Chapter Twenty-Four - Intermediate Filaments in Caenorhabditis elegans
Introduction
Caenorhabditis elegans cells contain the three filament systems: microtubules, microfilaments, and intermediate filaments (IFs), similar to mammalian cells. The genome of C. elegans contains 11 genes encoding for cytoplasmic IFs (Karabinos, Schmidt, Harborth, Schnabel, & Weber, 2001), and one gene encoding for a nuclear IF (Riemer, Dodemont, & Weber, 1993). Based on sequence comparisons, the IF proteins can be divided into six groups: Group 1 includes IFA-1,2,3,4; Group 2 includes IFB-1,2; Group 3 includes IFC-1,2; Group 4 includes IFD-1,2; Group 5 includes IFP-1; and Group 6 includes Ce-lamin (Carberry et al., 2009, Karabinos et al., 2001, Riemer et al., 1993). Like all other IFs, these C. elegans proteins have a short N-terminal head domain, a central rod domain that is highly conserved in length and includes four α-helical regions made of coiled-coils (termed 1A, 1B, 2A, and 2B) separated by three linker regions, and a C-terminal tail domain. Like all other invertebrate IFs, Coil 1B of all the C. elegans IF proteins contains six heptad repeats (42 amino acids) that are present in all lamins and are missing from all vertebrate cytoplasmic IF proteins, thus making invertebrate IFs more closely related to nuclear lamins than the vertebrate IFs. In addition, six C. elegans IF proteins contain a globular domain in their tail domain, similar to the globular domain in the lamin tail domains.
Section snippets
Essential Roles of Cytoplasmic IFs in C. elegans
Mammals have over 70 IF genes. With the exception of lamins, they are all expressed in a cell- and tissue-specific manner. For example, keratins are present in epithelial cells and vimentin is present in mesenchymal, endothelial, and hematopoietic cells. Muscle cells express desmin, neuronal cells express the neurofilament triplet proteins, neuroglia cells express glial fibrillary acidic protein (GAFP), and so on. Some cells have more than one filamentous IF network. The IF proteins have
Essential Roles of Lamin in C. elegans
Lamins are nuclear IF proteins that are conserved in all multicellular animals. They form a protein scaffold at the nuclear periphery, termed the nuclear lamina. A small fraction of lamins is also present in the nucleoplasm. Mammals have three lamin genes termed LMNA, LMNB1, and LMNB2 that encode four major isoforms: lamins A, lamin C (A-type lamins), lamin B1, and lamin B2 (B-type lamins) (Stuurman, Heins, & Aebi, 1998). The B-type lamins are permanently farnesylated at their C terminus, while
Assembly of C. elegans Lamins
Depending on the assembly conditions, bacterially expressed and purified Ce-lamin can form in vitro either 10-nm wide filaments or paracrystalline arrays (Foeger et al., 2006, Karabinos, Schunemann, et al., 2003). An in vitro assembly model of Ce-lamin, based on electron microscopy negative staining and cryo-electron tomography studies, suggested a hierarchal order of assembly wherein lamins first form dimers which then polymerize to form a polar head-to-tail linear polymer (Heitlinger et al.,
Assembly of C. elegans IFs
Karabinos, Schulze, et al. (2003) used bacterially expressed IFA-1, IFA-2, IFA-3, IFA-4, and IF-B1 to study the in vitro assembly properties of C. elegans IF proteins. Ten nanometer filaments formed when IFB-2 was mixed with equal amounts of IFA-1, IFA-2, IFA-3, or IFA-4, demonstrating assembly of filaments composed of heteropolymers (Fig. 1). The heteropolymer formation is supported by the coexpression in the same cells of IFB-1 with different IFA proteins and by the overlapping phenotypes
Ce-Lamin Assembly In Vitro
- 1.
Protein expression
BL21DE3 Codon Plus bacteria are transformed with a plasmid encoding Ce-lamin fused to 6 × His. To express the lamin protein, a bacterial culture grown overnight in LB, is diluted 1:10 in 500 ml 2YT medium
2YT medium (1 l):
Bacto tryptone 16 g
Bacto yeast extract 10 g
NaCl 5 g
Adjust pH to 7.0 with NaOH
The resulting culture is grown to an OD600 of 0.6–0.8, whereupon protein induction is initiated by the addition of IPTG to a final concentration of 0.5 mM. The bacterial culture is then
Summary
The 11 C. elegans genes that encode cytoplasmic IFs show tissue-specific patterns of expression and at least five of them are essential for the animal's survival. Many of the C. elegans IF proteins make heteropolymers, similar to mammalian cytoplasmic IFs. The single nuclear IF gene is evolutionary conserved and essential. This evolutionary conservation together with the relative simplicity of the system and the availability of the diverse genetics tools make C. elegans a powerful model to
Acknowledgments
We gratefully acknowledge funding from the Muscular Dystrophy Association (MDA), the Binational Israel-USA Science Foundation (BSF 2007215) and the Niedersachsen-Israeli Research Cooperation program.
References (30)
- et al.
The supramolecular organization of the C. elegans nuclear lamin filament
Journal of Molecular Biology
(2009) - et al.
Transmission electron microscope studies of the nuclear envelope in Caenorhabditis elegans embryos
Journal of Structural Biology
(2002) - et al.
Solubility properties and specific assembly pathways of the B-type lamin from Caenorhabditis elegans
Journal of Structural Biology
(2006) - et al.
mua-6, a gene required for tissue integrity in Caenorhabditis elegans encodes a cytoplasmic intermediate filament
Developmental Biology
(2003) - et al.
SUMO regulates the assembly and function of a cytoplasmic intermediate filament protein in C. elegans
Developmental Cell
(2009) - 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
Journal of Molecular Biology
(2003) - et al.
The single nuclear lamin of Caenorhabditis elegans forms in vitro stable intermediate filaments and paracrystals with a reduced axial periodicity
Journal of Molecular Biology
(2003) - et al.
Most genes encoding cytoplasmic intermediate filament (IF) proteins of the nematode Caenorhabditis elegans are required in late embryogenesis
European Journal of Cell Biology
(2004) - et al.
Nuclear lamins: Their structure, assembly, and interactions
Journal of Structural Biology
(1998) - et al.
Intermediate filaments are required for C. elegans epidermal elongation
Developmental Biology
(2004)
“Laminopathies”: A wide spectrum of human diseases
Experimental Cell Research
Caenorhabditis elegans as a model system for studying the nuclear lamina and laminopathic diseases
Nucleus
Ce-emerin and LEM-2: Essential roles in Caenorhabditis elegans development, muscle function, and mitosis
Molecular Biology of the Cell
Intermediate filaments in Caenorhabditis elegans
Cell Motility and the Cytoskeleton
Worms reveal essential functions for intermediate filaments
Proceedings of the National Academy of Sciences of the United States of America
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