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

Abstract

The in vitro polymerization and tissue-specific expression patterns of the four essential intermediate filament (IF) proteins (A1, A2, A3, B1) and the non-essential IF protein A4 were analyzed. Recombinant B1, used as a probe in blot overlay assays of the 11 Caenorhabditis elegans IF proteins, reacted strongly with proteins A1 to A4, indicating a heterotypic interaction. Obligate heteropolymeric filament assembly in vitro was confirmed by electron microscopy. Protein B1 formed long IF when mixed with an equimolar amount of A1, A2 or A3. Developmentally regulated coexpression of B1 and one or more members of the A family was found with GFP-promoter reporters. This coexpression pattern argues for a heteropolymer system in vivo. One or both splice variants of the B1 gene are always coexpressed in a tissue-specific manner with at least one member of the A family in hypodermis, pharynx, pharyngeal-intestinal valve, excretory cells, uterus, vulva and rectum. Interestingly, while the intestine normally lacks a B1/A pair, the dauer larva shows intestinal B1 and A4. These results are in line with similar postembryonic phenotypes of the hypodermis induced by RNA interference (RNAi) of genes B1, A2 and A3. Similarly, defects of the pharynx and its A1-GFP containing tonofilaments observed in the postembryonic B1 RNAi phenotype are consistent with the coexpression of B1 and A1 in the marginal cells. Thus RNAi analyses provide independent evidence for the existence of the B1/A obligate heteropolymer system in vivo. Proteins A1 and B1 have a similar and rather slow turnover rate in photobleaching experiments of the pharynx tonofilaments.

The making of hemidesmosome structures in vivo

“…They appear as electron-dense membrane plaques and are associated with C. elegans intermediate filaments IFA-2/MUA-6, IFA-3, and IFB-1 (Francis and Waterston, 1991;Bosher et al, 2003;Woo et al, 2004). C. elegans intermediate filaments are more closely related to nuclear lamins (Dodemont et al, 1994), yet can form IFB-1/IFA-2 and IFB-1/IFA-3 heterodimers (Karabinos et al, 2003). Fibrous organelles contain the sole plectin and BPAG1 homologue in the C. elegans genome, VAB-10A, which is generated by alternative splicing from the unique spectraplakin locus vab-10 and should thus play the combined role of both plakins in vertebrate hemidesmosomes (Table 1) (Bosher et al, 2003).…”

Intermediate filaments in Caenorhabditis elegans

“…Bars, 100 lm in (A); 10 lm in (A 0 ). also Karabinos et al, 2003]. In addition, the depicted worm synthesizes another fluorescent IF chimera which exhibits a very different distribution: the fluorescent proteins are present primarily in the subapical intestinal endotube where they are part of a dense filamentous network and accumulate to a lesser degree in the intestinal cytoplasm as granular structures [false pink color in Fig.…”

“…The first group is characterized by the obligatory presence of IFB-1 which forms heteropolymers with any IFA and is therefore co-expressed with different IFAs in various cell types [Karabinos et al, 2003; Table I]. IFA-1A is particularly abundant in the marginal cells of the pharynx where it is part of thick filament bundles that traverse the cells longitudinally from top to bottom (Fig.…”

“…We think that this and the fact that all IFCs, IFDs as well as IFP-1 and IFB-2 are present in the intestinal apical terminal web justify a common grouping. The IF-rich terminal web is a characteristic feature of the subapical region of adluminal metazoan intestinal cells [Hirokawa et al, 1982] consisting of a complex mesh of various cytoskeletal and [Karabinos et al, 2003] Early larval Essential for viability after L1 [Karabinos et al, 2001] IFA-1b: amphids, c some neurons, c pharyngeal-intestinal valve, c vulva, c rectum c [Karabinos et al, 2001[Karabinos et al, , 2003] IFA-2 (5 MUA-6) Hypodermis, b,c amphids, c uterus, b,c vulva, c rectum c [Hapiak et al, 2003;Karabinos et al, 2002Karabinos et al, , 2003 Early larval Hypodermal integrity, attachment of muscles to the body wall [Karabinos et al, 2001;Hapiak et al, 2003] IFA-3…”

“…Hypodermins (only embryonic and larval), c ventral nerve cord (only larval), c rectum c [Karabinos et al, 2001[Karabinos et al, , 2003 cytoskeleton-associated polypeptides [e.g., Ku et al, 1999]. Whether the intestinal C. elegans IF polypeptides also form heteropolymers has not been investigated.…”