Elsevier

Methods in Enzymology

Volume 568, 2016, Pages 661-679
Methods in Enzymology

Chapter Twenty-Four - Intermediate Filaments in Caenorhabditis elegans

https://doi.org/10.1016/bs.mie.2015.09.020Get rights and content

Abstract

More than 70 different genes in humans and 12 different genes in Caenorhabditis elegans encode the superfamily of intermediate filament (IF) proteins. In C. elegans, similar to humans, these proteins are expressed in a cell- and tissue-specific manner, can assemble into heteropolymers and into 5–10 nm wide filaments that account for the principal structural elements at the nuclear periphery, nucleoplasm, and cytoplasm. At least 5 of the 11 cytoplasmic IFs, as well as the nuclear IF, lamin, are essential. In this chapter, we will include a short review of our current knowledge of both cytoplasmic and nuclear IFs in C. elegans and will describe techniques used for their analyses.

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)

  • H.J. Worman et al.

    “Laminopathies”: A wide spectrum of human diseases

    Experimental Cell Research

    (2007)
  • E.M. Bank et al.

    Caenorhabditis elegans as a model system for studying the nuclear lamina and laminopathic diseases

    Nucleus

    (2011)
  • R. Barkan et al.

    Ce-emerin and LEM-2: Essential roles in Caenorhabditis elegans development, muscle function, and mitosis

    Molecular Biology of the Cell

    (2012)
  • K. Carberry et al.

    Intermediate filaments in Caenorhabditis elegans

    Cell Motility and the Cytoskeleton

    (2009)
  • R.D. Goldman

    Worms reveal essential functions for intermediate filaments

    Proceedings of the National Academy of Sciences of the United States of America

    (2001)
  • Cited by (6)

    View full text