Eckhard Kaufmann

Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins.

Mammalian neurofilament triplet proteins (68 K, 160 K and 200 K) have been correlated by a biochemical, immunological and protein chemical study. The 160 K and 200 K triplet proteins are intermediate filament proteins in their own right, since they reveal the alpha‐helical coiled‐coil rod domain analyzed in detail for the 68 K protein. Triplet proteins display two distinct arrays. Their amino‐terminal region built analogously to non‐neuronal intermediate filament proteins should allow a co‐polymerization process via the interaction of coiled‐coil domains. The extra mass of all triplet proteins is allocated to carboxy‐terminally located extensions of increasing size and unique amino acid sequences. These may provide highly charged scaffolds suitable for interactions with other neuronal components. Such a domain of 68 K reveals, in sequence analysis, 47 glutamic acids within 106 residues. The epitope recognized by a monoclonal antibody reacting probably with all intermediate filament proteins has been mapped. It is located within the last 20 residues of the rods, where six distinct intermediate filament proteins point to a consensus sequence.

Proteinchemical characterization of three structurally distinct domains along the protofilament unit of desmin 10 nm filaments

Limited chymotryptic cleavage of soluble chicken gizzard desmin protofilaments allows the characterization of three structurally distinct domains. A surface-exposed very basic amino-terminal region (the headpiece) with an amino acid sequence excluding alpha-helical organization (7.5 kd) is separated from the perhaps globular carboxy-terminal 48 residues (the tailpiece) by a distinctly different middle domain of approximately 330 residues. This 38 kd domain is very rich in alpha-helix (at least 83%), and electron microscopy reveals a thin rod with a length of 500 +/- 50 A. Amino acid sequence data also show that the rod domain is interrupted by a nonhelical portion. An alpha-helical array is able to form a coiled-coil spanning the carboxy-terminal half of the 38 kd domain. The alpha-type diffraction pattern of 10 nm filaments arises from a coiled-coil conformation displayed through most but not all of the middle domain of the protofilaments.

SDS—PAGE strongly overestimates the molecular masses of the neurofilament proteins

Direct molecular mass determination of the three porcine neurofilament proteins (H, M and L) was performed in 6 M guanidine—HCl using analytical gel filtration and sedimentation equilibrium centrifugation. The results show that SDS—PAGE strongly overestimates the values of the ‘higher molecular mass’ components H and M. This discrepancy stems from the carboxyterminal extensions known to have unusual amino acid composition.

Intermediate filament forming ability of desmin derivatives lacking either the amino-terminal 67 or the carboxy-terminal 27 residues☆

Amino acid sequence data and results from limited proteolytic digestion have been used to define the three-domain structure of intermediate filament proteins. A centrally located highly alpha-helical domain of about 310 residues well-conserved in sequence principles and length is flanked by the highly variable sequences of the non-alpha-helical headpiece and tailpiece. A direct involvement in filament formation of one or both terminal domains was previously proposed for desmin since chymotryptic removal of head and tailpiece provided a derivative unable to form filaments. In order to evaluate directly the importance of these regions we have prepared desmin derivatives lacking either the amino-terminal 67 (T-desmin) or carboxy-terminal 27 residues (L-desmin). Whereas the latter derivative is fully polymerization-competent the fragment lacking only the basic and arginine-rich headpiece cannot form filaments on its own and remains in a protofilamentous stage. These structures of T-desmin are not incorporated into filaments when mixed with protofilaments of desmin. If, however, the two proteins are mixed in 7 M-urea subsequent dialysis provides morphologically normal filaments containing T-desmin. The results suggest that at least certain hybrid protofilaments containing less than four headpieces are accepted in the filament. The removal of the 27 carboxy-terminal residues in L-desmin, although not interfering with filament formation, leads to a change in surface since filaments show lateral aggregation at 170 mM but not at 50 mM salt. The results are discussed in relation to current models of intermediate filament structure.

Antiparallel orientation of the two double-stranded coiled-coils in the tetrameric protofilament unit of intermediate filaments☆

The chymotryptically excised middle domain of desmin slightly exceeds in length the structurally conserved alpha-helical middle region documented in all intermediate filament proteins by amino acid sequence data. This rod domain is a protofilament derivative with a tetrameric organization, thus indicating the presence of two double-stranded coiled-coil units. We now show by immunoelectron microscopy that Fab fragments of a desmin-specific monoclonal antibody mixed with the rod lead to dumb-bell-shaped structures. The tagging of both ends together with the length of the rod (48 nm) argues for an antiparallel orientation of the two coiled-coils without a major stagger. This information combined with the lateral 21 nm periodicity of the intermediate filament observed by us and others leads to a structural hypothesis similar to those entertained from X-ray data on wool alpha-keratins, although here an antiparallel tetrameric unit of some 60 to 66 nm is invoked, which has never been isolated. The structure that we discuss allows for the existence of both the particles, and the antibody experiment strongly supports the antiparallel orientation postulated in both approaches. The tube-like filament structure proposed for the intermediate filament agrees with recent mass per unit length measurements and allows for two minor classes of intermediate filaments with different values in this property as also found experimentally.

The Drosophila fork head domain protein crocodile is required for the establishment of head structures.

The fork head (fkh) domain defines the DNA‐binding region of a family of transcription factors which has been implicated in regulating cell fate decisions across species lines. We have cloned and molecularly characterized the crocodile (croc) gene which encodes a new family member from Drosophila. croc is expressed in the head anlagen of the blastoderm embryo under the control of the anterior, the dorsoventral and the terminal maternal organizer systems. The croc mutant phenotype indicates that the croc wild‐type gene is required to function as an early patterning gene in the anterior‐most blastoderm head segment anlage and for the establishment of a specific head skeletal structure that derives from the non‐adjacent intercalary segment at a later stage of embryogenesis. As an early patterning gene, croc exerts unusual properties which do not allow it to be grouped among the established segmentation genes. A single‐site mutation within the croc fkh domain, which causes a replacement of the first out of four conserved amino acid residues thought to be involved in the coordinate binding of Mg2+, abolishes the DNA binding of the protein in vitro. In view of the resulting lack‐of‐function mutant phenotype, it appears likely that metal binding by the affected region of the fkh domain is crucial for proper folding of the DNA‐binding structure.

The POU Factor Oct-25 Regulates the Xvent-2B Gene and Counteracts Terminal Differentiation in Xenopus Embryos

The Xvent-2B promoter is regulated by a BMP-2/4-induced transcription complex comprising Smad signal transducers and specific transcription factors. Using a yeast one-hybrid screen we have found that Oct-25, a Xenopus POU domain protein related to mammalian Oct-3/4, binds as an additional factor to the Xvent-2B promoter. This interaction was further confirmed by both in vitro and in vivo analyses. The Oct-25 gene is mainly transcribed during blastula and gastrula stages in the newly forming ectodermal and mesodermal germ layers. Luciferase reporter gene assay demonstrated that Oct-25 stimulates transcription of the Xvent-2B gene. This stimulation depends on the Oct-25 binding site and the bone morphogenetic protein-responsive element. Furthermore, Oct-25 interacts in vitro with components of the Xvent-2B transcription complex, like Smad1/4 and Xvent-2. Overexpression of Oct-25 results in anterior/posterior truncations and lack of differentiation for neuroectoderm- and mesoderm-derived tissues including blood cells. This effect is consistent with an evolutionarily conserved role of class V POU factors in the maintenance of an undifferentiated cell state. In Xenopus, the molecular mechanism underlying this process might be coupled to the expression of Xvent proteins.

Xvent-1 mediates BMP-4-induced suppression of the dorsal-lip-specific early response gene XFD-1' in Xenopus embryos.

Ectopic expression of the ventralizing morphogen BMP‐4 (bone morphogenetic protein‐4) in the dorsal lip (Spemann organizer) of Xenopus embryos blocks transcription of dorsal‐lip‐specific early response genes. We investigated the molecular mechanism underlying the BMP‐4‐induced inhibition of the fork head gene XFD‐1′. The promoter of this gene contains a BMP‐triggered inhibitory element (BIE) which prevents activation of this gene at the ventral/vegetal side of the embryo in vivo. In the present study, we show that BMP‐4‐induced inhibition is not direct but indirect, and is mediated by Xvent homeobox proteins. Micro‐injections of Xvent‐1 RNA and XFD‐1′ promoter deletion mutants demonstrate that Xvent‐1 mimics the effect of BMP‐4 signalling not only by suppression of the XFD‐1′ gene, but also by utilizing the BIE. Suppression could be reverted using a dominant‐negative Xvent‐1 mutant. The repressor domain was localized to the N‐terminal region of the protein. Gel‐shift and footprint analyses prove that Xvent‐1 binds to the BIE. Moreover, PCR‐based target‐site selection for the Xvent‐1 homeodomain confirms distinct motifs within the BIE as preferential binding sites. Thus, biological and molecular data suggest that Xvent‐1 acts as direct repressor for XFD‐1′ transcription and mediates BMP‐4‐induced inhibition.

The homeodomain transcription factor Xvent-2 mediates autocatalytic regulation of BMP-4 expression in Xenopus embryos.

Like other genes of the transforming growth factor-β family, the BMP-4 gene is regulated by an autocatalytic loop. In Xenopus embryos this loop can be ectopically induced by injection of BMP-2 RNA. However, cycloheximide treatment subsequent to BMP-2 overexpression revealed that BMP signaling is not direct but requires additional factor(s). As putative mediator we have identified Xvent-2 which is activated by BMP-2/4 signaling and, in turn, activates BMP-4 transcription. Using promoter/reporter assays we have delineated Xvent-2 responsive elements within the BMP-4 gene. We further demonstrate that Xvent-2 which has recently been characterized as a transcriptional repressor can also act, context dependent, as an activator binding two copies of a 5′-CTAATT-3′ motif in the second intron of theBMP-4 gene. Replacement of Xvent-2 target sites within thegoosecoid (gsc) promoter by theBMP-4 enhancer converts Xvent-2 caused repression ofgsc to strong activation. This switch is obviously due to adjacent nucleotides probably binding a transcriptional co-activator interacting with Xvent-2. A model is presented describing the mechanism of BMP-4 gene activation in Xenopus embryos at the early gastrula stage.

Antagonistic actions of activin A and BMP-2/4 control dorsal lip-specific activation of the early response gene XFD-1' in Xenopus laevis embryos.

Transcription of the early response gene XFD‐1′ (XFKH1) in the dorsal lip (Spemann organizer) of Xenopus embryos is activated by dorsal mesoderm inducing factors. Promoter studies revealed the presence of an activin A response element (ARE) which is both necessary and sufficient for transcriptional activation of reporter genes in animal cap explants incubated with activin A. Surprisingly, this ARE is also active within vegetal explants in the absence of exogenously added inducers, but an additional inhibitory response element prevents transcription of the XFD‐1′ gene in the ventral/vegetal region of the embryo in vivo. This element is located upstream of the ARE, it responds to bone morphogenic proteins 2 and 4 (BMP‐2/4) triggered signals and it overrides the activating properties of the ARE. Expression patterns of BMP‐2 and BMP‐4 in the late blastula stage embryo and, especially, their absence from the dorsal blastopore lip may thus control the spatial transcription of the XFD‐1′ gene. Accordingly, the temporal activation and the spatial restriction of XFD‐1′ gene activity to the Spemann organizer is regulated by antagonistic actions of two distinct members of the TGF‐beta family (activin and BMP) which act on different promoter elements.