Thirty years ago, morphological evidence revealed that the thin filaments and desmin intermediate filaments comprise a “collar” surrounding the Z-discs of adjacent myofibrils (Granger and Lazarides, 1978
). Biochemical support for this became available when it was shown that the giant actin filament–binding protein, nebulin, interacts with desmin at the periphery of the Z-discs (Bang et al., 2002
). Nevertheless, the functional significance of the desmin–nebulin interaction in live myocytes and the potential role of this interaction in the context of muscle disease was previously unexplored.
In this study, we established a functional link between desmin and nebulin. Our results revealed that expression of a disease-associated point mutation at amino acid 245 (E245D) within the coil-IB region of desmin resulted in disruption of the endogenous desmin and nebulin Z-disc localization and promoted cytoplasmic aggregate formation, mirroring a major histological feature found in human DRM muscle biopsies. Furthermore, our data demonstrated that expression of the desmin E245D mutation dramatically altered actin filament lengths in myocytes. Our findings support a model in which the functional properties of nebulin associated with thin filament length regulation and/or maintenance appear to be, at least in part, modulated by its interaction with desmin. Thus, our data give novel insights into potential molecular consequences underlying DRMs.
Our targeting experiments showed that of all the GFP desmin regions tested, the coil IB region of desmin was consistently the most efficient at localizing to the Z-discs in cardiac and skeletal myocytes, in agreement with its interaction with C-terminal nebulin (, Supplemental Figure S1). Because of this result as well as our observation that overexpression of the full-length molecule itself alters thin filament lengths and result in the accumulation of cytoplasmic aggregates, we utilized the wild-type coil IB fragment (which displays little, or no, detectable cellular perturbations) in most of our studies to focus on the interaction of desmin at the Z-disc. A possible explanation for the observed cellular effects on full-length desmin overexpression is that it likely altered the stoichiometric ratios of desmin to nebulin and/or it may have influenced interactions with other desmin-binding partners within myocytes. Interestingly, expression of the wild-type coil IB fragment alone was found to “recruit” endogenous desmin to the Z-disc, suggesting that it has the ability to self-associate with the full-length molecule. Additionally, we observed clear M-line targeting of both the coil IIB and tail regions of desmin. This raises the exciting possibility that desmin is indeed a component of the transverse M-line associated filaments connecting adjacent myofibrils observed in electron micrographs and rarely visualized in confocal studies (Price, 1984
; O'Neill et al., 2002
); these filaments are thought to contribute to the transverse stability of myofibrils during muscle contraction-relaxation cycles (e.g., Wang and Ramirez-Mitchell, 1983
). Our data also suggest that the coil IIB and tail regions of desmin can target efficiently to the M-line in cultured cells because they are missing other regions of full-length desmin that contain interacting sites for non-M-line components that can mostly outcompete for M-line assembly in vivo. This interpretation is consistent with the near absence of GFP-desmin staining at the M-line in cultured myocytes. Based on the findings that the M-band protein EH-myomesin (skelemin), the major embryonic vertebrate heart isoform of myomesin, copurified with desmin and containing two “intermediate filament binding motifs,” it has been proposed that it may bind to desmin (Price, 1984
; Steiner et al., 1999
; Agarkova et al., 2000
). The significance of the M-line localization of desmin and identification of its M-line interacting partner(s) remain to be fully elucidated.
Our biochemical evidence revealed that the coil IB region of desmin is important for its interaction with the C-terminal end of nebulin, because desmin coil IB binds with high affinity to nebulin M160-164 with a dissociation constant of Kd
~35 nM (C). Notably, the affinity of desmin coil-IB and nebulin M160-164 is similar to that reported for other nebulin-protein interactions (e.g., Tmod1 with nebulin, CapZ with nebulin: McElhinny et al., 2005
; Pappas et al., 2008
). The dissociation constants were not significantly different between wild-type coil IB versus mutant coil IB E245D in ELISAs. However, the addition of the desmin coil IB E245D mutation reproducibly increased its binding capacity for nebulin (the Bmax was >4-fold higher for the mutant: C). In agreement with these findings, a competition assay revealed that the coil IB E245D mutant was a more efficient competitor than nonmutated coil IB for the binding of nonmutated coil IB to nebulin M160-164 (the IC50
was ~13-fold lower for the mutant: data not shown). One possibility is that the increase in binding capacity of the mutant may alter its binding affinities to other sarcomeric components. Together, these experiments show that both wild-type and mutant desmin coil-IB bind with high affinity to nebulin M160-164 in vitro. Interestingly with respect to our data, based on binding data, Pappas et al., (2008)
showed that nebulin modules M160-164 must be present at the edge of the Z-disc and not projecting out from the Z-disc as previously reported. Thus, we propose a model in which desmin maintains the alignment of the myofibrils by directly binding to a segment of nebulin M160-164 facing the periphery of the Z-disc.
Here, we report that overexpression of full-length desmin itself or the coil IB region of desmin containing the E245D mutation results in a marked removal of endogenous desmin from the Z-disc with a concomitant increase in endogenous desmin in intracellular aggregates containing the mutant proteins ( and ). These aggregates appeared similar to those reported in muscle biopsies taken from the patients with the desmin E245D mutation. These findings suggest that our approach of overexpressing the desmin E245D mutation in a model of primary cultures of myocytes is valid, as it resembles the genetic background found in humans, because this particular desmin mutation is heterozygous with an autosomal dominant pattern of inheritance (Vrabie et al., 2005
). Interestingly, we also found that expression of the desmin E245D mutation resulted in striking alterations in actin filament organization and lengths as evidenced by 1) nonuniformity in phalloidin staining at the Z-disc, suggesting disorganization and/or filament elongation at their barbed ends; 2) significant increase in nonstriated (i.e., no visible gaps/absence of H-zones) phalloidin staining (without a decrease in sarcomere lengths), suggesting thin filament elongation from their pointed ends; and 3) thin filament shortening in cardiac sarcomeres that contained H-zones in phalloidin staining ( and and Supplemental Figure S6). Intriguingly, the alterations in thin filament lengths observed from expression of the E245D desmin mutant are reminiscent of the changes in actin filament lengths observed in nebulin −/− mouse models and in nebulin knockdown studies. For example, in nebulin −/− mouse models, thin filament lengths were significantly shorter (Bang et al., 2006
; Witt et al., 2006
), whereas nebulin knockdown by siRNA led to both nonstriated (longer) and shorter actin filaments (McElhinny et al., 2005
; Pappas et al., 2008
). These data, together with the fact that no major changes in the distribution of most (but not all, see below) sarcomeric components were observed as a result of expressing the desmin E245D mutant (), are consistent with our prediction that nebulin's actin filament length regulation functions are compromised in cells expressing the desmin E245D. This adds a new functional role of desmin IFs in regulating (at least some of) nebulin's length-regulating properties.
The significance of our findings is particularly noteworthy because perturbations in actin organization and/or lengths would be expected to directly influence the relative range of lengths over which the sarcomeres can generate force effectively. Interestingly, desmin does not require nebulin to localize to the Z-disc as observed in nebulin −/− mice (Bang et al., 2006
). However the interaction appears to be important for force transmission, based on the observations reported in nebulin −/− models in which the missing linkage of desmin to the Z-disc to nebulin results in lowered stress generation in the nebulin −/− mice (Bang et al., 2006
; Witt et al., 2006
). Thus, an interesting question arises as what is the mechanical relationship between desmin and nebulin and how nebulin might aid in transmission of force from the thin filament to the intermediate filament system via its association with desmin at the Z-discs. Consistent with this idea, the observed perturbations in the organization of myosin upon expression of DRM-associated desmin E245D mutation could be influenced by suboptimal interactions with actin filaments of altered lengths (i). Another interpretation is that the observed changes in actin filament morphology may result from a nebulin-based misregulation of actin filament–capping protein dynamics (Tmod1 and/or CapZ) at the ends of the thin filaments. A conundrum is that although there appears to be much lower levels of nebulin in cardiac myofibrils, in comparison to skeletal myofibrils (e.g., Kazmierski et al., 2003
; Bang et al., 2006
), we observed similar thin filament perturbations as a result of the expression of desmin E245D mutant. We, as well as others, speculate that many sarcomeric components contribute to the regulation of thin filament lengths, which may also be affected by the expression of the desmin E245D mutant. This prediction is supported by the fact that invertebrate organisms such as Caenorhabditis elegans
(do not have nebulin or desmin orthologues), yet still have uniform thin filament lengths. It is clear that more studies will be required to resolve how the levels of nebulin influence its thin filament length–regulating properties.
In conclusion, our results provide new evidence that the desmin–nebulin interaction plays an important role in muscle physiology and that variations in actin filament lengths may directly contribute to pathogenic molecular consequences underlying certain desmin-related myopathies.