Characterization of distinct early assembly units of different intermediate filament proteins¹

Abstract

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 °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.

Introduction

Intermediate filaments (IFs) owe their name the observation that by electron microscopy their apparent diameter was intermediate in size to that of microfilaments (6 nm) and microtubules (25 nm) Ishikawa et al 1968, Hynes and Destree 1978. Further studies revealed that different tissues harbored IFs with slightly different diameters, for example 8 nm in keratinocytes and glial cells, whereas neurofilaments as well as desmin and vimentin filaments were found to be approximately 10 nm in diameter (cf. Lazarides, 1982). The human IF multigene family by now contains more than 50 members, more than two-thirds are cytokeratins (Fuchs & Weber, 1994). It has further been shown that this family is evolutionarily very old, with complex expression patterns found in both protostomic and deuterostomic organisms, e.g. the nematode Caenorhabditis elegans, synthesizes at least eight IF proteins (Dodemont et al., 1994).

Apart from their diameter, IFs differ from the other two principal cellular filament systems, the microfilaments (MFs) and microtubules (MTs), by several important aspects. They are insoluble in buffers of high ionic strength (e.g. 0.6 M KI), which dissolve MFs and MTs Starger et al 1978, Franke et al 1978, Geisler and Weber 1980. Their fibrous subunits form extended dimeric coiled-coils of more than 40 nm length, whereas both actin and tubulin are globular proteins (cf. Schliwa, 1986). Moreover, unlike MFs and MTs, IFs are non-polar structures, since their dimeric subunits are most probably oriented in an antiparallel fashion (for reviews, see Geisler and Weber 1986, Aebi et al 1988, Parry and Steinert 1995). IFs differ significantly from MFs and MTs with regard to their viscoelastic properties, their flexible nature allowing them to withstand mechanical stress without breakage (Janmey et al., 1998). Moreover, following denaturation with high concentrations (6–8 M) of urea, IF proteins can be renatured and induced to assemble either by direct dialysis into physiological buffers or alternatively, by a two-step procedure, first dialyzing the urea-dissolved proteins into low-ionic strength/high pH buffers, thereby generating tetramers mainly, and then installing physiological conditions either by dialysis or by rapid dilution into filament buffer (cf. Herrmann et al 1996b, Herrmann and Aebi 1998a, Herrmann and Aebi 1998b).

Full-width IFs have been frequently described to exhibit diameters quite different from 10 nm, ranging between 7 and 14 nm (cf. Franke & Kartenbeck, 1993). This variation may in part be caused by the specimen preparation steps, while there may be intrinsic parameters that cause this variation: (i) in vivo tightly bound IF-associated proteins (IFAPs) such as plectin (Wiche et al., 1982) and epinemin (Lawson, 1983), or non-selfassembling IF proteins that constitutively integrate into the vimentin or desmin system such as paranemin (Hemken et al., 1997) may contribute to the apparent diameter; (ii) different types of IFs may indeed form in a particular and unique way, for example neurofilaments or beaded chain filaments (cf. Heins et al 1993, Goulielmos et al 1996), both kinds of IFs being assembled from several distinct subunit proteins, so that this heteropolymer situation may have indeed led to new organizational principles (for reviews, see Heins et al 1993, Georgatos et al 1997); (iii) in vitro assembly of individual IF proteins may critically depend on the conditions employed, e.g. dialysis versus salt addition procedures, and the exact ionic strength and pH conditions. We have recently shown that human vimentin IFs assembled by dialysis are remarkably uniform for several micrometers with regard to their mass-per-length (MPL) ratio, whereas those initiated to assemble by rapid dilution with buffered salt solutions exhibit extensive mass variation along one and the same filament (Herrmann et al., 1996b). This property indicates that IFs are indeed polymorphic structures, thus allowing elongation to occur with segments containing different numbers of molecules. This variable number of polypeptides along one and the same IF, in turn, is reminiscent of the observation by Ngai et al. (1990) that the addition of newly synthesized subunits could occur along the entire length of preexisting vimentin IFs. However, the mass-per-length was not determined in that study, so it cannot readily be decided if the small subunits integrated into the filament were added to or replaced endogenous subunits.

Performing a kinetic-type assembly procedure, we have recently demonstrated that vimentin IF assembly can be described as a three-phase process: (i) rapid (in the sub-second range) lateral aggregation of tetramers into “unit-length” filaments (ULFs) which we believe occurs by complex molecular associations; (ii) annealing of these ULFs to “immature” 16 nm diameter filaments; (iii) compaction into mature 11 nm diameter IFs (Herrmann et al., 1996b). Although a priori we could not exclude that the last step was caused by a mass loss rather than a compaction-type reorganization event, inspection of IFs in the process of reducing their diameter suggested the latter. Using quantitative scanning transmission electron microscopy (STEM), we have now performed mass determination of ULFs formed by members of all four classes of cytoplasmic IF proteins demonstrating that they all form ULF-type structures harboring the same mass-per-length as fully matured IFs of their kind. These experiments further revealed that different IF proteins assemble into IFs with distinct numbers of subunits per cross-section. Moreover, we document that desmin, irrespective of its assembly mode and origin, recombinant or authentic, forms comparatively heterogeneous IFs with major mass-per-length values ranging between 50 and 80 kDa/nm, very different from the MPL values obtained for vimentin, keratins or the low molecular mass neurofilament triplet protein (NF-L). This latter finding strongly indicates that this polymorphism of in vitro assembled IFs exhibits intrinsic specific properties of different IF proteins and may also be of considerable physiological relevance.