Analysis of the novel excretory cell expressed ECP-1 protein and its proposed ECP-1/IFC-2 fusion protein EXC-2 in the nematode Caenorhabditis elegans
Introduction
Intermediate filament (IF) proteins are important components of the cytoskeleton in many metazoan cells. All IF proteins show a common tripartite structure comprising variable head and tail domains flanking the central alpha-helical rod domain, which can form a parallel, double-stranded coiled coil. Five major subfamilies of IF have been identified in vertebrates. The largest encode the type I and type II cytoplasmic keratins, which give rise via a heterodimeric coiled coil to the obligate heteropolymeric keratin filaments of epithelia and epidermal appendages. The type III IF subfamily includes desmin, vimentin and glial fibrillary acidic proteins, which can form, at least in part, homopolymeric IFs. The type IV IF subfamily is represented, for example, by neurofilament chains and α-internexin, while the type V IF proteins are the nuclear lamins. It is assumed that lamins represent an ancestral sequence of cytoplasmic IFs (for reviews see Fuchs and Weber, 1994; Parry and Steinert, 1995; Erber et al., 1998; Herrmann et al., 2009; Peter and Stick, 2015).
The primary (but not sole) function of the cytoplasmic IFs is to provide resistance against mechanical stress. This view is supported by a variety of epidermal fragility syndromes of mutated human keratins (Irvine and McLean, 1999), by knock-out experiments in mice (i.e. Hesse et al., 2000; Vijayaraj et al., 2009), as well as by (reverse) genetics (RNAi) in Caenorhabditis elegans (C. elegans; Karabinos et al., 2001, 2003, 2004; Hapiak et al., 2003; Woo et al., 2004; Hüsken et al., 2008; Zhang et al., 2011; Geisler et al., 2016).
The nematode C. elegans has a single and essential nuclear lamin (LMN-1; Liu et al., 2000; Fraser et al., 2000), and eleven cytoplasmic IF genes (ifa-1 to ifa-4, ifb-1, ifb-2, ifc-1, ifc-2, ifd-1, ifd-2, ifp-1), some of which give rise to alternatively spliced variants (Dodemont et al., 1994; Karabinos et al., 2001, 2003; Woo et al., 2004). Interestingly, the C. elegans IF proteins IFA-1 and IFB-1 represent a basic heteropolymeric IF cytoskeleton in all investigated nematode clades, in contrast to the remaining nine C. elegans IF sequences restricted to the clade III-V (IFA-2, IFA-4) and V (IFB-2) taxa, or even to the Caenorhabditis genus (IFA-3, IFC-1, IFC-2, IFD-1, IFD-2, IFP-1; Karabinos, 2019). Using RNAi on all eleven genes, we identified that the ifa-1, ifa-2, ifa-3 and ifb-1 genes, which form an obligate heteropolymeric IF system, are essential for the nematode development (Karabinos et al., 2001) due to a) transmission of a muscle force to the cuticle and b) maintaining of correct hypodermis/muscle relationship in development (Francis and Waterston, 1991; Hresko et al., 1994, 1999; Karabinos et al., 2002, 2003; Woo et al., 2004). In addition, the RNAi experiments using the ifb-1 gene cause morphogenetic defects and defective outgrowth of the excretory cells (Karabinos et al., 2003; Woo et al., 2004; Kolotuev et al., 2013) and the same holds also for its excretory cells' expressed dimerization partner IFA-4 (Karabinos et al., 2003; Al-Hashimi et al., 2018).
Previously, we and others have also reported that the other C. elegans IF proteins IFB-2, IFC-1, IFC-2, IFD-1, IFD-2 and IFP-1 (originally referred to as IFE-1; Karabinos et al., 2001) are expressed in the intestinal terminal web during different developmental stages and reveal various differently penetrant RNAi phenotypes (Karabinos et al., 2001, 2002; 2004; Hüsken et al., 2008; Geisler et al., 2016). Moreover, using the recombinantly expressed IFB-2, IFC-1, IFC-2, IFD-1, IFD-2 and IFP-1 proteins and the overlay assay as a tool, we found that the latter proteins form another heteropolymeric IFB-2/IFCDP-1 IF system in C. elegans and proposed a simple model for the intestinal IF meshwork (Karabinos et al., 2017). Interestingly, the alternatively spliced IFC-2 variant, called EXC-2, was recently found also to be needed for a normal excretory system maturation in C. elegans (Al-Hashimi et al., 2018). In order to better understand the IFC-2 function in the nematode tissue, we retrieved from the WormBase its multiple predicted alternatively spliced transcripts and analysed them using the molecular, immunofluorescence and RNAi approaches. We found that the 21-exon long genomic fragment encodes, besides the two different intestinal IFC-2a and IFC-2b IF proteins, also the novel excretory cell/IF unrelated protein ECP-1 and probably also the large ECP-1/IFC-2 fusion proteins EXC-2a/b, which all seem to be tissue-specific regulated from different promoters.
Section snippets
Analysis of different predicted alternatively spliced IFC-2 transcripts
Using the IFC-2-specific antibody, we previously detected two polypeptides of about 55 and 66 kDa in the whole nematode protein extract, which we named IFC-2a and IFC-2b, respectively, in agreement with the nomenclature used on other IF genes (Karabinos et al., 2004). The 502 (IFC-2a) and 622 (IFC-2b) aa residue long IFC-2 protein is encoded by the IFC-2-L and IFC-2-H cDNA (Fig. 1A), respectively, previously cloned by Dodemont et al. (1994). Interestingly, only the IFC-2b sequence is presented
Conclusion
Taken together, our presented immunofluorescence and promoter/GFP-reporter studies may suggest that the ecp-1, ifc-2 and probably also the large fusion exc-2 are independent genes which might be regulated from different promoters. Such an interpretation would be in agreement a) with the major ∼1,9 kb transcripts found in the IFC-2 Northern analysis (Dodemont et al., 1994), which represent the two ifc-2 mRNAs, described above, as well as with the minor ∼4 kb transcripts found in the same
Nucleic acid and RNA interference methods
Amplification of almost the whole coding regions of C. elegans IFC-2 (Karabinos et al., 2001) was performed using the cDNA library and experimental conditions previously described (Karabinos et al., 2004). The genomic 1680 nt long ecp-1 DNA fragment (the WormBase sequence IFC-2c/M6.1c) was PCR (polymerase chain reaction) amplified from the C. elegans genomic DNA. The empty RNAi plasmid L4440 was used in this study as a negative RNAi control. The primers used for amplification of the DNA
Conflicts of interest
The authors proclaim no conflict of interests.
Acknowledgements
We thank Tiemo Klisch for help with immunofluorescence experiments and Ruth Jähne for the cloning of GFP-reporters and RNAi plasmids. This work was funded by the European Regional Development OPVaV-2009/2.2/05-SORO (ITMS code:26220220143).
References (35)
- et al.
Form of the worm: genetics of epidermal morphogenesis in C. elegans
Trends Genet.
(2000) - et al.
Tissue integrity: hemidesmosomes and resistance to stress
Curr. Biol.
(2001) - et al.
mua-6, a gene required for tissue integrity in Caenorhabditis elegans, encodes a cytoplasmic intermediate filament
Dev. Biol.
(2003) Intermediate filament (IF) proteins IFA-1 and IFB-1 represent a basic heteropolymeric IF cytoskeleton of nematodes: a molecular phylogeny of nematode IFs
Gene
(2019)- et al.
Expression profiles of the essential intermediate filament (IF) protein A2 and the IF protein C2 in the nematode Caenorhabditis elegans
Mech. Dev.
(2002) - et al.
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
J. Mol. Biol.
(2003) - et al.
Most genes encoding cytoplasmic intermediate filament (IF) proteins of the nematode Caenorhabditis elegans are required in late embryogenesis
Eur. J. Cell Biol.
(2004) - et al.
Evolutionary aspects in intermediate filament proteins
Curr. Opin. Cell Biol.
(2015) - et al.
Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans
Gene
(2001) - et al.
Intermediate filaments are required for C. elegans epidermal elongation
Dev. Biol.
(2004)
A tension-induced mechanotransduction pathway promotes epithelial morphogenesis
Nature
Tubular excretory canal structure depends in intermediate filaments EXC-2 and IFA-4 in Caenorhabditis elegans
Genetics
Caenorhabditis elegans operons: form and function
Nat. Rev. Genet.
Intermediate filaments and polarization in the intestinal epithelium
Cells
Eight genes and alternative RNA processing pathways generate an unexpectedly large diversity of cytoplasmic intermediate filament proteins in the nematode Caenorhabditis elegans
EMBO J.
Molecular phylogeny of metazoan intermediate filament proteins
J. Mol. Evol.
Muscle cell attachment in Caenorhabditis elegans
J. Cell Biol.
Cited by (2)
Epithelial morphogenesis, tubulogenesis and forces in organogenesis
2021, Current Topics in Developmental BiologyCitation Excerpt :Mutations underlying exc-2 were recently found to affect the ifc-2 locus (Al-Hashimi et al., 2018), which, as discussed in the “Lumen Extension” section, encodes an intermediate filament protein. Further analysis of the exc-2 locus suggested the existence of a second gene encoding a novel protein, which has been called ecp-1, that could be affected by exc-2 mutations (Karabinos, Schulze, & Baumeister, 2019). However, there is uncertainty as to whether ecp-1 encodes a separate protein, or is an isoform of IFC-2 (termed ifc-2c) that lacks IF motifs.