disruption.In concert with adhesive integrin interactions and costimulatory receptor signaling, TCRs orchestrate a reorganization of the T-cell plasma membrane that may begin with a handful of receptors and eventually encompasses the entire contact face with the APC (some 50–100 µm²). TCRs first aggregate into micron scale clusters of TCRs, then flow to the center of the contact face, generating the central supramolecular activating complex (cSMAC) of the immune synapse (Monks et al. 1998; Grakoui et al. 1999; Krummel et al. 2000). Underscoring the importance of the cytoskeleton, the actin depolymerizing toxins latrunculin A and cytochalasin D are potent inhibitors of T-cell activation and block both TCR microcluster formation and cSMAC coalescence (Wulfing et al. 1998; Grakoui et al. 1999; Krummel et al. 2000; Varma et al. 2006). Ultimately, it is the coordination of the local interactions between receptors and effectors with the cell morphological level rearrangements that determines the nature and magnitude of T-cell responses to pathogens. Regulation of TCR signaling lifecycles and T-cell responses, therefore, falls squarely on the actin cytoskeleton.     

Polarized growth in fungi--interplay between the cytoskeleton, positional markers and membrane domains

One kind of the most extremely polarized cells in nature are the indefinitely growing hyphae of filamentous fungi. A continuous flow of secretion vesicles from the hyphal cell body to the growing hyphal tip is essential for cell wall and membrane extension. Because microtubules (MT) and actin, together with their corresponding motor proteins, are involved in the process, the arrangement of the cytoskeleton is a crucial step to establish and maintain polarity. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, actin-mediated vesicle transportation is sufficient for polar cell extension, but in S. pombe, MTs are in addition required for the establishment of polarity. The MT cytoskeleton delivers the so-called cell-end marker proteins to the cell pole, which in turn polarize the actin cytoskeleton. Latest results suggest that this scenario may principally be conserved from S. pombe to filamentous fungi. In addition, in filamentous fungi, MTs could provide the tracks for long-distance vesicle movement. In this review, we will compare the interaction of the MT and the actin cytoskeleton and their relation to the cortex between yeasts and filamentous fungi. In addition, we will discuss the role of sterol-rich membrane domains in combination with cell-end marker proteins for polarity establishment.     

UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph Polarize F-Actin during Embryonic Morphogenesis by Regulating the WAVE/SCAR Actin Nucleation Complex

Many cells in a developing embryo, including neurons and their axons and growth cones, must integrate multiple guidance cues to undergo directed growth and migration. The UNC-6/netrin, SLT-1/slit, and VAB-2/Ephrin guidance cues, and their receptors, UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph, are known to be major regulators of cellular growth and migration. One important area of research is identifying the molecules that interpret this guidance information downstream of the guidance receptors to reorganize the actin cytoskeleton. However, how guidance cues regulate the actin cytoskeleton is not well understood. We report here that UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph differentially regulate the abundance and subcellular localization of the WAVE/SCAR actin nucleation complex and its activator, Rac1/CED-10, in the Caenorhabditis elegans embryonic epidermis. Loss of any of these three pathways results in embryos that fail embryonic morphogenesis. Similar defects in epidermal enclosure have been observed when CED-10/Rac1 or the WAVE/SCAR actin nucleation complex are missing during embryonic development in C. elegans. Genetic and molecular experiments demonstrate that in fact, these three axonal guidance proteins differentially regulate the levels and membrane enrichment of the WAVE/SCAR complex and its activator, Rac1/CED-10, in the epidermis. Live imaging of filamentous actin (F-actin) in embryos developing in the absence of individual guidance receptors shows that high levels of F-actin are not essential for polarized cell migrations, but that properly polarized distribution of F-actin is essential. These results suggest that proper membrane recruitment and activation of CED-10/Rac1 and of WAVE/SCAR by signals at the plasma membrane result in polarized F-actin that permits directed movements and suggest how multiple guidance cues can result in distinct changes in actin nucleation during morphogenesis.     

The epidermal growth factor receptor is associated with actin filaments.

In this paper we describe our investigations on the association of receptors for the epidermal growth factor (EGF) with the cytoskeleton of A431 cells. In order to determine which filamentous system the EGF receptors are associated to, the cytoskeletal fraction to which these receptors bind was isolated. Second, the possible colocalization of EGF receptors with different cytoskeletal elements was examined in A431 cells. By selective extractions of the A431 cytoskeletons, it is shown that more than 90% of the cytoskeleton-associated EGF receptors are removed from the cytoskeletons together with the actin filamentous system. During several cycles of poly- and depolymerization of actin isolated from A431 cells, the EGF receptor precipitates together with the actin containing filaments, indicating that EGF receptors are able to bind in vitro to actin filaments. With immunofluorescence studies we show that EGF receptors especially colocalize with actin filaments. These results demonstrate that the EGF receptor is associated specifically with actin filaments in A431 cells.     

The Conformational State of Actin Filaments Regulates Branching by Actin-related Protein 2/3 (Arp2/3) Complex

Actin is a highly ubiquitous protein in eukaryotic cells that plays a crucial role in cell mechanics and motility. Cell motility is driven by assembling actin as polymerizing actin drives cell protrusions in a process closely involving a host of other actin-binding proteins, notably the actin-related protein 2/3 (Arp2/3) complex, which nucleates actin and forms branched filamentous structures. The Arp2/3 complex preferentially binds specific actin networks at the cell leading edge and forms branched filamentous structures, which drive cell protrusions, but the exact regulatory mechanism behind this process is not well understood. Here we show using in vitro imaging and binding assays that a fragment of the actin-binding protein caldesmon added to polymerizing actin increases the Arp2/3-mediated branching activity, whereas it has no effect on branch formation when binding to aged actin filaments. Because this caldesmon effect is shown to be independent of nucleotide hydrolysis and phosphate release from actin, our results suggest a mechanism by which caldesmon maintains newly polymerized actin in a distinct state that has a higher affinity for the Arp2/3 complex. Our data show that this new state does not affect the level of cooperativity of binding by Arp2/3 complex or its distribution on actin. This presents a novel regulatory mechanism by which caldesmon, and potentially other actin-binding proteins, regulates the interactions of actin with its binding partners.     

Patterning of the membrane cytoskeleton by the extracellular matrix

The extracellular matrices of different tissues contain components which affect the migration, morphology and differentiation of many types of cells. These forms of cell behavior often involve dramatic changes in cytoskeletal organization. Extracellular matrix components are recognized by specific cell surface receptors which span the membrane and interact with the actin cytoskeleton. In cultured cells, the matrix receptors are concentrated in sites of cell attachment called focal adhesions. Information that is conveyed from the extracellular matrix to the cytoskeleton may involve matrix components, cell surface receptors, as well as the proteins at the cytoplasmic face of the focal adhesion which link the receptors to the actin cytoskeleton.     

Thyroxine-dependent modulation of actin polymerization in cultured astrocytes. A novel, extranuclear action of thyroid hormone

Actin depolymerization specifically blocks the rapid thyroid hormone-dependent inactivation of type II iodothyronine 5'-deiodinase. Thyroid hormone appears to regulate enzyme inactivation by modulating actin-mediated internalization of this plasma membrane-bound protein. In this study, we examined the interrelationships between thyroxine-dependent enzyme inactivation and the organization of the actin cytoskeleton in cultured astrocytes. Steady-state enzyme levels were inversely related to actin content in dibutyryl cAMP-stimulated astrocytes, and increases in filamentous actin resulted in progressively shorter enzyme half-lives without affecting enzyme synthesis. In the absence of thyroxine, filamentous actin decreased by approximately 40% and soluble actin correspondingly increased; thyroxine normalized filamentous actin levels without changing total cell actin. Thyroxine treatment for only 10 min resulted in an approximately 50% loss of enzyme and increased filamentous actin 2-fold. Neither cycloheximide nor actinomycin D affected the thyroxine-induced actin polymerization. Astrocytes grown without thyroxine also showed a disorganized actin cytoskeleton, and 10 nM thyroxine or 10 nM reverse triiodothyronine normalized the actin cytoskeleton appearance within 20 min; 10 nM 3,3',5-triiodothyronine had no effect. These data show that thyroxine modulates the organization of the actin cytoskeleton in astrocytes and suggest that regulation of actin polymerization may contribute to thyroid hormone's influence on arborization, axonal transport, and cell-cell contact in the developing brain.     

The central role of septa in the basidiomycete Schizophyllum commune hyphal morphogenesis

The purpose of the present research was to observe in the filamentous basidiomycete Schizophyllum commune, the connection between the nuclear division and polymerization of the contractile actin ring with subsequent formation of septa in living hyphae. The filamentous actin was visualized using Lifeact-mCherry and the nuclei with EGFP tagged histone 2B (H2B). Time-lapse fluorescence microscopy confirmed that in monokaryotic and dikaryotic hyphae, the first signs of the contractile actin ring occur at the site of the nuclear division, in one to two minutes after division. At this stage, the telophase nuclei have moved tens of micrometers from the division site. The actin ring is replaced by the septum in six minutes. The apical cells treated with filamentous actin disrupting drug latrunculin A, had swollen tips but the cells were longer than in control samples due to the absence of the actin rings. The nuclear pairing and association with clamp cell development as well as the clamp cell fusion with the subapical cell was disrupted in latrunculin-treated dikaryotic hyphae, indicating that actin filaments are involved in these processes, also regulated by the A and B mating-type genes. This suggests that the actin cytoskeleton may indirectly be a target for mating-type genes.     

Changes in the organization of the actin cytoskeleton during preimplantation development of the pig embryo

The organization of the actin cytoskeleton was studied in unfertilized porcine oocytes and preimplantation stage embryos from Day 1 through Day 8 of development. Fixed and detergent-extracted oocytes and embryos were analyzed by fluorescence microscopy after staining with either rhodamine-phalloidin to localize filamentous actin or with affinity-purified anti-actin antibodies to localize the total immunodetectable actin. Whereas unfertilized oocytes contain immunoreactive cytoplasmic actin, rhodamine-phalloidin binding is not detected until fertilization when a prominent cortical staining pattern becomes apparent. In early cleavage stage embryos, filamentous actin is concentrated in the cell cortex of blastomeres especially at sites of cell-cell contact. Compacting morulae exhibit a marked accumulation of actin at the margins of blastomeres where numerous interdigitating cell processes are located. The predominantly pericellular distribution of actin becomes a distinguishing feature of trophectodermal cells in the expanding blastocyst at Day 6 of development; these cells form a prominent actin-limited zone circumscribing the inner cell mass. In Day 8 blastocysts, three cell types are present that are readily distinguishable based upon their actin displays among other cytological features. Trophectodermal cells exhibit continuous actin-rich lateral borders and stress fibers along their basal surface. Inner cell mass cells contain a discontinuous actin boundary and prominent foci of actin along their blastocoelic surface. Lining the blastocoel are patches of endodermal cells in which the actin is exclusively cortical. The data are discussed with respect to differences between species and the chronology of actin rearrangements during preimplantation development of the porcine embryo.     

The effect of methylglyoxal on actin

Studying the gelation of Ehrlich ascites tumor cell extracts at various methyglyoxal concentrations, an increase of the gelled protein fraction, composed mainly of actin, was found at 10^?7 M to 10^?5 M. When methylglyoxal was added to intact tumor cells, the filamentous portion of cytoplasmic actin was increased at 10^?7 M to 10^?6 M concentrations. Furthermore, certain functional properties of purified skeletal muscle actin were also affected by μM concentrations of methylglyoxal; the speed of actin polymerization was facilitated and more filamentous actin formed in polymerizing conditions. The possible mode of methylglyoxal action is discussed.     

Ezrin, Radixin and Moesin: key regulators of membrane-cortex interactions and signaling

The cell cortex serves as a critical nexus between the extracellular environment/cell membrane and the underlying cytoskeleton and cytoplasm. In many cells, the cell cortex is organized and maintained by the Ezrin, Radixin and Moesin (ERM) proteins, which have the ability to interact with both the plasma membrane and filamentous actin. Although this membrane-cytoskeletal linkage function is critical to stability of the cell cortex, recent studies indicate that this is only a part of what ERMs do in many cells. In addition to their role in binding filamentous actin, ERMs regulate signaling pathways through their ability to bind transmembrane receptors and link them to downstream signaling components. In this review we discuss recent evidence in a variety of cells indicating that ERMs serve as scaffolds to facilitate efficient signal transduction on the cytoplasmic face of the plasma membrane.     

N-cadherin function is required for differentiation-dependent cytoskeletal reorganization in lens cells in vitro

Members of the cadherin family of cell adhesion molecules participate in calcium-dependent cell-cell adhesions that are necessary for the cell sorting events that regulate early developmental processes. Although individual cadherin molecules have been shown to participate in tissue histogenesis, the regulation of function of these receptors in cell differentiation has been more difficult to identify. We have determined that N-cadherin linkage to the cytoskeleton is correlated with lens cell differentiation in vivo. Through the use of a chick embryo lens culture system that mimics differentiation in vivo, we have determined that N-cadherin linkage to the cytoskeleton is altered and lens differentiation is blocked by function-blocking antibodies to N-cadherin. In the presence of the N-cadherin function-blocking antibody, NCD-2, both N-cadherin and filamentous actin are prevented from organizing at the cortical membranes. This correlates with an inhibition of lens morphogenesis and differentiation. These results are paralleled by changes in the expression of the molecular components of the cadherin-catenin complex and their linkage to the actin cytoskeleton. In the presence of NCD-2, expression of N-cadherin, alpha-catenin, and beta-catenin is inhibited and their association with the cytoskeleton blocked. Overall cadherin expression, however, remains unchanged as demonstrated by studies with a pan-cadherin antibody. This is accompanied by an increase in expression of the cadherin cytoskeletal protein plakoglobin. Although the cells have tried to compensate for the loss of N-cadherin by up-regulation of another cadherin(s) and plakoglobin, this is unable to compensate for N-cadherin function. The data strongly suggest that N-cadherin and its associated cytoskeleton play an important role in the differentiation process that leads to the formation of the crystalline lens.     

Dynamin at the actin-membrane interface

Many important cellular processes such as phagocytosis, cell motility and endocytosis require the participation of a dynamic and interactive actin cytoskeleton that acts to deform cellular membranes. The extensive family of non-traditional myosins has been implicated in linking the cortical actin gel with the plasma membrane. Recently, however, the dynamins have also been included in these cell processes as a second family of mechanochemical enzymes that self-associate and hydrolyze nucleotides to perform 'work' while linking cellular membranes to the actin cytoskeleton. The dynamins are believed to form large helical polymers from which extend many interactive proline-rich tail domains, and these domains bind to a variety of SH3-domain-containing proteins, many of which appear to be actin-binding proteins. Recent data support the concept that the dynamin family might act as a 'polymeric contractile scaffold' at the interface between biological membranes and filamentous actin.     

GFP-FABD2 fusion construct allows in vivo visualization of the dynamic actin cytoskeleton in all cells of Arabidopsis seedlings

In vivo visualization of filamentous actin in all cells of Arabidopsis thaliana seedlings is essential for understanding the numerous roles of the actin cytoskeleton in diverse processes of cell differentiation. A previously introduced reporter construct based on the actin-binding domain of mouse talin proved to be useful for unravelling some of these aspects in cell layers close to the organ surface. However, cells more deeply embedded, especially stelar cells active in polar transport of auxin, show either diffuse or no fluorescence at all due to the lack of expression of the fusion protein. The same problem is encountered in the root meristem. Recently introduced actin reporters based on fusions between A. thaliana fimbrin 1 and GFP gave brilliant results in organs from the root differentiation zone upwards to the leaves, however failed to depict the filamentous actin cytoskeleton in the transition zone of the root, in the apical meristem and the root cap. To overcome these problems, we have prepared new transgenic lines for the visualization of F-actin in vivo. We report here that a construct consisting of GFP fused to the C-terminal half of A. thaliana fimbrin 1 reveals dynamic arrays of F-actin in all cells of stably transformed A. thaliana seedlings.     

Stable, soluble T-cell receptor molecules for crystallization and therapeutics

Antibody and T-cell receptors (TCRs) are the primary recognition molecules of the adaptive immune system. Antibodies have been extensively characterized and are being developed for a large number of therapeutic applications. This has been possible because of the ability to manufacture stable, soluble, monoclonal antibodies which retain the antigen specificity of B cells. Unlike antibodies, TCRs are not expressed in a soluble form, but are anchored to the T-cell surface by an insoluble trans-membrane domain. Characterization and development of TCRs has been hampered by the lack of suitable methods for producing them as soluble and stable proteins. Here we report the engineering of soluble human TCRs suitable for crystallization studies and potentially for in vivo therapeutic use.