Journal of Molecular Biology
In Vivo and in Vitro Evidence that the Four Essential Intermediate Filament (IF) Proteins A1, A2, A3 and B1 of the Nematode Caenorhabditis elegans Form an Obligate Heteropolymeric IF System
Introduction
Intermediate filament (IF) proteins are important components of the cytoskeleton in many metazoa. Man has more than 65 different IF genes.1 All IF proteins show a common tripartite structure. Variable head and tail domains flank the central α-helical rod domain, which can form a parallel double-stranded coiled coil. On the basis of sequence similarities, biochemical properties, tissue-specific expression patterns and the organization of the corresponding genes, five major subfamilies are identified. Human type I and II proteins are, with 25 members each, by far the largest subfamilies. They encode the keratins I and II, which give rise via a hetero coiled coil to the obligate heteropolymeric keratin filaments of epithelia and epidermal appendages. The four type III proteins can form, at least in part, homopolymeric IF. The seven genes coding for type IV proteins show a unique intron pattern totally unrelated to that of type I to III genes. Finally, type V covers the nuclear lamins.2., 3., 4.
Point mutations in many IF genes are connected to human diseases. Mutations in at least 14 epidermal keratin genes cause various fragility syndromes of the skin,5 and indicate that one function of IF is connected with cellular resistance to mechanical stress. Similar mutations in the type III genes desmin and GFAP are associated with myopathies and Alexander's disease, respectively.6., 7. Ablation of murine genes gives sometimes corresponding results but often only mild phenotypes are observed.8
There are two prototype sequences of cytoplasmic IF proteins that parallel metazoan phylogeny. Compared with nuclear lamins, the first prototype, originally defined in the vertebrates, lacks 42 residues (six heptads) in the coil 1b subdomain of the central rod. It accounts for the currently known 13 sequences of the cephalochordate Branchiostoma9 and the five sequences of the urochordate Ciona.10., 11. The early chordates have orthologs of type I to III proteins and some IF proteins without an obvious vertebrate counterpart. Neurofilament type IV proteins emerged only with the vertebrate lineage. Thus, the first sequence prototype is specific for the chordates.
The second prototype of cytoplasmic IF proteins covers all sequences established from 12 different protostomic phyla.12., 13. Here, the coil 1b subdomain has always the same length as in nuclear lamins and the tail domain displays often a lamin homology segment. The nematode C. elegans has a single nuclear lamin, which is an essential protein of the early embryo,14., 15. and 11 genes encoding cytoplasmic IF proteins. Using RNA interference (RNAi) with microinjected double-stranded RNA (dsRNA) on all 11 genes, we identified four genes essential for nematode development (A1, A2, A3 and B1) and a mild adult phenotype for one gene (C2).16 The phenotypes for the IF proteins A2 and A3 indicate an essential role of the hypodermal IF in the transmission of muscle force to the cuticle and the maintenance of the correct hypodermis/muscle relationship in development.17., 18., 19., 20.
Using GFP-promoter reporters, we have analyzed the expression pattern of the four essential IF genes as well as of the A4 gene during C. elegans development. A strict coexpression of B1 and at least one member of the A1 to A4 group is observed. This coexpression pattern relates to the in vitro polymerization properties of the proteins. In gel overlays of all 11 recombinant IF proteins, B1 strongly decorates only A1 to A4, while filament assembly studies show that B1 gives rise to obligate heteropolymeric IF when mixed with equal molar amounts of A1, A2 or A3. Using B1 RNAi by feeding21 on wild-type as well as on transgenic worms expressing the A1-GFP protein we observed a postembryonic phenotype similar to that found earlier for A2 and A3. This result fits the hypodermal coexpression of B1 with A2 and A3. The postembryonic B1 RNAi phenotype obtained by feeding shows additional defects of the A1-GFP-containing tonofilament bundles in marginal cells in line with the coexpression of B1 and A1 in the pharynx.
Section snippets
Expression patterns of A1a, B1a and A4 promoter/gfp reporters
The four genes A1 to A4 and the two genes B1 and B2 form the A and B protein subgroups in the collection of the 11 C. elegans IF proteins (Figure 1A and B).16 In addition, the A1 and the B1 genes each produce two alternative splice variants designated A1a, A1b and B1a, B1b (Figure 1A).16., 22. The protein A1b (F38B2.1a) differs from the shorter splice variant A1a (F38B2.1b) by an additional 25 residues on the N-terminal end that are encoded by the separate A1b-specific exon (Figure 1C).
Discussion
C. elegans has 11 genes encoding cytoplasmic IF proteins. Using RNA interference with microinjection on all 11 genes, we identified four genes (A1, A2, A3, B1) as essential for nematode development.16 Here, we have defined the expression pattern of these genes during development using GFP-promoter reporters and have analyzed the in vitro assembly properties of the corresponding recombinant proteins. A strict coexpression pattern of B1 and the four members of the A subfamily (A1 to A4) is seen.
Nucleic acid techniques
C. elegans strain N2 Bristol was cultured and harvested using standard procedures.28 The full-length cDNAs of the A1b (F38B2.1a) and A3 (F52E10.5) IF proteins22 were amplified by PCR from the cDNA library as described.16 The following primers were used:
A1b sense 5′-CAATCAACATTTCAAAACATATGGAGATTACCAGAGAAAG-3′, antisense 5′-GTTAATTGTTGGGAAGAAGCTTGATTAGGCAGTGCTC-3′; A3 sense 5′- CCATGGCCGATCCAGATTCCTACCGCAGCTC-3′, antisense 5′-GATTTATGAAGTTGTAG-TCTGTTGGCTTTGGCG-3′.
The A1b and A3 PCR fragments were
Acknowledgements
We thank Uwe Plessmann and Wolfgang Berning-Koch for expert technical assistance. This work was supported, in part, by the Deutsche Forschungsgemeinschaft grant SCHU 1033/3-4 to E.S.
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