Michael W. Klymkowsky

Epithelial-Mesenchymal Transition: A Cancer Researcher's Conceptual Friend and Foe

Epithelial-mesenchymal transition (EMT) describes a series of rapid changes in cellular phenotype. During EMT, epithelial cells down-modulate cell-cell adhesion structures, alter their polarity, reorganize their cytoskeleton, and become isolated, motile, and resistant to anoikis. The term EMT is often applied to distinct biological events as if it were a single conserved process, but in fact EMT-related processes can vary in intensity from a transient loss of cell polarity to the total cellular reprogramming, as found by transcriptional analysis. Based on clinical observations, it is more appropriate in most cases to describe the emergence of an EMT-like phenotype during tumor progression. Although EMT implies complete trans-differentiation, EMT-like emphasizes the intermediary phenotype associated with tumor cell renewal and adaptation to specific microenvironments. Here, we categorize the various EMT-like phenotypes found in human carcinomas that, depending on the tumor type, may or not represent analogous stages in tumor progression. We based these categories on the global tumor phenotype. The tumor microenvironment, which is associated with stromal reactions, hypoxia, paucity of nutrients, impaired differentiation, and activation of various EMT-associated pathways, modulates overall tumor phenotype and leads to tumor heterogeneity.

Regulation of Wnt Signaling by Sox Proteins: XSox17α/β and XSox3 Physically Interact with β-catenin

Abstract Using a functional screen in Xenopus embryos, we identified a novel function for the HMG box protein XSox17β. Ectopic expression of XSox17β ventralizes embryos by inhibiting the Wnt pathway downstream of β-catenin but upstream of the Wnt-responsive gene Siamois . XSox17β also represses transactivation of a TCF/LEF-dependent reporter construct by Wnt and β-catenin. In animal cap experiments, it both activates transcription of endodermal genes and represses β-catenin-stimulated expression of dorsal genes. The inhibition activity of XSox17β maps to a region C-terminal to the HMG box; this region of XSox17β physically interacts with the Armadillo repeats of β-catenin. Two additional Sox proteins, XSox17α and XSox3, likewise bind to β-catenin and inhibit its TCF-mediated signaling activity. These results reveal an unexpected mechanism by which Sox proteins can modulate Wnt signaling pathways.

Chapter 22 Whole-Mount Staining of Xenopus and Other Vertebrates

Publisher Summary This chapter discusses whole-mount staining of xenopus and other vertebrates. Whole-mount staining makes the analysis of normal and experimentally manipulated embryos much simpler. It can be used in the assay of cellular differentiation in induction and tissue recombination experiments. It should be possible not only to assay for the indication of specific tissues, but to characterize the three-dimensional relationships between the tissue types. Whole-mount staining greatly simplifies the characterization of the expressions patterns of exogenous DNAs. Similarly, the effects of injected antibodies, antisense reagents, or the ecotopic expression of specific molecules on development can be analyzed rapidly. The prerequisite for any whole-mount analysis is that one is able to see through the specimen. This means either that the specimen must be naturally transparent, a rare feature among higher metazoans, or that it must be possible to “clear” it. Clearing generally involves two steps: extracting material from the specimen, then matching the refractive index of the bulk of the specimen remaining, thereby rendering it transparent. Primary considerations in selecting a clearing agent are how closely it matches the refractive index of the specimen, its inherent toxicity, and its compatibility with the staining reagents used.

Structural studies of a membrane-bound acetylcholine receptor from Torpedo californica☆

Abstract Membranes prepared from Torpedo californica electroplax containing acctylcholine receptors have been studied by X-ray diffraction and electron microscopy. X-ray diffraction data suggest that acetylcholine receptor molecules traverse the endplate membrane, extending 15 ± 5 A on one side of the bilayer and some 55 ± 5 A on the other, with an overall length normal to the membrane of 110 A. Lattices of acetylcholine receptor have the symmetry of the crystallographic plane group p 1, with one molecule per unit cell. A low-resolution projection of the surface structure of receptor arrays was determined by reconstruction of images from electron micrographs. The resolution of the image is ~ 20 A in the plane of the membrane. The electron density profile through the membrane, derived from X-ray diffraction of vesicle dispersions and of oriented membranes, has been analyzed to resolutions of 20 and 13 A, respectively. The high-angle X-ray scattering pattern was observed to a resolution of 1.7 A. Maxima in the scattering pattern were analyzed in terms of the state of the lipids and secondary structure in the membranes. Sharp maxima in the scattering pattern indicate that long stretches of secondary structure are present in the receptor-containing membranes. The receptor membranes contain repeating structural units of length 80 A (5.2 A repeat) oriented perpendicular to the membrane plane, and uninterpreted components greater than 90 A in length with a basic repeat of 6.3 A.

Understanding Randomness and its Impact on Student Learning: Lessons Learned from Building the Biology Concept Inventory (BCI)

While researching student assumptions for the development of the Biology Concept Inventory (BCI; http://bioliteracy.net), we found that a wide class of student difficulties in molecular and evolutionary biology appears to be based on deep-seated, and often unaddressed, misconceptions about random processes. Data were based on more than 500 open-ended (primarily) college student responses, submitted online and analyzed through our Eds Tools system, together with 28 thematic and think-aloud interviews with students, and the responses of students in introductory and advanced courses to questions on the BCI. Students believe that random processes are inefficient, whereas biological systems are very efficient. They are therefore quick to propose their own rational explanations for various processes, from diffusion to evolution. These rational explanations almost always make recourse to a driver, e.g., natural selection in evolution or concentration gradients in molecular biology, with the process taking place only when the driver is present, and ceasing when the driver is absent. For example, most students believe that diffusion only takes place when there is a concentration gradient, and that the mutational processes that change organisms occur only in response to natural selection pressures. An understanding that random processes take place all the time and can give rise to complex and often counterintuitive behaviors is almost totally absent. Even students who have had advanced or college physics, and can discuss diffusion correctly in that context, cannot make the transfer to biological processes, and passing through multiple conventional biology courses appears to have little effect on their underlying beliefs.

The appearance of acetylated alpha-tubulin during early development and cellular differentiation in Xenopus.

Early development in Xenopus is characterized by dramatic changes in the organization of the microtubule cytoskeleton. We have used whole-mount immunocytochemistry to follow the expression of the acetylated form of alpha-tubulin during early Xenopus development. In the egg and early embryo, the monoclonal anti-acetylated tubulin antibody 6-11B-1 stained meiotic and mitotic spindles, midbody microtubules, and what appears to be the central region of the sperm aster; the antibody did not stain the sperm aster itself or the cortical microtubule system associated with the rotation of the fertilized egg. Following gastrulation, acetylated tubulin disappeared from all but mitotic midbody microtubules. During the course of neurulation high levels of acetylated tubulin reappeared in the precursors of the ciliated epidermal cells (stage 15), transiently in neural folds (stage 16/17), in neuronal processes (stage 18/19), and in somas (stage 21). The changing pattern of anti-acetylated tubulin staining during Xenopus development raises intriguing questions as to the physiological significance of tubulin acetylation.

Immunospecific identification and three-dimensional structure of a membrane-bound acetylcholine receptor from Torpedo californica

Abstract Antibodies, raised against affinity column-purified acetylcholine receptor from Torpedo californica, were used as a basis for immunospecific identification of the receptor in membrane fragments. Rabbit and goat anti-receptor antibodies were coupled directly or indirectly via goat anti-rabbit antibody to colloidal gold spheres or to ferritin. The labeled membranes were visualized by negative stain electron microscopy, and show that the receptor corresponds to the 85 A diameter rosette seen in membranes derived from electroplaques. Electron micrographs of immunospecifically labeled receptor, in the plane perpendicular to the membrane surface, confirm and extend our previous conclusions based on X-ray diffraction analysis, that the molecule extends above the extracellular membrane surface by approximately 55 A, and little on the cytoplasmic side. Calculated molecular volumes based on X-ray diffraction and electron microscopy indicate that the membrane receptor has a molecular weight in the range of 250,000 to 310,000, a range consistent with current estimates of detergent-solubilized monomer molecular weight.

The β-catenin/VegT-regulated early zygotic gene Xnr5 is a direct target of SOX3 regulation

In Xenopus laevis, β-catenin-mediated dorsal axis formation can be suppressed by overexpression of the HMG-box transcription factor XSOX3. Mutational analysis indicates that this effect is due not to the binding of XSOX3 to β-catenin nor to its competition with β-catenin-regulated TCF-type transcription factors for specific DNA binding sites, but rather to SOX3 binding to sites within the promoter of the early VegT- andβ -catenin-regulated dorsal-mesoderm-inducing gene Xnr5. Although B1-type SOX proteins, such as XSOX3, are commonly thought to act as transcriptional activators, XSOX3 acts as a transcriptional repressor of Xnr5 in both the intact embryo and animal caps injected with VegT RNA. Expression of a chimeric polypeptide composed of XSOX3 and a VP16 transcriptional activation domain or morpholino-induced decrease in endogenous XSOX3 polypeptide levels lead to an increase in Xnr5 expression, as does injection of an anti-XSOX3 antibody that inhibits XSOX3 DNA binding. These observations indicate that maternal XSOX3 acts in a novel manner to restrict Xnr5 expression to the vegetal hemisphere.

Intermediate filaments: new proteins, some answers, more questions

The past year has seen significant progress in the characterization of intermediate filament proteins. New proteins have been identified and physiologically significant differences between known proteins have been revealed. Changes in intermediate filament organization have been linked to changes in cell behavior, and mutational analyses are beginning to reveal the connection between intermediate filament expression, network formation, cellular behavior and disease.

The body language of cells: The intimate connection between cell adhesion and behavior

Michael W. Klymkowsky’ and Brian Parrt* *Molecular, Cellular and Developmental Biology University of Colorado, Boulder Boulder, Colorado 80309-0347 fMolecular and Cellular Biology Harvard University Cambridge, Massachusetts 02138 A simple touch between organisms can convey a wide range of different messages, from affection to hostility. Touch appears critical for both social intercourse and the achievement of normal developmental milestones. The analogy of the cells in a metazoan to the individuals in a society has been, perhaps, overused. Yet, in a fundamen- tal way, it is extremely apt. For metazoan cells the im- portance of touching (and the associated “juxtacrine sig- naling”) as a source of information in both embryonic morphogenesis and the maintenance of tissue integrity and organ function is becoming increasingly obvious. Cells touch one another through a number of different surface molecules; among the most intriguing are the cadherins and their associated proteins. Together with jux- tacrine signals, most notably those involving proteins of the Wnt family, these proteins generate a range of adhe- sive and signaling interactions between neighboring cells. Juxtacrine signaling requires that cells be brought into close apposition. A major class of cell-cell adhesion junc- tions (AJs) are those mediated by the cadherins. As cells approach one another and touch, cadherins begin to clus- ter and connect, through their cytoplasmic domains and associated proteins (catenins), with the cytoskeleton. Within 20 sof cell-cell contact, discrete AJs, characterized by cytoplasmic plaques and associated cytoskeletal fi- bers, are evident (Heaysman and Pegrum, 1973) (Fig- ure 1). The first step in the assembly of an AJ (Figures 2A and 2B) is the interaction between the extracellular domains of cadherins on neighboring cells. The extracellular por- tion of cadherins typically consists of five tandem repeats of an - 110 amino acid homology domain, the cadherin repeat. X-ray crystallographic studies of the isolated N-terminal domain of N-cadherin, together with deduc- tions based on the crystal packing of polypeptides (Sha- piro et al., 1995), suggest that these cadherin domains stack on one another and that this stacking is stabilized by CaZ+ positioned at the interface between domains. Lateral interactions between the stacks of cadherin repeats lead to the formation a cadherin dimer. Interactions between cells involve the N-terminal cadherin repeat domain and are proposed to generate a “cadherin zipper” whose strength depends on the number of cadherin molecules involved. It is not known whether the cadherin dimer exists prior to the formation of the cadherin zipper and whether simple dimerization generates signals in a manner similar to the signaling induced by the dimerization of other mem- brane receptors. Prior to their clustering, cadherins associate with the cytoplasmic proteins 8-catenin or plakoglobin (PKG) through their cytoplasmic domains (Hinck et al., 1994); it is not clear, however, whether all cadherin molecules are associated with catenins prior to clustering. 8-Catenin and PKG are closely related to one another and to the product of the Drosophila segment polarity gene armadillo (arm) (see Peifer, 1995). 8-Catenin binds to the tail domain of nondesmosomal cadherins, whereas PKG (sometimes called r-catenin) binds to both desmosomal and nondes- mosomal cadherins (Cowin, 1994). The clustered cad- herin+-catenin/PKG complex appears to act as a nucleus for the formation of a cytoplasmic “plaque” composed of other catenins (Figure 2C). If cadherin clustering gener- ates a signaling response (see above), it seems likely that the association 8-catenin/PKG with cadherins could act to enhance this signal. At adherens junctions, the plaque associated with cad- herin tail domains mediates the “end-on” anchoring of mi- crofilaments via vinculin-like a-catenin polypeptide and a-actinin (Knudsen et al., 1995). At desmosomes, cadherins interact with other polypeptides (e.g., desmo- plakins) that mediate an en passant interaction with inter- mediate filaments. In the absence of functional catenins, AJs do not form, presumably because the cadherin zipper is not adequately stabilized by the assembly of cyto- plasmic plaque. In addition to linking cadherins and thereby stabilizing AJs, 8-catenin and PKG also mediate interactions be- tween cadherins and other membrane receptor proteins, notably the epidermal growth factor (EGF) receptor (Hos- chuetzky et al., 1994) and c-ErbB (Kanai 1995). There are also indications that N-cadherin interacts with fibroblast growth factor receptors during neurite outgrowth (Williams et al., 1994). How these interactions modulate receptor activity is unclear. It firmly established, how- ever, that f3-catenin and PKG mediate the effects of several juxtacrine signaling systems, most notably those involving Wnt proteins. The diffusion of secreted Wnts is severely limited through their binding to extracellular matrix and cell surfaces; this effectively restricts the effects of Wnt signals to the immediate neighbors of the Wnt-secreting cell. Wnt signaling plays a key role in patterning inverte- brate and vertebrate embryos during processes such as the determination of segment polarity in Drosophila, pri- mary axis formation in Xenopus, and limb development in mice and chickens (Moon, 1993; Perrimon, 1994; Yang and Niswander, 1995; Parr and McMahon, 1995). In Drosophila, the segment polarity gene wingless (wg) encodes a Wnt polypeptide. Genetic studies (see Perri- mon, 1994; Peifer, 1995) have identified a number of the gene products involved in the Wnt signaling pathway. The 8-catenin/PKG homolog arm appears to play a key role