Benjamin Geiger

The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells

Roland Mall, Werner W. Franke and Dorothea L. Schiller Division of Membrane Biology and Biochemistry institute of Cell and Tumor Biology German Cancer Research Center D-6900 Heidelberg, Federal Republic of Germany Benjamin Geiger Department of Chemical Immunology The Weizmann Institute of Science Rehovot, Israel Reinhard Krepler Department of Pathology University of Vienna School of Medicine A-l 090 Vienna, Austria Introduction A large proportion of the cytoplasm of vertebrate cells, normal or transformed, is represented by components of the cytoskeleton, including actin-containing micro- filaments, tubulin-containing microtubules and fila- ments of intermediate size, with diameters of 7-l 1 nm. Although such structures have a widespread oc- currence in diverse cell types, examples have been reported in which they are formed in different cell types from different proteins of a multigene family of proteins, or from different subunit polypeptides of a class of related proteins. For example, differentiation specificity of expression of different actins has been described in different cell types of mammals (Vande- kerckhove and Weber, 1979). By far the most striking differentiation specificity of composition has been ob- served for the intermediate-sized filaments. Although all filaments of this category are morphologically iden- tical in different cell types, are insoluble in solutions of a broad range of low or high salt concentrations and non-ionic detergents and seem to share some common assembly properties (Steinert et al., 1981 b) and antigenic determinants (Pruss et al., 1981) im- munological and biochemical criteria allow us to dis- tinguish at least five different types of intermediate filaments (Bennett et al., 1978; Franke et al., 1978a, 1981f; Hynes and Destree, 1978; Lazarides, 1980; Anderton, 1981 ; Holtzer et al., 1981; Osborn et al., 1981). First, filaments containing keratin-like proteins (“cytokeratins”) are characteristic of epithelial cells. Second, vimentin filaments occur in mesenchymally derived cells, in astrocytes, in Sertoli cells, in vascular smooth muscle cells and in many cultured cell lines. Third, desmin filaments are typical of most types of myogenic cells. Fourth, neurofilaments are typical of neuronal cells. Fifth, glial filaments are typical of as- trocytes. During cell transformation and tumor devel- opment this cell type specificity of intermediate fila- ments is largely conserved’ (Franke et al., 1978a, 1978b, 1979a; Hynes and Destree, 1978; Sun and Green, 1978a; Sun et al., 1979; Bannasch et al., 1980; Battifora et al., 1980; Schlegel et al., 1980a; Altmannsberger et al., 1981; Gabbiani et al., 1981; Denk et al., 1982) and classification of tumors by their specific type of intermediate filaments has re- cently become very valuable in clinical histodiagnosis (see, for example, Schlegel et al., 1980a; Gabbiani et al., 1981; Ramaekers et al., 1981). The intermediate filaments of the vimentin, desmin or glial types all consist usually of only one type of subunit protein (desmin and vimentin can occur in the same filament in BHK cells and vascular smooth mus- cle cells; Steinert et al., 1981 a; Quinlan and Franke, 1982). In contrast with these, the cytokeratin fila- ments, which are composed of proteins related to, but not identical with, epidermal (Y keratins, are a complex family of many different polypeptides. These cytoker- atins, which show biochemical and immunological re- lationships of various degrees, are expressed, in dif- ferent epithelia, in different combinations polypep- tides ranging in their isoelectric pH values from 5 to 8 and in their apparent molecular weights from 40,000 to 68,000 (Doran et al., 1980; Winter et al., 1980; Fuchs and Green, 1980, 1981; Franke et al., 1981 a, 1981 b, 1981 c; Milstone and McGuire, 1981; Wu and Rheinwald, 1981). A given epithelium or epithelial cell can therefore be characterized by the specific pattern of its cytokeratin components. Human Cytokeratin Polypeptides and Their Tissue Distribution Cytoskeletal preparations from epithelial tissues ex- tracted in high salt buffer and Triton X-l 00 are highly enriched in intermediate-sized filaments containing proteins that react specifically with antibodies to au- thentic epidermal [Y keratin (see, for example, Sun and Green, 1977; Fuchs and 1978, 1980, 1981; Franke et al., 1978b, 1980, 1981a, 1981 b, 1981~; Wu and Rheinwald, 1981) and that are recovered in filaments reconstituted in vitro from denatured mono- mers (Tezuka and Freedberg, 1972; Lee and Baden, 1976; Steinert et al., 1976, 1981 a; Sun and Green, 1978b; Gipson and Anderson, 1980; Milstone, 1981; Franke et al., 1981 b, 1981~; Renner et al., 1981). When such preparations are made from different hu- man tissues and examined by two-dimensional gel electrophoresis, with the aid of isoelectric focusing as well as nonequilibrium pH gradient electrophoresis for better resolution of basic polypeptides, complex pat- terns of cytokeratin polypeptides are found. The dis- tinct cytokeratin polypeptides that we have so far identified in various human tissues are schematically summarized and arranged according to their specific coordinates on two-dimensional gel electrophoresis in Figure 1, and the corresponding tissue distribution is shown in Table 1 A. Typically, the cytokeratin polypep- tides appear in series of isoelectric variants; all but the most basic spot usually represent phosphorylated

Transmembrane crosstalk between the extracellular matrix--cytoskeleton crosstalk.

Integrin-mediated cell adhesions provide dynamic, bidirectional links between the extracellular matrix and the cytoskeleton. Besides having central roles in cell migration and morphogenesis, focal adhesions and related structures convey information across the cell membrane, to regulate extracellular-matrix assembly, cell proliferation, differentiation, and death. This review describes integrin functions, mechanosensors, molecular switches and signal-transduction pathways activated and integrated by adhesion, with a unifying theme being the importance of local physical forces.

Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates

Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism underlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of focal adhesions. A novel approach was developed for real-time, high-resolution measurements of forces applied by cells at single adhesion sites. This method combines micropatterning of elastomer substrates and fluorescence imaging of focal adhesions in live cells expressing GFP-tagged vinculin. Local forces are correlated with the orientation, total fluorescence intensity and area of the focal adhesions, indicating a constant stress of 5.5 ± 2 nNμm-2. The dynamics of the force-dependent modulation of focal adhesions were characterized by blocking actomyosin contractility and were found to be on a time scale of seconds. The results put clear constraints on the possible molecular mechanisms for the mechanosensory response of focal adhesions to applied force.

Environmental sensing through focal adhesions.

Recent progress in the design and application of artificial cellular microenvironments and nanoenvironments has revealed the extraordinary ability of cells to adjust their cytoskeletal organization, and hence their shape and motility, to minute changes in their immediate surroundings. Integrin-based adhesion complexes, which are tightly associated with the actin cytoskeleton, comprise the cellular machinery that recognizes not only the biochemical diversity of the extracellular neighbourhood, but also its physical and topographical characteristics, such as pliability, dimensionality and ligand spacing. Here, we discuss the mechanisms of such environmental sensing, based on the finely tuned crosstalk between the assembly of one type of integrin-based adhesion complex, namely focal adhesions, and the forces that are at work in the associated cytoskeletal network owing to actin polymerization and actomyosin contraction.

Transmembrane crosstalk between the extracellular matrix and the cytoskeleton

Integrin-mediated cell adhesions provide dynamic, bidirectional links between the extracellular matrix and the cytoskeleton. Besides having central roles in cell migration and morphogenesis, focal adhesions and related structures convey information across the cell membrane, to regulate extracellular-matrix assembly, cell proliferation, differentiation, and death. This review describes integrin functions, mechanosensors, molecular switches and signal-transduction pathways activated and integrated by adhesion, with a unifying theme being the importance of local physical forces.

Molecular interactions in cell adhesion complexes

Cell adhesions consist of multimolecular protein complexes of transmembrane adhesion receptors anchoring intracellular cytoskeletal structural proteins and signal transduction molecules. Recent advances reveal that components of cell adhesion complexes display multiple interactions and functions, which cooperate to mediate both cell adhesion and signaling. Cell-matrix and cell-cell adhesions can serve as both recipients and generators of signaling information, using hierarchical and synergistic molecular interactions regulated by aggregation, conformational changes, phosphorylation, and tension.

Dynamics and segregation of cell–matrix adhesions in cultured fibroblasts

Here we use time-lapse microscopy to analyse cell–matrix adhesions in cells expressing one of two different cytoskeletal proteins, paxillin or tensin, tagged with green fluorescent protein (GFP). Use of GFP–paxillin to analyse focal contacts and GFP–tensin to study fibrillar adhesions reveals that both types of major adhesion are highly dynamic. Small focal contacts often translocate, by extending centripetally and contracting peripherally, at a mean rate of 19 micrometres per hour. Fibrillar adhesions arise from the medial ends of stationary focal contacts, contain α5β1 integrin and tensin but not other focal-contact components, and associate with fibronectin fibrils. Fibrillar adhesions translocate centripetally at a mean rate of 18 micrometres per hour in an actomyosin-dependent manner. We propose a dynamic model for the regulation of cell–matrix adhesions and for transitions between focal contacts and fibrillar adhesions, with the ability of the matrix to deform functioning as a mechanical switch.

Hierarchical assembly of cell-matrix adhesion complexes

The adhesion of cells to the extracellular matrix is a dynamic process, mediated by a series of cell-surface and matrix-associated molecules that interact with each other in a spatially and temporally regulated manner. These interactions play a major role in tissue formation, cellular migration and the induction of adhesion-mediated transmembrane signals. In this paper, we show that the formation of matrix adhesions is a hierarchical process, consisting of several sequential molecular events. One of the earliest steps in surface recognition is mediated, in some cells, by a 1 microm-thick cell-surface hyaluronan coat, which precedes the establishment of stable, cytoskeleton-associated adhesions. The earliest forms of these integrin-mediated contacts are dot-shaped FXs (focal complexes), which are formed under the protrusive lamellipodium of migrating cells. These adhesions recruit, sequentially, different anchor proteins that are involved in binding the actin cytoskeleton to the membrane. Conspicuous in its absence from FXs is zyxin, which is recruited to these sites only on retraction of the leading edge and the transformation of the FXs into a focal adhesion. Continuing application of force to focal adhesions results in the formation of fibrillar adhesions and reorganization of the extracellular matrix. The formation of these adhesions depends on actomyosin contractility and matrix pliability.

Exploring the Neighborhood: Adhesion-Coupled Cell Mechanosensors

Here we discuss recent studies addressing adhesion-coupled mechanosensory processes and consider their molecular nature. Are cells using stretch-activated ion channels to explore the extracellular environment surrounding them, or do they use for that purpose the submembrane protein network that interconnects integrin receptors with the actin cytoskeleton?

Differential molecular interactions of β-catenin and plakoglobin in adhesion, signaling and cancer

Plakoglobin and beta-catenin are homologous proteins functioning in cell adhesion and transactivation. Their activities are controlled by three types of interactions: those with cadherins in adherens junctions, linking them to the actin cytoskeleton; interactions in the nucleus, where they bind to transcription factors and stimulate gene expression; interactions of free cytoplasmic beta-catenin with axin and adenomatous polyposis coli (APC) protein which target it for degradation. Studies in the past year have demonstrated the complex interplay between these three types of interactions and the different behavior of beta-catenin and plakoglobin in their involvement in morphogenesis and tumorigenesis strongly suggesting that catenins play key roles in adhesion-mediated signaling.