In our quest to identify binding partners for the giant sarcomeric protein UNC-89 (obscurin), we found that two portions of UNC-89 interact with the MEL-26, a BTB–domain containing substrate recognition protein known to interact with CUL-3 (Cul3)-based E3 ubiquitin ligase. Furthermore, we found that interaction of UNC-89 with MEL-26 occurs through the MATH domain of MEL-26 ( and ), the same domain of MEL-26 that had been identified previously as interacting with a known substrate of the CUL-3/MEL-26 complex in C. elegans embryos, the MEI-1 microtubule-severing protein (katanin). Using antibodies, we found that muscle MEL-26 is localized to M-lines, colocalizing with UNC-89, and to I-bands (). To our knowledge this is the first time that a cullin-associated protein or a cullin has been localized in the sarcomere. Loss of function of unc-89 results in a characteristic pattern of disorganization of muscle thick filaments, including myosin aggregates, which suggests a role for unc-89 in thick-filament assembly and/or maintenance. We found that loss or gain of function of mel-26 or loss of function or gain of function of mei-1 results in thick-filament disorganization similar to that of unc-89 loss of function ( and ). Because a mutant β-tubulin that is resistant to cleavage by MEI-1 (katanin) can partly suppress the thick-filament disorganization of a mel-26 mutant (), at least one function of MEI-1 in muscle is to sever microtubules. We observed that loss of function of unc-89 results in a reduction in the level of MEI-1 protein (). MEI-1, previously reported to be ubiquitinated in vitro, is likely to be degraded in the proteasome based on inhibiting proteasome function by rpt-2(RNAi) (). However, compensating for decreased levels of MEI-1 caused by unc-89 by inhibiting the proteasome did not improve the organization of thick filaments in unc-89(su75) versus unc-89(su75); rpt-2(RNAi) (unpublished data). This is consistent with our observations that rpt-2(RNAi) in the unc-89-mutant background still resulted in a rise of MEI-1 beyond wild-type levels and that gain of function of MEI-1 () causes disorganization of thick filaments, as does MEI-1 loss of function.
To account for our findings, we propose a model in which the CUL-3/MEL-26 complex is localized to the M-line through the interaction of UNC-89 with MEL-26, and the function of this interaction is to inhibit ubiquitin-mediated degradation of MEI-1 (). Given that both UNC-89 and MEI-1 bind to the MATH domain of MEL-26, we tested the hypothesis that UNC-89 and MEI-1 compete for binding to MEL-26. However, we could not demonstrate release of MEI-1 from MEL-26 in the presence of excess UNC-89 fragments (). We could show that both MEI-1 and UNC-89 bound to MEL-26, but we do not know whether a single MEL-26 molecule can simultaneously bind to both MEI-1 and UNC-89 (e.g., using opposite surfaces of the MATH domain). Nevertheless, although there may not be direct competition for binding, the interaction of UNC-89 influences the activity of the CUL-3/MEL-26 complex toward MEI-1 and, given our data, most likely by inhibiting its activity. At the very least, the interaction of UNC-89 with MEL-26 localizes the activity of the CUL-3/MEL-26 complex at the sarcomeric M-line.
If our model that UNC-89 inhibits the complex is correct, then normally the activity of MEL-26 is reduced and the protein level of MEI-1 is elevated. Thus the similarity in phenotypes for loss of function of unc-89
, for gain of function of mel-26
, and for loss of function of mei-1
is consistent with the model. The fact that we observed that both gain and loss of function of mei-1
are also similar to loss of function of unc-89
is more difficult to explain. Perhaps proper thick-filament assembly and/or maintenance depend on tightly controlled activities of mei-1
, restricting it to a certain range of activity. This idea is not unprecedented: proper thick-filament assembly/maintenance is known to depend on precise levels of UNC-96 and UNC-45, as loss or gain of function of each gene results in similar effects on thick-filament organization (Barral et al., 1998
; Mercer et al., 2006
; Landsverk et al., 2007
; Qadota et al., 2007
). During meiosis, mei-1
activity must also be kept within a specific range. Increased or decreased mei-1
activity results in defective meiotic spindles, too short for increased activity or too long for decreased activity. In both cases, these abnormal spindles missegregate chromosomes, resulting in aneuploid gametes (Johnson et al., 2009
We also found that loss of function of cul-3 results in a somewhat more severe disorganization of thick filaments than unc-89, mel-26 or mei-1 (). This perhaps suggests that in body-wall muscle, CUL-3 can associate with additional BTB adaptor proteins besides MEL-26. Indeed, our analysis reveals that the C. elegans genome contains 49 BTB domain–containing proteins. By SAGE data (on WormBase), 20 of the 49 are expressed in body-wall muscle, four of these probably at a high level of expression (including mel-26).
The ability of tbb-2(sb26)
to partly suppress the thick-filament disorganization of mel-26
is consistent with β-tubulin being at least one substrate for MEI-1 in muscle. However, since the phenotype is not entirely suppressed, there is the possibility that there may be additional substrates for MEI-1 muscle. Indeed, in the embryo the preference of MEI-1 for the TBB-2 β-tubulin isotype is not absolute (Lu et al., 2004
) and muscle may express additional tubulin isotypes. In addition, other MEI-1 functions, such as microtubule bundling (McNally and McNally, 2011
), might be relevant in muscle. Nevertheless, we observed an additional aspect of the mei-1(RNAi)
phenotype that is consistent with a role for microtubules in striated muscle: the spindle-shaped body-wall muscle cells are shorter in two dimensions. Disruption of the microtubule network in these cells may lead to a change in cell shape either during development or as a result of hypercontraction.
Little is known about the potential role of microtubules in muscle cell shape or sarcomeric organization in C. elegans
. To our knowledge, the localization of microtubules in nematode body-wall muscle has not been reported. Nevertheless, the C. elegans
echinoderm microtubule-associated protein–like protein ELP-1, which binds to microtubules in vitro, is localized in a criss-crossing network resembling microtubules, and the network is sensitive to the microtubule disruptor nocodazole (Hueston et al., 2008
). Of interest, RNAi-mediated knockdown of elp-1
in a dystrophin (dys-1
)-null animal results in adult worms that have hypercontracted muscle cells (Hueston and Suprenant, 2009
), similar to what we observed for mei-1(RNAi)
. In mammals, electron microscopy of cardiac muscle revealed a fairly extensive network of microtubules; microtubules surround myofibrils and sarcoplasmic reticulum in a helical arrangement (Goldstein and Entman, 1979
). In myotubes induced to regenerate their myofibrils, in the presence of the microtubule-stabilizing drug Taxol, A-bands are formed, but thin filaments and Z-bands are not formed; in the presence of the microtubule-depolymerizing drug Colcemid, complete sarcomeres are formed, but they are not laterally aligned (Toyama et al., 1982
). Generation of a stable, postranslationally modified microtubule array is an early event in the differentiation of myotubes (Gundersen et al., 1989
). During cardiac hypertrophy, there is an increase in microtubule network density associated with sarcomere dysfunction (Tsutsui et al., 1993
The results presented here are not the first to implicate the ubiquitin proteasome system in controlled degradation of sarcomeric proteins in C. elegans
. The RING finger protein RNF-5, not known to be associated with cullins, is localized to dense bodies (Z-disks) and regulates the levels of the LIM domain protein UNC-95 (Broday et al., 2004
). A report from our laboratory demonstrated a connection to CRLs. We showed that CSN-5 interacts with two sarcomeric M-line proteins, UNC-98 and UNC-96 (Miller et al., 2009
). CSN-5 is one of eight highly conserved subunits of the COP9 signalosome complex, which regulates CRLs in a complicated manner, primarily through deneddylation (Cope and Deshaies, 2003
; Schwechheimer, 2004
). Antibodies to CSN-5 localize the protein to A-bands in wild type and colocalize with abnormal accumulations of paramyosin found in unc-98
, and unc-15
(paramyosin) mutants. Knockdown of csn-5
results in an increase in the level of UNC-98 protein and a slight reduction in the level of UNC-96 protein, suggesting that normally CSN-5 promotes the degradation of UNC-98 and stabilizes UNC-96.
Cullins and BTB-domain proteins are associated with human diseases. Litterman et al. (2011)
reported that in humans, obscurin-like 1 (OBSL1) directly interacts with Cul7, and both proteins are crucial for Golgi morphogenesis and dendrite growth of neurons. Cul7 and OBSL1 also cause human 3M syndrome, an autosomal recessive disorder characterized by growth retardation, characteristic facial features, and skeletal anomalies. The majority of patients have mutations in Cul7, whereas others have null mutations in OBSL1 (Hanson et al., 2009
). Cirak et al. (2010)
reported a family in which early-onset autosomal-dominant distal myopathy is associated with a heterozygous missense mutation in Kelch-like homologue 9 (KLHL9), which is a substrate recognition protein that interacts with Cul3. Of interest, the mutation, L95F, is located in the conserved BTB domain of KLHL9, which mediates interaction with Cul3.
Finally, in Lange et al. (2012)
, our colleagues studying obscurin in the mammalian heart show that the turnover of the small ankyrin protein sAnk1.5, previously known to interact with obscurin, is regulated by ubiquitylation mediated by the BTB-domain protein KCTD6. KCTD6, like MEL-26, is a substrate recognition protein for Cul3. Furthermore, these authors demonstrate that in the absence of obscurin, degradation of sAnk1.5 is increased, and conversely, RNAi-mediated knockdown of KCTD6 results in increased levels of sAnk1.5. Thus, although the ultimate substrates identified by our study and that of Lange et al.
are different—MEI-1 and sAnk1.5, respectively—the mechanism is similar: apparent inhibition of a Cul3 complex in striated muscle by UNC-89 (obscurin).