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.

Giant axonal neuropathy: a conditional mutation affecting cytoskeletal organization.

Giant axonal neuropathy (GAN) results from autosomal recessive mutations (gan-) that affect cytoskeletal organization; specifically, intermediate filaments (IFs) are found collapsed into massive bundles in a variety of different cell types. We studied the gan- fibroblast lines WG321 and WG139 derived from different GAN patients. Although previous studies implied that the gan- IF phenotype was constitutive, we find that it is conditional. That is, when cells were grown under the permissive condition of medium containing over 2% fetal calf serum, most cells had normal IF organization. IF bundles formed when gan- cells were transferred to the nonpermissive condition of low (0.1%) serum. Microtubule organization appeared normal in the presence or absence of serum. The effect of serum starvation was largely blocked or reversed by the addition of BSA to the culture media. We found no evidence that the gan- phenotype depends upon progress through the cell cycle. We discuss the possible role of serum effects in the etiology of GAN and speculate as to the molecular nature of the gan- defect.

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.

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.

A nontetrameric species is the major soluble form of keratin in Xenopus oocytes and rabbit reticulocyte lysates.

Inside the interphase cell, approximately 5% of the total intermediate filament protein exists in a soluble form. Past studies using velocity gradient sedimentation (VGS) indicate that soluble intermediate filament protein exists as an approximately 7 S tetrameric species. While studying intermediate filament assembly dynamics in the Xenopus oocyte, we used both VGS and size-exclusion chromatography (SEC) to analyze the soluble form of keratin. Previous studies (Coulombe, P. A., and E. Fuchs. 1990. J. Cell Biol. 111:153) report that tetrameric keratins migrate on SEC with an apparent molecular weight of approximately 150,000; the major soluble form of keratin in the oocyte, in contrast, migrates with an apparent molecular weight of approximately 750,000. During oocyte maturation, the keratin system disassembles into a soluble form (Klymkowsky, M. W., L. A. Maynell, and C. Nislow. 1991. J. Cell Biol. 114:787) and the amount of the 750-kD keratin complex increases dramatically. Immunoprecipitation analysis of soluble keratin from matured oocytes revealed the presence of type I and type II keratins, but no other stoichiometrically associated polypeptides, suggesting that the 750-kD keratin complex is composed solely of keratin. To further study the formation of the 750-kD keratin complex, we used rabbit reticulocyte lysates (RRL). The 750-kD keratin complex was formed in RRLs contranslating type I and type II Xenopus keratins, but not when lysates translated type I or type II keratin RNAs alone. The 750-kD keratin complex could be formed posttranslationally in an ATP-independent manner when type I and type II keratin translation reactions were mixed. Under conditions of prolonged incubation, such as occur during VGS analysis, the 750-kD keratin complex disassembled into a 7 S (by VGS), 150-kD (by SEC) form. In urea denaturation studies, the 7 S/150-kD form could be further disassembled into an 80-kD species that consists of cofractionating dimeric and monomeric keratin. Based on these results, the 750-kD species appears to be a supratetrameric complex of keratins and is the major, soluble form of keratin in both prophase and M-phase oocytes, and RRL reactions.

Microinjection of a monoclonal antibody against a 37-kD protein (tropomyosin 2) prevents the formation of new acetylcholine receptor clusters.

We have shown previously that chick muscle cells transformed with Rous sarcoma virus are unable to form clusters of acetylcholine receptors (AChRs) (Anthony, D. T., S. M. Schuetze, and L. L. Rubin. 1984. Proc. Natl. Acad. Sci. USA. 81:2265-2269) and are missing a 37-KD tropomyosin-like protein (TM-2) (Anthony, D. T., R. J. Jacobs-Cohen, G. Marazzi, and L. L. Rubin. 1988. J. Cell Biol. 106:1713-1721). In an attempt to clarify the role of TM-2 in the formation and/or maintenance of AChR clusters, we have microinjected a monoclonal antibody specific for TM-2 (D3-16) into normal chick muscle cells in culture. D3-16 injection blocks the formation of new clusters but does not affect the preexisting ones. In addition, TM-2 is concentrated at rat neuromuscular junctions. These data suggest that TM-2 may play an important role in promoting the formation of AChR clusters.

Morphology, behavior, and interaction of cultured epithelial cells after the antibody-induced disruption of keratin filament organization.

The organization of intermediate filaments in cultured epithelial cells was rapidly and radically affected by intracellularly injected monoclonal antikeratin filament antibodies. Different antibodies had different effects, ranging from an apparent splaying apart of keratin filament bundles to the complete disruption of the keratin filament network. Antibodies were detectable within cells for more than four days after injection. The antibody-induced disruption of keratin filament organization had no light-microscopically discernible effect on microfilament or microtubule organization, cellular morphology, mitosis, the integrity of epithelial sheets, mitotic rate, or cellular reintegration after mitosis. Cell-to-cell adhesion junctions survived keratin filament disruption. However, antibody injected into a keratinocyte-derived cell line, rich in desmosomes, brought on a superfasciculation of keratin filament bundles, which appeared to pull desmosomal junctions together, suggesting that desmosomes can move in the plane of the plasma membrane and may only be 'fixed' by their anchoring to the cytoplasmic filament network. Our observations suggest that keratin filaments are not involved in the establishment or maintenance of cell shape in cultured cells.

Protease effects on the structure of acetylcholine receptor membranes from Torpedo californica.

Protease digestion of acetylcholine receptor-rich membranes derived from Torpedo californica electroplaques by homogenization and isopycnic centrifugation results in degradation of all receptor subunits without any significant effect on the appearance in electron micrographs, the toxin binding ability, or the sedimentation value of the receptor molecule. Such treatment does produce dramatic changes in the morphology of the normally 0.5- to 2-microns-diameter spherical vesicles when observed by either negative-stain or freeze-fracture electron microscopy. Removal of peripheral, apparently nonreceptor polypeptides by alkali stripping (Neubig et al. 1979, Proc. Natl. Acad. Sci. U. S. A. 76:690-694) results in increased sensitivity of the acetylcholine receptor membranes to the protease trypsin as indicated by SDS gel electrophoretic patterns and by the extent of morphologic change observed in vesicle structure. Trypsin digestion of alkali-stripped receptor membranes results in a limit degradation pattern of all four receptor subunits, whereupon all the vesicles undergo the morphological transformation to minivesicles. The protein-induced morphological transformation and the limit digestion pattern of receptor membranes are unaffected by whether the membranes are prepared so as to preserve the receptor as a disulfide bridged dimer, or prepared so as to generate monomeric receptor.

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.

Membrane-anchored plakoglobins have multiple mechanisms of action in Wnt signaling.

In Wnt signaling, beta-catenin and plakoglobin transduce signals to the nucleus through interactions with TCF-type transcription factors. However, when plakoglobin is artificially engineered to restrict it to the cytoplasm by fusion with the transmembrane domain of connexin (cnxPg), it efficiently induces a Wnt-like axis duplication phenotype in Xenopus. In Xenopus embryos, maternal XTCF3 normally represses ventral expression of the dorsalizing gene Siamois. Two models have been proposed to explain the Wnt-like activity of cnxPg: 1) that cnxPg inhibits the machinery involved in the turnover of cytosolic beta-catenin, which then accumulates and inhibits maternal XTCF3, and 2) that cnxPg directly acts to inhibit XTCF3 activity. To distinguish between these models, we created a series of N-terminal deletion mutations of cnxPg and examined their ability to induce an ectopic axis in Xenopus, activate a TCF-responsive reporter (OT), stabilize beta-catenin, and colocalize with components of the Wnt signaling pathway. cnxPg does not colocalize with the Wnt pathway component Dishevelled, but it does lead to the redistribution of APC and Axin, two proteins involved in the regulation of beta-catenin turnover. Expression of cnxPg increases levels of cytosolic beta-catenin; however, this effect does not completely explain its signaling activity. Although cnxPg and Wnt-1 stabilize beta-catenin to similar extents, cnxPg activates OT to 10- to 20-fold higher levels than Wnt-1. Moreover, although LEF1 and TCF4 synergize with beta-catenin and plakoglobin to activate OT, both suppress the signaling activity of cnxPg. In contrast, XTCF3 suppresses the signaling activity of both beta-catenin and cnxPg. Both exogenous XLEF1 and XTCF3 are sequestered in the cytoplasm of Xenopus cells by cnxPg. Based on these data, we conclude that, in addition to its effects on beta-catenin, cnxPg interacts with other components of the Wnt pathway, perhaps TCFs, and that these interactions contribute to its signaling activity.

A comparative evaluation of beta-catenin and plakoglobin signaling activity.

Vertebrates have two Armadillo-like proteins, beta-catenin and plakoglobin. Mutant forms of beta-catenin with oncogenic activity are found in many human tumors, but plakoglobin mutations are not commonly found. In fact, plakoglobin has been proposed to suppress tumorigenesis. To assess differences between beta-catenin and plakoglobin, we compared several of their biochemical properties. After transient transfection of 293T cells with an expression vector encoding either of the two proteins, soluble wild type beta-catenin does not significantly accumulate, whereas soluble wild type plakoglobin is readily detected. As anticipated, beta-catenin is stabilized by the oncogenic mutation S37A; however, the analogous mutation in plakoglobin (S28A) does not alter its half-life. S37A-beta-catenin activates a TCF/LEF-dependent reporter 20-fold more potently than wild type beta-catenin, and approximately 5-fold more potently than wild type or S28A plakoglobin. These differences may be attributable to an enhanced affinity of S37A beta-catenin for LEF1 and TCF4, as observed here by immunoprecipitation assays. We show that the carboxyl-terminal domain is largely responsible for the difference in signaling and that the Armadillo repeats account for the remainder of the difference. The relatively weak signaling by plakoglobin and the failure of the S28A mutation to enhance its stability, may explain why plakoglobin mutations are infrequent in malignancies.

Cadherins and catenins, Wnts and SOXs: embryonic patterning in Xenopus.

Wnt signaling plays a critical role in a wide range of developmental and oncogenic processes. Altered gene regulation by the canonical Wnt signaling pathway involves the cytoplasmic stabilization of beta-catenin, a protein critical to the assembly of cadherin-based cell-cell adherence junctions. In addition to binding to cadherins, beta-catenin also interacts with transcription factors of the TCF-subfamily of HMG box proteins and regulates their activity. The Xenopus embryo has proven to be a particularly powerful experimental system in which to study the role of Wnt signaling components in development and differentiation. We review this literature, focusing on the role of Wnt signaling and interacting components in establishing patterns within the early embryo.

Embryonic expression of Xenopus laevis SOX7.

SOX7, first described in Xenopus laevis by Shiozawa et al. [Biochim. Biophys. Acta 1309(1996)73], is a member, along with SOXs 17 and 18, of the F subgroup of SOX-type transcription factors. As part of a study of maternal SOX proteins that may modulate beta-catenin signaling, we isolated a XSOX7 cDNA from oocyte RNA and examined the pattern of XSOX7 expression during early development. While present maternally cell-type specific expression was first observed in the ciliated cells within the epidermis of early neurula stage embryos. As development proceeds, the pattern of XSOX7 expression becomes increasingly complex. XSOX7 is expressed in the aortic arch, the olfactory pit, the stomodeal depression, the procardiac tube, within cells of the developing embryonic vasculature, in the notochord, and within the hindbrain. XSOX7 expression continues within the hindbrain in 3-day old ( approximately stage 40) larvae. Given its widespread expression, XSOX7 is likely to be involved in a number of developmental processes.

Jaw muscle development as evidence for embryonic repatterning in direct-developing frogs.

The Puerto Rican direct-developing frog Eleutherodactylus coqui (Leptodactylidae) displays a novel mode of jaw muscle development for anuran amphibians. Unlike metamorphosing species, several larval-specific features never form in E. coqui; embryonic muscle primordia initially assume an abbreviated, mid-metamorphic configuration that is soon remodelled to form the adult morphology before hatching. Also lacking are both the distinct population of larval myofibres and the conspicuous, larval-to-adult myofibre turnover that are characteristic of muscle development in metamorphosing species. These modifications are part of a comprehensive alteration in embryonic cranial patterning that has accompanied life history evolution in this highly speciose lineage. Embryonic 'repatterning' in Eleutherodactylus may reflect underlying developmental mechanisms that mediate the integrated evolution of complex structures. Such mechanisms may also facilitate, in organisms with a primitively complex life cycle, the evolutionary dissociation of embryonic, larval, and adult features.

Plakophilin, armadillo repeats, and nuclear localization.

Plakophilins are armadillo-repeat containing proteins, identified through their localization to desmosomes. Expressed in a wide range of tissues, plakophilins are largely nuclear in most cell types [Schmidt et al. (1997) Cell Tissue Res 290:481; Mertens et al. (1996) J. Cell Biol 135:1009]. Using Xenopus embryos and cultured A6 cells, together with myc- and green fluorescent protein (GFP)-tags, we found that both the N-terminal, non-armadillo repeat "head" and the C-terminal armadillo repeat-containing regions can enter nuclei. The "arm" repeat domain is predominantly cytoplasmic and concentrated at the cell cortex, whereas the head and full-length polypeptides are concentrated in the nucleus. The head domain can also be seen to decorate and disrupt keratin filament network organization in some cells. In the course of these studies, we found that the distribution of the myc-epitope and green fluorescence differed in fixed cells, e.g., while the green fluorescence of a myc- and GFP-tagged head domain polypeptide was usually exclusively nuclear, a substantial fraction of the myc-immunoreactivity was cytoplasmic. Treating cells with the translation inhibitor cycloheximide reduces the cytoplasmic myc-signal, suggesting that it represented nascent polypeptides awaiting folding and nuclear import. Based on these types of experiments, GFP can be seen as a marker of the distribution of the mature form of the tagged polypeptide.

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.

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)

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.

Mitochondrial activity, embryogenesis, and the dialogue between the big and little brains of the cell.

While it is clear that mitochondria play integral roles in cellular homeostasis, adaptation, cellular and survival, recent studies suggest possible roles for mitochondria as modulators of what were previously considered cytoplasmic/nuclear signaling systems. Embryonic patterning has been linked to asymmetries in mitochondria-based respiratory activity. As outlined by Coffman (2009), defining the role of mitochondria as modulators of embryonic patterning is inherently difficult, given their essential metabolic roles. This review attempts to place mitochondrial-transcription factor interactions in the context of the early development of the tetrapod Xenopus laevis, where a number of the proteins and signaling systems known to play critical roles in embryonic patterning, e.g. ß-catenin, NF-κB, p53, and STAT3, have been found to localize to mitochondria.