Catenins: playing both sides of the synapse

Synapses of the central nervous system (CNS) are specialized cell-cell junctions that mediate intercellular signal transmission from one neuron to another. The directional nature of signal relay requires synaptic contacts to be morphologically asymmetric with distinct protein components, while changes in synaptic communication during neural network formation require synapses to be plastic. Synapse morphology and plasticity require a dynamic actin cytoskeleton. Classical cadherins, which are junctional proteins associated with the actin cytoskeleton, localize to synapses and regulate synaptic adhesion, stability and remodeling. The major intracellular components of cadherin junctions are the catenin proteins, and increasing evidence suggests that cadherin-catenin complexes modulate an array of synaptic processes. Here we review the role of catenins in regulating the development of pre- and postsynaptic compartments and function in synaptic plasticity, with particular focus on their role in regulating the actin cytoskeleton.     

Regulation of cytoskeletal structure in human mammary carcinoma cells MCF-7 by culture substrata

Three-dimensional cellular structures formed by MCF-7 human mammary carcinoma cells within collagen gels were isolated with collagenase and cultivated on plastic substratum to examine whether the cytoskeleton specific for cells forming cellular structures (S-type) changes to that specific for cells grown as monolayers (M-type). The cytoskeleton isolated as 0.05% Triton-insoluble fraction from the cellular structures after culture for 1 day on plastic was exclusively S-type. However, both types of cytoskeletons were observed in the cellular structures cultivated for 7 days on plastic as well as in the cells grown as monolayers for 2 days after dissociation of the cellular structures with trypsin. By use of an antibody raised against a 65-kD polypeptide that was specific for the M-type cytoskeleton, the presence of the polypeptide was found to be restricted to the cells grown out as monolayers from the edge of the cellular structures. In the cells grown for 2 days as monolayers, a mixture of cells both having and lacking the polypeptide was observed. After a 7-day culture of the dissociated cells as monolayers on plastic, however, most of the cells had M-type cytoskeletons. The present results show that the apparent change in the cytoskeleton of MCF-7 cells from S-type to M-type does not occur in cells involved in the three-dimensional cellular structures even in the absence of collagen gels, but that it occurs in cells which are grown as monolayers for at least 7 days on plastic substratum.     

Mechanical aspects of cell shape regulation and signaling

Physical forces play a critical role in cell integrity and development, but little is known how cells convert mechanical signals into biochemical responses. This mini-review examines potential molecular mediators like integrins, focal adhesion proteins, and the cytoskeleton in the context of a complex cell structure. These molecules-when activated by cell binding to the extracellular matrix-associate with the skeletal scaffold via the focal adhesion complex. Vinculin is presented as a mechanical coupling protein that contributes to the integrity of the cytoskeleton and cell shape control, and examples are given of how mechanical signals converge into biochemical responses through force-dependent changes in cell geometry and molecular mechanics.     

The effect of sub-lethal ALA-PDT on the cytoskeleton and adhesion of cultured human cancer cells

5-Aminolevulinic acid (ALA), a precursor of the endogenous photosensitizer protoporphyrin IX, is used in the photodynamic therapy (PDT) of cancer. Sub-lethal ALA-PDT (1-min irradiation with 370-450 nm blue light, 0.6 mW/cm(2) after 2-h incubation with 1 mM ALA) has been earlier shown to change cell morphology and to inhibit both trypsin-induced detachment of cultured cancer cells from the plastic substrata and cell attachment to the bottom of the plastic well plates. In the present study, we found that such treatment of human adenocarcinoma WiDr cells grown in dense colonies stimulated the formation of actin cortex between cells in the colonies and increased the number of actin stress fibres in some, but not in all, cells. However, ALA-PDT did not change the microtubular cytoskeleton in these cells. A similar treatment of glioblastoma D54Mg cells, which grow separately and communicate by protrusions, caused loss of fibrillar actin structures in growth cones, retraction of protrusions, and surface blebbing in some cells. The application of the cytoskeleton inhibitors cytochalasin D, colchicine or taxol showed that the inhibition of trypsin-induced detachment of photosensitized WiDr cells was related to ALA-PDT-induced changes in actin and microtubular cytoskeleton. Some signal transduction processes are suggested to be involved in ALA-PDT-induced changes in cytoskeleton, cell shape, and adhesion.     

Plasticity of cadherin-catenin expression in the melanocyte lineage

Cadherins are calcium-dependent cell adhesion receptors with strong morphoregulatory functions. To mediate functional adhesion, cadherins must interact with actin cytoskeleton. Catenins are cytoplasmic proteins that mediate the interactions between cadherins and the cytoskeleton. In addition to their role in cell-cell adhesion, catenins also participate in signaling pathways that regulate cell growth and differentiation. Cadherins and catenins appear to be involved in melanocyte development and transformation. Here, we investigated the function of cadherin-catenin complexes in the normal development and transformation of melanocytes by studying the patterns of expression of the cell-cell adhesion molecules, E-, N- and P-cadherin, and the expression of their cytoplasmic partners, alpha-, beta- and gamma-catenin during murine development. Similar analyses were performed in vitro using murine melanoblast, melanocyte, and melanoma cell lines in the presence and absence of keratinocytes, the cells with which melanocytes interact in vivo. Overall, the results suggest that the expression of cadherins and catenins is very plastic and depends on their environment as well as the transformation status of the cells. This plasticity is important in fundamental cellular mechanisms associated with normal and pathological ontogenesis, as well as with tumorigenesis.     

Driving actin dynamics under the influence of alcohol

The actin cytoskeleton plays a pivotal role in regulating neuronal development and activity, and dysregulation of actin dynamics has been linked to impaired cognitive function. In this issue of Cell Rothenfluh et al. (2006) , and Offenh?user et al. (2006) show that actin dynamics can also affect the cellular and behavioral responses of flies and mice to alcohol.     

Regulation of cytoskeleton‐associated protein activities: Linking cellular signals to plant cytoskeletal function

The plant cytoskeleton undergoes dynamic remodeling in response to diverse developmental and environmental cues. Remodeling of the cytoskeleton coordinates growth in plant cells, including trafficking and exocytosis of membrane and wall components during cell expansion, and regulation of hypocotyl elongation in response to light. Cytoskeletal remodeling also has key functions in disease resistance and abiotic stress responses. Many stimuli result in altered activity of cytoskeleton-associatedproteins,microtubuleassociated proteins(MAPs) and actin-binding proteins(ABPs). MAPs and ABPs are the main players determining the spatiotemporally dynamic nature of the cytoskeleton, functioning in a sensory hub that decodes signals to modulate plant cytoskeletal behavior. Moreover, MAP and ABP activities and levels are precisely regulated during development and environmental responses, but our understanding of this process remains limited. In this review, we summarize the evidence linking multiple signaling pathways, MAP and ABP activities and levels, and cytoskeletal rearrangements in plant cells. We highlight advances in elucidating the multiple mechanisms that regulate MAP and ABP activities and levels, including calcium and calmodulin signaling, ROP GTPase activity, phospholipid signaling, and post-translational modifications.     

Cytoskeletal responses during early development of the downy mildew of grapevine (Plasmopara viticola)

A host-free system was established to induce the early development of the obligate biotrophic pathogen Plasmopara viticola, the downy mildew of grapevine. This system was used to study cytoskeletal responses during encystation and germ tube formation. During these processes, both the actin and the tubulin cytoskeleton show a stage-specific pattern of distribution. Elimination of the cytoskeleton by the actin drug latrunculin B and the microtubule drug ethyl-N-phenyl-carbamate did not affect the release of mobile zoospores from the sporangia, nor the encystation process, but efficiently inhibited the formation of a germ tube. The data are discussed with respect to a role of both actin and microtubules for the establishment of the cell polarity guiding the emergence and the growth of the germ tube.     

Plasticity of Cadherin–Catenin Expression in the Melanocyte Lineage

Cadherins are calcium‐dependent cell adhesion receptors with strong morphoregulatory functions. To mediate functional adhesion, cadherins must interact with actin cytoskeleton. Catenins are cytoplasmic proteins that mediate the interactions between cadherins and the cytoskeleton. In addition to their role in cell–cell adhesion, catenins also participate in signaling pathways that regulate cell growth and differentiation. Cadherins and catenins appear to be involved in melanocyte development and transformation. Here, we investigated the function of cadherin–catenin complexes in the normal development and transformation of melanocytes by studying the patterns of expression of the cell–cell adhesion molecules, E‐, N‐ and P‐cadherin, and the expression of their cytoplasmic partners, α‐, β‐ and Γ‐catenin, during murine development. Similar analyses were performed in vitro using murine melanoblast, melanocyte, and melanoma cell lines in the presence and absence of keratinocytes, the cells with which melanocytes interact in vivo. Overall, the results suggest that the expression of cadherins and catenins is very plastic and depends on their environment as well as the transformation status of the cells. This plasticity is important in fundamental cellular mechanisms associated with normal and pathological ontogenesis, as well as with tumorigenesis.     

Cytoskeleton in human mammary carcinoma cells forming three-dimensional cellular structures within collagen gels

Human mammary carcinoma cell line MCF-7 cells grown on type I collagen gels floating in a medium occasionally invaginated into the gels as a cell mass and formed cylindrical or domed structures within it. The 0.05% Triton-insoluble cytoskeleton of such cellular structures sedimented as a white flocculent layer at the boundary between 60 and 70% sucrose layers by ultracentrifugation, and consisted of 4 basal components: 54-kD (beta-tubulin), 45-kD, 42-kD (actin), and 39-kD polypeptides. By contrast, the isolated cytoskeleton of MCF-7 cells grown as monolayers on plastic substratum formed a finer cytoskeletal network with a smaller buoyant density and consisted of two distinct polypeptides with apparent molecular sizes of 80-kD and 65-kD in addition to the 4 basal components found in the morphologically developing cells. The present results indicate that the cytoskeleton of MCF-7 cells forming the three-dimensional cellular structures within collagen gels is lacking in these two polypeptides, and that it has a coarser cytoskeletal network with a greater buoyant density than that of the monolayered cells on plastic.     

Control of T lymphocyte morphology by the GTPase Rho

Background  

Rho family GTPase regulation of the actin cytoskeleton governs a variety of cell responses. In this report, we have analyzed the role of the GTPase Rho in maintenance of the T lymphocyte actin cytoskeleton.     

Barley MLO modulates actin-dependent and actin-independent antifungal defense pathways at the cell periphery

Cell polarization is a crucial process during plant development, as well as in plant-microbe interactions, and is frequently associated with extensive cytoskeletal rearrangements. In interactions of plants with inappropriate fungal pathogens (so-called non-host interactions), the actin cytoskeleton is thought to contribute to the establishment of effective barriers at the cell periphery against fungal ingress. Here, we impeded actin cytoskeleton function in various types of disease resistance using pharmacological inhibitors and genetic interference via ectopic expression of an actin-depolymerizing factor-encoding gene, ADF. We demonstrate that barley (Hordeum vulgare) epidermal cells require actin cytoskeleton function for basal defense to the appropriate powdery mildew pathogen Blumeria graminis f. sp. hordei and for mlo-mediated resistance at the cell wall, but not for several tested race-specific immune responses. Analysis of non-host resistance to two tested inappropriate powdery mildews, Erysiphe pisi and B. graminis f. sp. tritici, revealed the existence of actin-dependent and actin-independent resistance pathways acting at the cell periphery. These pathways act synergistically and appear to be under negative control by the plasma membrane-resident MLO protein.     

The adaptor protein Nck-1 couples the netrin-1 receptor DCC (deleted in colorectal cancer) to the activation of the small GTPase Rac1 through an atypical mechanism

Netrins are a family of secreted proteins that guide the migration of cells and axonal growth cones during development. DCC (deleted in colorectal cancer) is a receptor for netrin-1 implicated in mediating these responses. Here, we show that DCC interacts constitutively with the SH3/SH2 adaptor Nck in commissural neurons. This interaction is direct and requires the SH3 but not SH2 domains of Nck-1. Moreover, both DCC and Nck-1 associate with the actin cytoskeleton, and this association is mediated by DCC. A dominant negative Nck-1 inhibits the ability of DCC to induce neurite outgrowth in N1E-115 cells and to activate Rac1 in fibroblasts in response to netrin-1. These studies provide evidence for an important role of mammalian Nck-1 in a novel signaling pathway from an extracellular guidance cue to changes in the actin-based cytoskeleton responsible for axonal guidance.     

Interaction of the low-molecular-weight GTP-binding protein rap2 with the platelet cytoskeleton is mediated by direct binding to the actin filaments

The interaction of the low-molecular-weight GTP-binding protein rap2 with the cytoskeleton from thrombin-aggregated platelets was investigated by inducing depolymerization of the actin filaments, followed by in vitro-promoted repolymerization. We found that the association of rap2 with the cytoskeleton was spontaneously restored after one cycle of actin depolymerization and repolymerization. Exogenous rap2, but not unrelated proteins, added to depolymerized actin and solubilized actin-binding proteins, was also specifically incorporated into the in vitro reconstituted cytoskeleton. The incorporation of exogenous rap2 was also observed when the cytoskeleton from resting or thrombin-activated platelets was subjected to actin depolymerization-repolymerization. Moreover, such interaction occurred equally well when exogenous rap2 was loaded with either GDP or GTPgammaS. We also found that polyhistidine-tagged rap2 immobilized on Ni(2+)-Sepharose and loaded with either GDP or GTPgammaS, could specifically bind to cytoskeletal actin. Moreover, when purified monomeric actin was induced to polymerize in vitro in the presence of rap2, the small G-protein specifically associated with the actin filaments. Finally, rap2 loaded with either GDP or GTPgammaS was able to bind to purified F-actin immobilized on a plastic surface. These results demonstrate that rap2 interacts with the platelet cytoskeleton by direct binding to the actin filaments and that this interaction is not regulated by the activation state of the protein.     

Optimal antigen localization in human tissues using aldehyde-fixed plastic-embedded sections

Although the utility of antigen labeling techniques in frozen tissues is well known, it is generally acknowledged that an improvement in morphologic preservation is desirable. Conventionally processed paraffin-embedded tissues are limited in the range of antigens that can be detected and newer plastic embedding techniques have been even more restricted. By using cold (4 degrees C) processing and limited fixation a wide range of antigens (including T and B markers) has been demonstrated in 2 mu plastic sections. The morphologic preservation and antigen localization are superior to other techniques. The combination of precise antigen localization and excellent morphologic preservation should expand the diagnostic and investigative uses of immunohistology.     

The POLARIS peptide of Arabidopsis regulates auxin transport and root growth via effects on ethylene signaling

The rate and plane of cell division and anisotropic cell growth are critical for plant development and are regulated by diverse mechanisms involving several hormone signaling pathways. Little is known about peptide signaling in plant growth; however, Arabidopsis thaliana POLARIS (PLS), encoding a 36-amino acid peptide, is required for correct root growth and vascular development. Mutational analysis implicates a role for the peptide in hormone responses, but the basis of PLS action is obscure. Using the Arabidopsis root as a model to study PLS action in plant development, we discovered a link between PLS, ethylene signaling, auxin homeostasis, and microtubule cytoskeleton dynamics. Mutation of PLS results in an enhanced ethylene-response phenotype, defective auxin transport and homeostasis, and altered microtubule sensitivity to inhibitors. These defects, along with the short-root phenotype, are suppressed by genetic and pharmacological inhibition of ethylene action. PLS expression is repressed by ethylene and induced by auxin. Our results suggest a mechanism whereby PLS negatively regulates ethylene responses to modulate cell division and expansion via downstream effects on microtubule cytoskeleton dynamics and auxin signaling, thereby influencing root growth and lateral root development. This mechanism involves a regulatory loop of auxin-ethylene interactions.     

Colloidal gold immunostaining on deplasticized ultra-thin sections

We localized tissue antigens on ultra-thin sections by deplasticizing the sections while on the grid, incubating in primary antiserum followed by immunoglobulin-conjugated colloidal gold, and ultimately re-embedding in dilute Epon. This procedure permitted ultrastructural localization of tissue antigens that were previously masked by the embedding plastic surrounding tissue components. In addition, replacement of the plastic matrix on the thin section after immunostaining prevented development of the drying artifacts that occur in unsupported tissue sections. Optimal preservation of components in the tissue sections was achieved despite extensive steps involved in plastic removal and immunostaining. This method may be useful in situations where the number of exposed epitopes on the surface of a thin section is low. The procedure also allows the use of antisera at greater dilutions and provides enhanced immunostaining specificity with low background.     

The success of assisted colonization and assisted gene flow depends on phenology

Global warming will jeopardize the persistence and genetic diversity of many species. Assisted colonization, or the movement of species beyond their current range boundary, is a conservation strategy proposed for species with limited dispersal abilities or adaptive potential. However, species that rely on photoperiodic and thermal cues for development may experience conflicting signals if transported across latitudes. Relocating multiple, distinct populations may remedy this quandary by expanding genetic variation and promoting evolutionary responses in the receiving habitat – a strategy known as assisted gene flow. To better inform these policies, we planted seeds from latitudinally distinct populations of the annual legume, Chamaecrista fasciculata, in a potential future colonization site north of its current range boundary. Plants were exposed to ambient or elevated temperatures via infrared heating. We monitored several life history traits and estimated patterns of natural selection to determine the adaptive value of plastic responses. To assess the feasibility of assisted gene flow between phenologically distinct populations, we counted flowers each day and estimated the degree of temporal isolation between populations. Increased temperatures advanced each successive phenological trait more than the last, resulting in a compressed life cycle for all but the southern‐most population. Warming altered patterns of selection on flowering onset and vegetative biomass. Population performance was dependent on latitude of origin, with the northern‐most population performing best under ambient conditions and the southern‐most performing most poorly, even under elevated temperatures. Among‐population differences in flowering phenology limited the potential for genetic exchange among the northern‐ and southern‐most populations. All plastic responses to warming were neutral or adaptive; however, photoperiodic constraints will likely necessitate evolutionary responses for long‐term persistence, especially when involving populations from disparate latitudes. With strategic planning, our results suggest that assisted colonization and assisted gene flow may be feasible options for preservation.