Chapter Twenty-Five - Mechanical Probing of the Intermediate Filament-Rich Caenorhabditis Elegans Intestine
Introduction
Cells experience a wide range of mechanical stresses in their native tissue environment. This is especially true for epithelial cells, which are responsible for efficient and continuous barrier formation while being subjected to forces from the outside and inside of the body. Major players in the integration and dissipation of these forces are the intermediate filaments together with their plasma membrane attachment sites by acting as mechanical shock absorbers, especially at large deformations (Beil et al., 2003, Fudge et al., 2008). The in vitro observations, that single-intermediate filaments can be stretched up to 3.6-fold before breaking (Kreplak, Bär, Leterrier, Herrmann, & Aebi, 2005) and that intermediate filaments respond to high strains by hardening (e.g., Janmey et al., 1991, Lin et al., 2010), are ideal properties to support this function. Observations on genetically modified cells provide further evidence. Thus, deletion of vimentin intermediate filaments leads to reduced stiffness and impaired mechanical stability of fibroblasts resulting in reduced migration and contraction (Brown et al., 2001, Eckes et al., 1998, Wang and Stamenović, 2000). Expression of different desmin intermediate filament mutants in rat fibroblasts leads to altered cell stiffness as determined by atomic force microscopy (Plodinec et al., 2011). Similarly, epithelial cells expressing keratin mutants (Lulevich, Yang, Rivkah Isseroff, & Liu, 2010) and keratin-free keratinocytes are less stiff but become more motile (Ramms et al., 2013, Seltmann, Fritsch, et al., 2013, Seltmann, Roth, et al., 2013). The occurrence of blister-forming diseases in patients carrying point mutations in keratin genes is probably the most compelling evidence for a mechanical function of epithelial intermediate filaments (recent review in Homberg & Magin, 2014). However, the underlying mechanisms are still not fully understood. Besides a direct mechanical contribution of keratin filaments themselves, perturbed signaling and adhesion are also considered to be important factors in the development of mechanical deficiencies (Kröger et al., 2013, Liovic et al., 2008; Russell et al., 2010, Seltmann et al., 2015).
In the recent past considerable efforts have been undertaken to measure mechanical properties of the cellular intermediate filament system. Most emphasis has been on the investigation of single cells using microchannels (Rolli, Seufferlein, Kemkemer, & Spatz, 2010), microfluidic optical stretchers (Seltmann, Fritsch, et al., 2013), and atomic force microscopy (Lulevich et al., 2010, Ramms et al., 2013, Walter et al., 2011) to probe whole-cell mechanical properties from the outside, while particle-tracking microrheology (Sivaramakrishnan, DeGiulio, Lorand, Goldman, & Ridge, 2008) and magnetic tweezers (Ramms et al., 2013) have been used for testing cytoplasmic viscoelasticity. These studies were complemented by analyses of the keratin cytoskeleton in fixed and partially extracted cells (e.g., Beil et al., 2003, Paust et al., 2013, Walter et al., 2011). Besides encountering a very high degree of cell-to-cell variability, these analyses neglected the tight coupling of epithelial cells to each other and the extracellular matrix. For epithelial cell sheets, analyses have been restricted to reconstituted cell monolayers using dispase assays to examine tissue cohesion (Kröger et al., 2013) and to the study of keratin network dynamics in cells grown on flexible substrates (Beriault et al., 2012, Felder et al., 2008, Fois et al., 2013, Fudge et al., 2008, Hecht et al., 2012).
Thus, a major current need is the investigation of the mechanical properties of the epithelial intermediate filament cytoskeleton in its native tissue environment. Such an enterprise should taken into account the multidimensional intermediate filament network organization, which is determined by its cell type-specific 3D arrangement, turnover dynamics, association with other filament systems, attachment to defined adhesion sites, and the multiple dynamic interactions with other cellular components. The complex nature of vertebrate tissues and the substantial efforts needed to isolate, cultivate, and genetically modify them are major obstacles. Therefore, using the genetic model organism Caenorhabditis elegans may offer a simplified approach. Its highly invariant body plan, the simplicity and speed to grow and propagate clonal worm lines and the easiness of genetic manipulation together with the abundance of intermediate filaments in the epithelial tissues of C. elegans are all in favor of using this very well-characterized model organism. A proof of principle was recently provided by the elegant work of Michel Labouesse and his group which uncovered an epidermal mechanotransduction pathway in the epidermis resulting in intermediate filament phosphorylation (Zhang et al., 2011). Remarkably, they were able to show that application of mechanical pressure on C. elegans embryos can trigger this pathway. Our own interest is in the epithelial cytoskeleton of the C. elegans intestine (Carberry et al., 2012, Carberry et al., 2009). The approximately 800 μm long tube-like intestine (midgut) extends from the pharynx (foregut) to the hindgut and consists of 20 cells, which are involved in nutrient uptake and secretion. They are arranged as a single-cell layer forming nine intestinal rings (ints) surrounding the ellipsoid lumen (recent review in McGhee, 2013).
The C. elegans genome contains 11 cytoplasmic intermediate filament genes coding for at least 14 polypeptides because of differential splicing (Carberry et al., 2009, Dodemont et al., 1994, Karabinos et al., 2001) and a single gene for a nuclear lamin (Gruenbaum et al., 2002, Liu et al., 2000). Of the 11 genes encoding cytoplasmic intermediate filament polypeptides, six are predominantly if not exclusively transcribed in the intestine. They encode for IFB-2, IFC-1, IFC-2, IFD-1, IFD-2, and IFP-1 (Carberry et al., 2009). All are characterized by a central α-helical rod domain with subdomains L1, L12, and L2 encompassing the characteristic heptad repeat needed to form the stable coiled-coil dimers (Carberry et al., 2009, Dodemont et al., 1994). The polymerization properties of the intestinal intermediate filaments have not been investigated to date. A unique feature of the cytoplasmic C. elegans intermediate filament polypeptides is the presence of a 42 amino acid insertion in coil 1b that is typical for protostomia and the nuclear lamins. The B-type intermediate filament polypeptides also contain an Ig-like domain in their carboxytermini. Evolutionary analyses show that the intestinal intermediate filament polypeptides of C. elegans are unique and differ from those expressed in other nematodes, invertebrates, and vertebrates (Table 1).
The presence of the C. elegans intestinal intermediate filaments seems to be linked to the endotube, a prominent and mechanically resilient structure that is characteristic for C. elegans. The endotube is a clearly discernable dense region in electron micrographs, which is located just below the organelle-free terminal web that anchors the microvillar actin bundles (Bossinger et al., 2004, McGhee, 2013, Munn and Greenwood, 1984). The intestinal intermediate filaments are highly enriched in the subapical region and have been localized to the endotube by immunoelectron microscopy (Bossinger et al., 2004). The endotube is anchored to the C. elegans apical junction (CeAJ). This apical cell–cell adhesion site is ultrastructurally homogenous but encompasses several molecular subdomains (recent review in Pásti & Labouesse, 2014).
In this communication, we will first describe, how the intermediate filaments and cell junctions are organized as a contiguous sheath surrounding the intestinal lumen and will then go on to describe a simple method to isolate functionally intact intestines, whose mechanical properties can be quantitatively examined using a dual pipette assay. Possible applications of this system will be briefly outlined at the end.
Section snippets
Imaging of Intermediate Filaments in C. elegans Intestines by Epifluorescence Microscopy
The following protocol is routinely used to examine the intermediate filament distribution in worms producing fluorescently labeled reporters. Figure 1 shows a live L4 larva, in which IFB-2::CFP fusion proteins localize specifically to the subapical periluminal domain of the intestine (Carberry et al., 2009, Hüsken et al., 2008). The continuous tube-like sleeve surrounding the intestinal lumen from the first to ninth int is readily seen.Materials Viable L4 larvae of strain BJ49 kcIs6 [ifb2::cfp]IV
Outline of Intestinal Rings in C. elegans by a Fluorescent Apical Junction Reporter
For understanding the mechanical coupling of the cytoplasmic intermediate filament cytoskeleton in the intestine, a detailed characterization of the spatial arrangement of the CeAJ is crucial. Using fluorescently labeled components of the CeAJ intermediate filament attachment sites can be easily outlined. The example shown in Fig. 2A and B presents the fluorescence of an mCherry-labeled reporter for the CeAJ component DLG-1 revealing a ladder-type pattern in the intestine (Bossinger et al., 2001
Dissection of Intestines and Vitality Testing
Tissue mechanics are dependent on the functioning of all cellular components. It is therefore critical to prepare viable intestinal tissue and to assess its functionality. With comparatively little experience, viable intestines can be prepared with this simple procedure. We prefer to use L4 larvae because they can be easily identified in the dissection stereo microscope. In addition, the well-defined L4 stage is rather short (~ 10 h at 22 °C) and thereby improves comparability between different
Experimental Setup for Micropipette Measurements
Micropipettes are well-established tools for mechanical probing (Evans, 1989, Mitchison and Swann, 1954, Needham, 1993). Careful preparation of micropipettes is essential for high-precision measurements. They have to be tailored to fully attach to the isolated intestine without any leakage and without damaging the brittle cells. The setup for the dual pipette assay needs to be calibrated for each new pipette and needs to be readjusted in between measurements (Ligezowska et al., 2011).
Outlook
The examples presented highlight the advantages of the dissected C. elegans intestine as a model system for the examination of tissue mechanics and reveal its unique properties. They further highlight the extreme mechanical resilience of the C. elegans intermediate filament system, which withstands forces up to 1 μN and can be stretched to more than 150% without rupture even when the rest of the cell is pulled away. Our data further demonstrate that the intestine does not fully resume its
Acknowledgments
This work was supported by a scholarship from the Jürgen Manchot-Stiftung to O.J. and by grants from the German Research Council to R.M. (ME 1458/6-3), O.B. (BO 1061/11-3), and R.L. (LE566 14-3).
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Current address: Molecular Cell Biology, Institute of Anatomy I, University of Cologne, 50937 Cologne, Germany.