Cary RB and Klymkowsky MW (1995), Disruption of intermediate filament organizatio...

XB-ART-19876

Development 1995 Apr 01;1214:1041-52.

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Disruption of intermediate filament organization leads to structural defects at the intersomite junction in Xenopus myotomal muscle.

Cary RB , Klymkowsky MW .

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In mature striated muscle, intermediate filaments (IFs) are associated with the periphery of Z-discs and sites of myofibril-membrane attachment. Previously T. Schultheiss, Z. X. Lin, H. Ishikawa, I. Zamir, C. J. Stoeckert and H. Holtzer (1991) J. Cell Biol. 114, 953) reported that the disruption of IF organization in cultured chick myotubes had no detectable effect on muscle cell structure. Cultured muscle is not, however, under the mechanical loads characteristic of muscle in situ. The dorsal myotomal muscle (DMM) of the Xenopus tadpole provides an accessible model system in which to study the effects of mutant IF proteins on an intact, functional muscle. DNAs encoding truncated forms of Xenopus vimentin or desmin were injected into fertilized Xenopus eggs. Embryos were allowed to develop to the tadpole stage and then examined by confocal or electron microscopy. DMM cells containing the truncated IF polypeptides displayed disorganized IF systems. While the alignment of Z-lines appeared unaffected, cells accumulating mutant IF polypeptides displayed abnormal organization at the intersomite junction. Myocyte termini are normally characterized by deep invaginations of the sarcolemma. In myocytes expressing mutated IF polypeptides, these membrane invaginations were reduced or completely absent. Furthermore, the attachment of myofibrils to the junctional membrane was often aberrant or completely disrupted. These results suggest that in active muscle IFs play an important role in the organization and/or stabilization of myofibril-membrane attachment sites.

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Species referenced: Xenopus laevis
Genes referenced: myc vim

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	Fig. 1. A schematic of the system and the reagents used to study it. (A) In the stage 40 tadpole, there is a DMM present on both the left and right sides of the animal. Each DMM is composed of blocks of muscle cells, the somites, which are attached to one another at the intersomite junction (ISJ). (B) At the ISJ, finger-like projections of the muscle cells extend into, and attach to, the extracellular matrix that separates the somites. To measure the effects of mutated IF proteins on the attachment of myofibrils to the ISJ, we measured the distance from the center of the ISJ (‘medial line’) to the first Z-line. Since there is not a single myofibril in the cell, each myofibril was measured separately and averaged together as described in equation 1 (see Materials and Methods). Two types of constructs were used in our studies: embryos (or cultured cells) were injected with DNA encoding either an epitope-tagged full-length form of Xenopus vimentin-1 or desmin (C) or truncated forms of these polypeptides (D). The truncated proteins lack the highly conserved helix termination region (‘HTR’) of the central rod domain and the entire C-terminal tail domain. For both full-length and truncated polypeptides, the sequence of the junction between IF polypeptide and the myc-tag is shown.
	Fig. 2. Truncated vimentin and desmin disrupt IF organization. Xenopus XR-1 cells were injected with VIMDC (A,B) or DESDC DNA (C,D) and stained with anti-tag and anti-vimentin antibodies. (A) In cells injected with VIMDC DNA, the truncated polypeptide appears as either fine punctate material (arrow) or larger irregular aggregates (arrowhead). (B) Anti-vimentin staining reveals the extended vimentin filament system in an uninjected cell; in the cells expressing the VIMDC polypeptide, the endogenous vimentin filament system is disrupted. Similar behavior was seen in cells expressing DESDC. (C) The DESDC polypeptide was found in punctate structures, while anti-vimentin staining (D) revealed the disruption of the endogenous vimentin filament system. Bar in A marks 10 mm for all parts.
	Fig. 3. EM analysis of exogenous proteins in DMM cells. Embryos, injected at the 1-cell stage with plasmid DNA encoding either (A,C,D) VIMDC or (B) full-length desmin, were fixed and examined by immunoelectron microscopy at stage 38 or stage 48. Unlike the aggregates of exogenous desmin (marked ‘a’), which are associated with discrete 10 nm filaments (arrowheads in B), the aggregates of VIMDC protein (marked ‘a’) have no associated filamentous material (A). When examined at lower magnification, it is apparent that the presence of the VIMDC polypeptide has no drastic effect on the lateral alignment of myofibrils and Z-lines (open arrows) in stage 38 DMM cells (C). In this picture, an aggregate of mutant polypeptide is visible under the sarcolemma (solid arrow). (D) A myocyte from a stage 48 tadpole. This cell contains a large subsarcolemmal VIMDC aggregate (marked ‘a’) located near the nucleus (marked ‘N’). Despite the apparent distention of the sarcolemma near the aggregate, myofibrils appear normal and well aligned (arrows). Bar in A marks 200 nm for A and B. Bar in C marks 0.5 mm. Bar in D marks 1.5 mm.
	Fig. 4. VIMDC effects on ISJ structure. There are drastic changes in the region of the ISJ associated with VIMDC expression. The inset in A shows the ISJ region that contains two VIMDC-expressing cells, shown at higher magnification in A and B. Both cells lack the invaginations typical of DMM cells. On the intracellular side, myofibrils fail to attach to the sarcolemma. In these cells, the myofibrils appear severed (arrows) and one myofibril fragment appears attached to the membrane in an aberrant ‘end-on’ configuration (arrowhead in A). An aggregate of mutant protein is visible near the sarcolemma (marked ‘a’ in A). Bar in A marks 0.5 mm for A and B.
	Fig. 5. Over-expression of full-length desmin. In cells expressing full-length, myc-tagged vimentin- 1 (not shown) or desmin (illustrated here), the accumulation of exogenous protein does not produce any apparent distortion of ISJ morphology or myofibril-membrane attachment. The invaginations of the ISJ membrane appear normal (arrows). Bar marks 0.5 mm.
	Fig. 6. Confocal microscopy of pDESDC-expressing myocytes. To use confocal microscopy to analyze the effects of truncated desmin on myocyte structure, fertilized eggs were injected with DESDC DNA and tadpoles were fixed and stained in whole-mount to reveal the exogenous DESDC polypeptide (fluorescein) and skeletal muscle myosin (Texas Red). Images are displayed in ‘anaglyph’ mode, such that myosin staining is in red-orange and DESDC staining is in yellow-green; serial sections of two cells (A-C,D,E) are shown. In the cell in A-C, a few myofibrils can be seen extending to the ISJ membrane. At the ISJ (arrows), fragments of myofibrils are visible (curved arrows, A-C). These fragments of myofibril material are presumably analogous to those observed in electron micrographs (Fig. 4). There are also myofibrils that fail to extend to the ISJ membrane (arrowheads in B). Aggregates of DESDC are visible. (C,D) Another DESDC-expressing cell; again the ISJ region is clearly aberrant (compare with flanking cells, marked ‘wt’). In many sections (example in D), no myofibrils are visible and aggregates of DESDC are present near the cell end (curved arrows). In other regions of the cell (E), a small number of myofibrils extend to the ISJ membrane (curved arrows) though they occupy only a small percentage of the cell’s width (large arrows). No ‘pulled away’ myofibrils are visible. Bar in E marks 5 mm for all parts.
	Fig. 7. Confocal micrographs of myocytes expressing full-length desmin. For comparison, the morphology of two termini of a cell expressing the full-length, myc-tagged form of desmin is illustrated in pairs of serial section images (A and C are one end, B and D the other; inset in C provides a low-magnification view of much of this cell). The rectangularity of the terminal membranes and the proximity of myofibrils to the ISJ appear normal. The ISJ is marked by small arrows (A-D). (A) The terminal Z-line of the cell is marked with a curved arrow. Exogenous desmin is localized to the sarcolemma, the cell end, and Z-lines; in addition, there are aggregates of the exogenous protein scattered throughout the cell (see inset, C). Some myofibrils appear to fall short of the ISJ membrane (C – curved arrow); these myofibrils appear to be moving out of the section of focus. Bar in D marks 5 mm for all parts.