Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis.

Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm-repeat protein family. They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they occur in the nuclei of cells and, in epithelial cells, at the plasma membrane within the desmosomal plaques. We have shown by solid phase-binding assays that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epithelial cells and, to some extent, to vimentin. In line with this we show that recombinant PKP1a binds strongly to IFs assembled in vitro from CKs 8/18, 5/14, vimentin or desmin and integrates them into thick (up to 120 nm in diameter) IF bundles extending for several microm. The basic amino-terminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation experiments. In particular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio. In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by co-immunoprecipitation and sucrose density fractionation. The significance of these interactions of PKP1a with IF proteins on the one hand and desmoplakin on the other is discussed in relation to the fact that PKP1a is not bound - and does not bind - to extended IFs in vivo. We postulate that (1) effective cellular regulatory mechanisms exist that prevent plakophilins from unscheduled IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recruitment of plakophilins to the desmosomal plaques.

The intermediate filament protein consensus motif of helix 2B: its atomic structure and contribution to assembly.

Nearly all intermediate filament proteins exhibit a highly conserved amino acid motif (YRKLLEGEE) at the C-terminal end of their central alpha-helical rod domain. We have analyzed its contribution to the various stages of assembly by using truncated forms of Xenopus vimentin and mouse desmin, VimIAT and DesIAT, which terminate exactly before this motif, by comparing them with the wild-type and tailless proteins. It is surprising that in buffers of low ionic strength and high pH where the full-length proteins form tetramers, both VimIAT and DesIAT associated into various high molecular weight complexes. After initiation of assembly, both VimIAT and DesIAT aggregated into unit-length-type filaments, which rapidly longitudinally annealed to yield filaments of around 20 nm in diameter. Mass measurements by scanning transmission electron microscopy revealed that both VimIAT and DesIAT filaments contained considerably more subunits per cross-section than standard intermediate filaments. This indicated that the YRKLLEGEE-motif is crucial for the formation of authentic tetrameric complexes and also for the control of filament width, rather than elongation, during assembly. To determine the structure of the YRKLLEGEE domain, we grew crystals of peptides containing the last 28 amino acid residues of coil 2B, chimerically fused at its amino-terminal end to the 31 amino acid-long leucine zipper domain of the yeast transcription factor GCN4 to facilitate appropriate coiled-coil formation. The atomic structure shows that starting from Tyr400 the two helices gradually separate and that the coiled coil terminates with residue Glu405 while the downstream residues fold away from the coiled-coil axis.

Characterization of distinct early assembly units of different intermediate filament proteins.

We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from the four cytoplasmic sequence homology classes, and compared these values with those of the corresponding mature filaments. After two seconds under standard assembly conditions (i.e. 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 37 degrees C), vimentin, desmin and the neurofilament triplet protein NF-L aggregated into similar types of "unit-length filaments" (ULFs), whereas cytokeratins (CKs) 8/18 already yielded long IFs at this time point, so the ionic strength had to be reduced. The number of molecules per filament cross-section, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 at two seconds compared to 16 and 21 at one hour. NF-L exhibited corresponding values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, with major peak values of 32 and 45 at two seconds and 30, 37 and 44 after one hour. Desmin formed filaments of distinctly higher mass with 47 molecules per cross-section, at two seconds and after one hour of assembly. This indicates that individual types of IF proteins generate filaments with distinctly different numbers of molecules per cross-section. Also, the observed significant reduction of apparent filament diameter of ULFs compared to the corresponding mature IFs is the result of a "conservative" radial compaction-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subunits per cross-section. Moreover, the MPL composition of filaments is strikingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significantly increased number of molecules per cross-section (56 and 84) compared to assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature "pathological" ULFs form that are arrested in this state, indicating that the system is forced into strong but unproductive interactions between subunits. Similar "dead-end" structures were obtained with vimentins mutated to introduce principal alterations in subdomains presumed to be of general structural importance, indicating that these sequence changes led to new modes of intermolecular interactions.

Structure and assembly properties of the intermediate filament protein vimentin: the role of its head, rod and tail domains.

We have investigated the functional role of the non-helical end domains of vimentin on its assembly properties using truncated Xenopus and human recombinant proteins. Removal of the amino-terminal "head" domain yielded a molecule that did not assemble into 10 nm filaments but remained in a soluble oligomeric particle form with a sedimentation coefficient considerably smaller than that of wild-type vimentin (Vim(wt)). In contrast, removal of the carboxy-terminal "tail" domain had no obvious effect on the sedimentation characteristics. In particular, sedimentation equilibrium analysis under low ionic strength conditions yielded oligomeric particle species of Mr 135,000 to 360,000, indistinguishable from those obtained with Vim(wt). When induced to form filaments from this state by rapid dilution into filament forming buffer, Vim(wt) and Vim(deltaT) protein generated similar viscosity profiles. However, as determined by scanning transmission electron microscopy, under these conditions Vim(deltaT) formed filaments of heterogeneous diameter, corresponding to various distinct mass-per-length (MPL) values: whereas Vim(wt) yielded MPL values peaking between 40 and 45 kDa/nm, Vim(deltaT) filaments produced histograms which could be fitted by three Gaussian curves peaking between 37 and 131 kDa/nm. In contrast, when dialyzed against, instead of being rapidly diluted into, filament forming buffer, Vim(deltaT) gave histograms with one major peak at about 54 kDa/nm. The MPL heterogeneity observed for Vim(deltaT) was already evident at the earliest stages of assembly. For example, ten seconds after initiation, "unit-length" filament segments (58 to 63 nm) were formed with both wt and deltaT proteins, but the diameters were considerably larger for Vim(deltaT) compared to Vim(wt) (20(+/- 3) nm versus 16(+/- 3)nm), indicating a distinct role of the carboxy-terminal tail domain in the width control during unit-length filament formation. Despite this difference both Vim(deltaT) and Vim(wt) filaments appeared to grow stepwise in a modular fashion from such unit-length filament segments. This suggests that assembly occurred by a principally similar mechanism involving the end-on-fusion or annealing of unit-length filaments.

Vimentin in a cold-water fish, the rainbow trout: highly conserved primary structure but unique assembly properties.

We have isolated from a rainbow trout (Oncorhynchus mykiss) spleen cDNA library a clone coding for vimentin. The deduced amino acid sequence reveals a high degree of identity with vimentin from carp (81%), frog (71%), chick and human (73% each). Large stretches in the central alpha-helical rod are identical within all four classes of vertebrates, but in 17 residues spread over the entire rod, the two fish differ distinctly from the tetrapod species. In addition, in the more diverged non-helical head domain, a nonapeptide motif previously shown to be important for regular filament formation is conserved. Recombinant trout vimentin assembles into bona fide filaments in vitro, with a temperature optimum between 18 and 24 degrees C. Above 27 degrees C, however, filament assembly is abruptly abolished and short filaments with thickened ends as well as structures without typical intermediate filament appearance are formed. This distinguishes its assembly properties significantly from amphibian, avian and mammalian vimentin. Also in vivo, after cDNA transfection into vimentin-free mammalian epithelial cells, trout vimentin does not form typical intermediate filament arrays at 37 degrees C. At 28 degrees C, and even more pronounced at 22 degrees C, the vimentin-positive material in the transfected cells is reorganized in the perinuclear region with a partial fibrillar appearance, but typical intermediate filament arrays are not formed. Together with immunoblotting and immunolocalization data from trout tissues, where vimentin is predominantly found in glial and white blood cells, we conclude that vimentin is indeed important in its filamentous form in fish and other vertebrates, possibly fulfilling cellular functions not directly evident in gene targeting experiments carried out in mice.

Temperature-sensitive intermediate filament assembly. Alternative structures of Xenopus laevis vimentin in vitro and in vivo.

In assembly assays of intermediate filaments (IFs) from vimentin of the amphibian species Xenopus laevis we have observed the formation of so far unknown structures at temperatures above 28 degrees C. Upon assembly in vitro at temperatures above 34 degrees C massive aggregates, partly with a protofilamentous substructure, were found and their formation correlated with drastically reduced end-viscosity. Large spheroidal, dense aggregates with a complex suborganization were also seen to form at 37 degrees C in the cytoplasm of living mammalian cells devoid of endogenous vimentin upon transfection with cDNA encoding the amphibian vimentin, and this was also true for vimentin forced to accumulate in the nucleoplasm by the introduction of a "nuclear localization signal". Upon shift from the non-permissive (37 degrees C) to the permissive (28 degrees C) temperature, such aggregates of non-IF vimentin structures gradually disappeared and a normal-looking IF meshwork formed. The results, which are discussed in relation to other structures assembled by IF proteins, indicate a marked thermosensitivity in the amino acid sequence of the vimentin which seems to have been reduced during evolution of warm-blooded animals. They further show that members of the multigene gene family of IF proteins can occur in structures totally different from IFs.