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Mechanical probing of the intermediate filament-rich C. elegans intestine

Jahnel O, Hoffmann B, Merkel R, Bossinger O, Leube RE, 2016.

It is commonly accepted that intermediate filaments have an important mechanical function. This function relies not only on intrinsic material properties but is also determined by dynamic interactions with other cytoskeletal filament systems, distinct cell adhesion sites and cellular organelles which are fine-tuned by multiple signaling pathways. While aspects of these properties and processes can be studied in vitro, their full complexity can only be understood in a viable tissue context. Yet, suitable and easily accessible model systems for monitoring tissue mechanics at high precision are rare. We show that the dissected intestine of the genetic model organism C. elegans fulfils this requirement.


The 20 intestinal cells, which are arranged in an invariant fashion, are characterized by a dense subapical mesh of intermediate filaments that are attached to the C. elegans apical junction (CeAJ). We present procedures to visualize details of the characteristic intermediate filament-junctional complex arrangement in living animals. We then report on methods to prepare intestines with a fully intact intermediate filament cytoskeleton and detail procedures to assess their viability. A dual micropipette-assay is described to measure mechanical properties of the dissected intestine while monitoring the spatial arrangement of the intermediate filament system. Advantages of this approach are (i) the high reproducibility of measurements because of the uniform architecture of the intestine and (ii) the high degree of accessibility allowing not only mechanical manipulation of an intact tissue but also control of culture medium composition and addition of drugs as well as visualization of cell structures. With this method examination of worms carrying mutations in the intermediate filament system, its interacting partners and regulators will become feasible.

Animated 3D-representation of DLG-1::mCherry fluorescence in the intestine and pharynx of an intact worm. Scale bar: 50 µm.


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Schematic 3D animation of a part of the C. elegans intestine. The scheme shows the CeAJ in red, the intermediate filament-rich endotube in yellow, nuclei in blue and transparent cell borders.


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Recording of the fluorescent wave prior to cell death in a dissected intestine. Time-lapse fluorescence microscopy and phase contrast microscopy (bottom) of a dissected intestine detecting characteristic autofluorescent intestinal granules in the cytoplasm at the beginning of the recording period. The recording was started 8 min after dissection. At 13 min diffuse fluorescence appears in cells of the anterior part that is propagated toward the back as a fluorescent wave. Subsequently, the diffuse fluorescence is transformed into another kind of granular fluorescence before all fluorescence is lost upon cell death.


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Recording of the fluorescent wave prior to cell death in an intestine 120 min after dissection. Time-lapse fluorescence microscopy (corresponding Fig. 3B) detects characteristic autofluorescent intestinal granules in the cytoplasm at the beginning of the recording period. Note the appearance of diffuse fluorescence that is propagated toward the back as a fluorescent wave. Subsequently, the diffuse fluorescence is transformed into another kind of granular fluorescence before all fluorescence is lost upon cell death.


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Uptake of trypan blue into dying cells of a dissected intestine. The time-lapse series shows the overlays of fluorescence (white) and phase contrast recordings. Note the occurrence of a fluorescent wave starting at 57 min and the subsequent loss of fluorescence coincident with uptake of trypan blue (dark blue)..


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Recording of repeated and incrementally increasing stretching of a dissected intestine (see corresponding Fig. 7). The movie shows phase contrast recordings of an intestine attached to a holding pipette at right and a stretching pipette at left. The time-lapse series presents the first and the last of seven consecutive cycles, each at 0.18 µN, 0.24 µN and 0.31 µN, and a final cycle at 0.37 µN. Note the increasing maximum elongation and the increasing prestretch length as indications of the elastic and plastic properties of the intestine.


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