The body language of cells: the intimate connection between cell adhesion and behavior.
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Animals↗
Biomedical subjects
M W Klymkowsky
Publications and source records attributed to M W Klymkowsky.
Anterior axis duplication in Xenopus induced by the over-expression of the cadherin-binding protein plakoglobin.
Plakoglobin interacts with both classical and desmosomal cadherins. It is closely related to Drosophila aramadillo (arm) gene product; arm acts in the wingless (wg)-signaling pathway to establish segment polarity. In Xenopus, homologs of wg--i.e., wnts, can produce anterior axis duplications by inducing dorsal mesoderm. Studies in Drosophila suggest that wnt acts by increasing the level of cytoplasmic armadillo protein (arm). To test whether simply increasing the level of plakoglobin mimics the effects of exogenous wnts in Xenopus, we injected fertilized eggs with RNA encoding an epitope-tagged form of plakoglobin; this induced both early radial gastrulation and anterior axis duplication. Exogenous plakoglobin accumulates in the nuclei of embryonic cells. Plakoglobin binds to the tail domain of the desmosomal cadherin desmoglein 1. When RNA encoding the tail domain of desmoglein was coinjected with plakoglobin RNA, both the dorsalizing effect and nuclear accumulation of plakoglobin were suppressed. Mutational analysis indicates that the central arm repeat region of plakoglobin is sufficient to induce axis duplication and that this polypeptide accumulates in the nuclei of embryonic cells. These data show that increased plakoglobin levels can, by themselves, generate the intracellular signals involved in the specification of dorsal mesoderm.
Amino Acid Sequence↗
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.
Animals↗
Intermediate filament organization, reorganization, and function in the clawed frog Xenopus.
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Animals↗
Disruption of intermediate filament organization leads to structural defects at the intersomite junction in Xenopus myotomal muscle.
In mature striated muscle, intermediate filaments (IFs) are associated with the periphery of Z-discs and sites of myofibril-membrane attachment. Previously T. Schultheiss, Z. X. Lin, H. Ishikawa, I. Zamir, C. J. Stoeckert and H. Holtzer (1991) J. Cell Biol. 114, 953) reported that the disruption of IF organization in cultured chick myotubes had no detectable effect on muscle cell structure. Cultured muscle is not, however, under the mechanical loads characteristic of muscle in situ. The dorsal myotomal muscle (DMM) of the Xenopus tadpole provides an accessible model system in which to study the effects of mutant IF proteins on an intact, functional muscle. DNAs encoding truncated forms of Xenopus vimentin or desmin were injected into fertilized Xenopus eggs. Embryos were allowed to develop to the tadpole stage and then examined by confocal or electron microscopy. DMM cells containing the truncated IF polypeptides displayed disorganized IF systems. While the alignment of Z-lines appeared unaffected, cells accumulating mutant IF polypeptides displayed abnormal organization at the intersomite junction. Myocyte termini are normally characterized by deep invaginations of the sarcolemma. In myocytes expressing mutated IF polypeptides, these membrane invaginations were reduced or completely absent. Furthermore, the attachment of myofibrils to the junctional membrane was often aberrant or completely disrupted. These results suggest that in active muscle IFs play an important role in the organization and/or stabilization of myofibril-membrane attachment sites.
Animals↗
Morphogenesis and the cytoskeleton: studies of the Xenopus embryo.
The morphological transformation from oocyte to embryo is brought about by the structural components of the cell, the cytoskeleton. Cytoskeletal elements act to generate and maintain cellular asymmetries, cellular movement and morphologies, and to integrate signals and forces into morphogenetically coherent behavior. Because of its unique experimental accessibility, the Xenopus embryo provides a powerful model system in which to study the "body language" of early embryonic development.
Animals↗
Secretory pathway function, but not cytoskeletal integrity, is required in poliovirus infection.
We examined the importance of two interactions between poliovirus and its host cell: the putative association between viral proteins and a rearranged intermediate filament (IF) network and the apparent requirement for functional vesicle budding machinery within the host-cell secretory pathway. Poliovirus capsid proteins appeared to associate with reorganized IF proteins during infection. Treatment of cells with cytochalasin D and nocodazole in combination disrupted normal cytoskeletal organization and prevented the poliovirus-induced redistribution of IF proteins to a juxtanuclear location. However, this treatment had no effect on viral yields from single-cycle infections, indicating that neither cytoskeletal integrity nor a specific poliovirus-induced rearrangement of IF proteins is required in the poliovirus life cycle. In contrast, we report that the inhibition of poliovirus replication by brefeldin A (BFA), an inhibitor of secretory membrane traffic, is specific to the host cell. Polioviral yields were not affected by BFA in two BFA-resistant cell lines, demonstrating that BFA targets a host protein or process required by poliovirus. No BFA-resistant virus was detected in these experiments, further supporting the hypothesis that poliovirus replication requires secretory pathway function, perhaps for the generation of vesicles on which viral RNA replication complexes are assembled.
Animals↗
Type II collagen distribution during cranial development in Xenopus laevis.
Epithelially expressed type II collagen is thought to play a prominent role in the embryonic patterning and differentiation of the vertebrate skull, primarily on the basis of data derived from amniotes. We describe the spatiotemporal distribution of type II collagen in the embryonic head of the African clawed frog, Xenopus laevis, using whole-mount and serial-section immunohistochemical analysis. We studied embryos spanning Nieuwkoop and Faber (1967) stages 21-39, a period including cranial neural crest cell migration and ending immediately before the onset of neurocranial chondrogenesis. Xenopus displays a transient expression of type II collagen beginning at least as early as stage 21; staining is most intense and widespread at stages 33/34 and 35/36 and subsequently diminishes. Collagen-positive areas include the ventrolateral surface of the brain, sensory vesicles, notochord, oropharynx, and integument. This expression pattern is similar, but not identical, to that reported for the mouse and two bird species (Japanese quail, domestic fowl); thus epithelially expressed type II collagen appears to be a phylogenetically widespread feature of vertebrate cranial development. Consistent with the proposed role of type II collagen in mediating neurocranial differentiation, most collagen-positive areas lie adjacent to subsequent sites of chondrogenesis in the neurocranium but not the visceral skeleton. However, much of the collagen is expressed after the migration of cranial neural crest, including presumptive chondrogenic crest, seemingly too late to pattern the neurocranium by entrapment of these migrating cells.
Animals↗
Desmin organization during the differentiation of the dorsal myotome in Xenopus laevis.
The reorganization of desmin-type intermediate filaments during muscle differentiation has been studied primarily in cultured cell systems. Here we describe the process of desmin reorganization during the differentiation of the dorsal myotomal muscle of the clawed frog Xenopus laevis. This muscle differs from those described previously primarily in that the desmin system forms de novo, i.e., without the presence of a pre-existing vimentin filament system. The most striking observation is that prior to myotomal segmentation and rotation desmin is concentrated at the medial and lateral tips of the myocytes. It remains concentrated in these regions following somite rotation and is located primarily to the intersomite junctions as late as the stage 33-35 tadpole. As the muscle matures (stage 30 and later) desmin becomes increasingly associated with the sarcolemma and with the Z-discs. The concentration of desmin at the nascent intersomite junction suggests that desmin is involved in coupling somites to one another in the early Xenopus embryo.
Animals↗
Differential organization of desmin and vimentin in muscle is due to differences in their head domains.
In most myogenic systems, synthesis of the intermediate filament (IF) protein vimentin precedes the synthesis of the muscle-specific IF protein desmin. In the dorsal myotome of the Xenopus embryo, however, there is no preexisting vimentin filament system and desmin's initial organization is quite different from that seen in vimentin-containing myocytes (Cary and Klymkowsky, 1994. Differentiation. In press.). To determine whether the organization of IFs in the Xenopus myotome reflects features unique to Xenopus or is due to specific properties of desmin, we used the injection of plasmid DNA to drive the synthesis of vimentin or desmin in myotomal cells. At low levels of accumulation, exogenous vimentin and desmin both enter into the endogenous desmin system of the myotomal cell. At higher levels exogenous vimentin forms longitudinal IF systems similar to those seen in vimentin-expressing myogenic systems and massive IF bundles. Exogenous desmin, on the other hand, formed a reticular IF meshwork and non-filamentous aggregates. In embryonic epithelial cells, both vimentin and desmin formed extended IF networks. Vimentin and desmin differ most dramatically in their NH2-terminal "head" regions. To determine whether the head region was responsible for the differences in the behavior of these two proteins, we constructed plasmids encoding chimeric proteins in which the head of one was attached to the body of the other. In muscle, the vimentin head-desmin body (VDD) polypeptide formed longitudinal IFs and massive IF bundles like vimentin. The desmin head-vimentin body (DVV) polypeptide, on the other hand, formed IF meshworks and non-filamentous structures like desmin. In embryonic epithelial cells DVV formed a discrete filament network while VDD did not. Based on the behavior of these chimeric proteins, we conclude that the head domains of vimentin and desmin are structurally distinct and not interchangeable, and that the head domain of desmin is largely responsible for desmin's muscle-specific behaviors.
Amino Acid Sequence↗
Vimentin's tail interacts with actin-containing structures in vivo.
The tail domain of the intermediate filament (IF) protein vimentin is unnecessary for IF assembly in vitro. To study the role of vimentin's tail in vivo, we constructed a plasmid that directs the synthesis of a 'myc-tagged' version of the Xenopus vimentin-1 tail domain in bacteria. This polypeptide, mycVimTail, was purified to near homogeneity and injected into cultured Xenopus A6 cells. In these cells the tail polypeptide co-localized with actin even in the presence of cytochalasin. Two myc-tagged control polypeptides argue for the specificity of this interaction. First, a similarly myc-tagged lamin tail domain localizes to the nucleus, indicating that the presence of the myc tag did not itself confer the ability to co-localize with actin (Hennekes and Nigg (1994) J. Cell Sci. 107, 1019-1029). Second, a myc-tagged polypeptide with a molecular mass and net charge at physiological pH (i.e. -4) similar to that of the mycVimTail polypeptide, failed to show any tendency to associate with actin-containing structures, indicating that the interaction between mycVimTail and actin-containing structures was not due to a simple ionic association. Franke (1987; Cell Biol. Int. Rep. 11, 831) noted a similarity in the primary sequence between the tail of the type I keratin DG81A and vimentin. To test whether the DG81A tail interacted with actin-containing structures, we constructed and purified myc-tagged DG81A tail polypeptides. Unexpectedly, these keratin tail polypeptides were largely insoluble under physiological conditions and formed aggregates at the site of injection. While this insolubility made it difficult to determine if they associated with actin-containing structures, it does provide direct evidence that the tails of vimentin and DG81A differ dramatically in their physical properties. Our data suggest that vimentin's tail domain has a highly extended structure, binds to actin-containing structures and may mediate the interaction between vimentin filaments and microfilaments involved in the control of vimentin filament organization (Hollenbeck et al. (1989) J. Cell Sci. 92, 621; Tint et al. (1991) J. Cell Sci. 98, 375).
Actins↗
Evidence that the deep keratin filament systems of the Xenopus embryo act to ensure normal gastrulation.
To study the role of keratin filaments in Xenopus development, fertilized eggs were injected with anti-keratin monoclonal antibodies. The anti-keratin monoclonal antibodies AE1 and AE3 induce abnormal gastrulation; in the most severely affected embryos gastrulation fails completely. In contrast, embryos injected with the anti-keratin antibody 1h5 develop normally. Immunocytochemical data indicate that injected 1h5 binds to the dense superficial keratin filament system of the embryo but not to the deeper keratin filament networks of ectodermal and subectodermal cells. Injected AE1 and AE3 do not bind to the superficial keratin system but appear to interact preferentially with the deep keratin filament systems of the embryo. We conclude that the superficial keratin filament system is not involved in the process of gastrulation per se but may protect the embryo from mechanical damage. On the other hand, our results suggest that the integrity of the deeper keratin filament systems is required for the mechanical integration of the morphogenetic movements that underlie gastrulation in Xenopus.
Animals↗
Cranial ontogeny in the direct-developing frog, Eleutherodactylus coqui (Anura: Leptodactylidae), analyzed using whole-mount immunohistochemistry.
Direct development in amphibians is an evolutionarily derived life-history mode that involves the loss of the free-living, aquatic larval stage. We examined embryos of the direct-developing anuran Eleutherodactylus coqui (Leptodactylidae) to evaluate how the biphasic pattern of cranial ontogeny of metamorphosing species has been modified in the evolution of direct development in this lineage. We employed whole-mount immunohistochemistry using a monoclonal antibody against the extracellular matrix component Type II collagen, which allows visualization of the morphology of cartilages earlier and more effectively than traditional histological procedures; these latter procedures were also used where appropriate. This represents the first time that initial chondrogenic stages of cranial development of any vertebrate have been depicted in whole-mounts. Many cranial cartilages typical of larval anurans, e.g., suprarostrals, cornua trabeculae, never form in Eleutherodactylus coqui. Consequently, many regions of the skull assume an adult, or postmetamorphic, morphology from the inception of their development. Other components, e.g., the lower jaw, jaw suspensorium, and the hyobranchial skeleton, initially assume a mid-metamorphic configuration, which is subsequently remodeled before hatching. Thirteen of the adult complement of 17 bones form in the embryo, beginning with two bones of the jaw and jaw suspensorium, the angulosplenial and squamosal. Precocious ossification of these and other jaw elements is an evolutionarily derived feature not found in metamorphosing anurans, but shared with some direct-developing caecilians. Thus, in Eleutherodactylus cranial development involves both recapitulation and repatterning of the ancestral metamorphic ontogeny.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Host cell factors controlling vimentin organization in the Xenopus oocyte.
To study vimentin filament organization in vivo we injected Xenopus oocytes, which have no significant vimentin system of their own, with in vitro-synthesized RNAs encoding Xenopus vimentins. Exogenous vimentins were localized primarily to the cytoplasmic surface of the nucleus and to the subplasma membrane "cortex." In the cortex of the animal hemisphere, wild-type vimentin forms punctate structures and short filaments. In contrast, long anastomosing vimentin filaments are formed in the vegetal hemisphere cortex. This asymmetry in the organization of exogenous vimentin is similar to that of the endogenous keratin system (Klymkowsky, M. W., L. A. Maynell, and A. G. Polson. 1987. Development (Camb.). 100:543-557), which suggests that the same cellular factors are responsible for both. Before germinal vesicle breakdown, in the initial stage of oocyte maturation, large vimentin and keratin filament bundles appear in the animal hemisphere. As maturation proceeds, keratin filaments fragment into soluble oligomers (Klymkowsky, M. W., L. A. Maynell, and C. Nislow. 1991. J. Cell Biol. 114:787-797), while vimentin filaments remain intact and vimentin is hyperphosphorylated. To examine the role of MPF kinase in the M-phase reorganization of vimentin we deleted the conserved proline of vimentin's single MPF-kinase site; this mutation had no apparent effect on the prophase or M-phase behavior of vimentin. In contrast, deletion of amino acids 19-68 or 18-61 of the NH2-terminal "head" domain produced proteins that formed extended filaments in the animal hemisphere of the prophase oocyte. We suggest that the animal hemisphere cortex of the prophase oocyte contains a factor that actively suppresses the formation of extended vimentin filaments through a direct interaction with vimentin's head domain. During maturation this "suppressor of extended filaments" appears to be inactivated, leading to the formation of an extended vimentin filament system.
Amino Acid Sequence↗
Inhibition of poliovirus RNA synthesis by brefeldin A.
Brefeldin A (BFA), a fungal metabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was found to inhibit poliovirus replication 10(5)- to 10(6)-fold. BFA does not inhibit entry of poliovirus into the cell or translation of viral RNA. Poliovirus RNA synthesis, however, is completely inhibited by BFA. A specific class of membranous vesicles, with which the poliovirus replication complex is physically associated, is known to proliferate in poliovirus-infected cells. BFA may inhibit poliovirus replication by preventing the formation of these vesicles.
Adenoviridae↗
Intermediate filaments. Getting under the skin.
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Animals↗
Whole-mount staining of Xenopus and other vertebrates.
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Animals↗
Cytokeratin phosphorylation, cytokeratin filament severing and the solubilization of the maternal mRNA Vg1.
During meiotic maturation, the cortical cytokeratin filament system of the Xenopus oocyte disappears (Klymkowsky, M. W., and L. A. Maynell. 1989. Dev. Biol. 134:479). Here we demonstrate that this disappearance results from the severing of cytokeratin filaments into a heterogenous population of oligomers, with S- values ranging from 12S and greater. Cytokeratin filament severing correlates with the hyperphosphorylation of the type II cytokeratin of the oocyte. Both the severing of cytokeratin filaments and cytokeratin hyperphosphorylation are reversed by treatment with cycloheximide. These data suggest that fragmentation of cytokeratin filaments is controlled, at least in part, by the phosphorylation of the type II cytokeratin, and that the cytokeratin kinase activity responsible is biosynthetically labile. Cytokeratin filaments have been suggested to anchor the maternal mRNA Vg1 to the vegetal cortex of the oocyte (Pondel, M., and M. L. King. 1988. Proc. Natl. Acad. Sci. USA. 85:7216). By injecting fractions containing active maturation promoting factor or a purified, mutant cyclin protein, we find that the bulk of the Vg1 mRNA in the oocyte can be solubilized under conditions that block the fragmentation of cytokeratin filaments, and that the fragmentation of cytokeratin filaments itself leads to the solubilization of only a minor fraction of the Vg1 mRNA. Thus, at best, cytokeratin filaments directly anchor only a minor fraction of the Vg1 mRNA in the oocyte. Moreover, factors distinct from maturation promoting factor appear to be required for the complete solubilization of Vg1 mRNA during oocyte maturation.
Actin Cytoskeleton↗