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To gain further insight into the molecular architecture, assembly and maintenance of the sarcomere, we have carried out a molecular analysis of the UNC-96
and UNC-89 proteins in the muscle of C. elegans. Specifically, we are utilizing the yeast 2-hybrid system to identify myofibril proteins that interact with
UNC-96 or UNC-89. Functional analysis of these interacting proteins might in turn allow us to identify the mechanisms by which UNC-89 and UNC-96
affect muscle structure. UNC-96 is a novel 47 kDa polypeptide that has no recognizable protein domains. UNC-89 encodes a giant 732 kDa polypeptide
that is considered to have a mammalian counterpart known as obscurin. Both UNC-89 and UNC-96 mutants display significant defects in the organization
of M-lines, which results in a disrupted myofilament lattice observed by polarized light microscopy. Our working model is that UNC-96 is required in adult
muscle to maintain the architecture of already assembled myofibrils through its association with UNC-98, paramyosin and other unidentified proteins. The
UNC-89 protein has several isoforms, many of which contain paired protein kinase domains that have been shown to interact with a protein phosphatase
(B0379.4b). An exhaustive search for interactors of an UNC-89 protein kinase domain led to the identification of B0379.4b, which is now being used as a
bait to identify additional interactors. Full-length UNC-96 used as a bait gave too high of a background, making the difference between true and false positives
indistinguishable. Therefore, the UNC-96 cDNA for both halves of the protein were used as new baits. The C-terminal half of UNC-96, termed “B,” and B0379.
4b have given several positives. The N-terminal half, Segment “A,” has not given any positives, suggesting an absence of interaction in this region. The most
recently developed “C” segment is still in the process of undergoing a 2-hybrid screen.
In the nematode, C. elegans, muscle is organized into four body wall quadrants. Each muscle quadrant consists of multiple overlapping muscle cells, each of
which contains multiple myofibrils made up of repeating sarcomeres. Actin-containing thin filaments and their attachment structures, called dense bodies
(collectively called I bands), alternate with thick filaments and their attachment structures, M-lines (collectively, A-bands). Dense bodies and M-lines anchor
thin and thick filaments to the muscle cell membrane, allowing contractile forces to ultimately move the whole animal. C. elegans is an excellent model system
in which to study muscle due to the optical transparency of the worm. Additionally, this system allows us to test protein interactions at a genetic level and to study
muscle function in a whole animal.
In our lab, we have identified a protein, UNC-96, that is responsible for A-band integrity and is also likely to be involved in muscle maintenance. By polarized light
microscopy, unc-96 mutants display reduced myofibrillar organization and characteristic birefringent “needles.” These “needles” contain myofibrillar proteins that
are collecting or mislocalizing to the ends of the muscle cells, namely paramyosin (thick filament component) and UNC-98.
Another protein responsible for muscle structure, UNC-89, is much longer than UNC-96. UNC-89 mutants are thinner and more transparent than their wild-type
counterparts. In addition, by poloarized light microscopy, these mutants have a characteristic myofilament patterning that resembles a “basketweave” appearance.
Note that there is also a mild pharyngeal phenotype.
Isoforms of UNC-89 contain two protein kinase domains that have been shown in a yeast 2-hybrid screen to interact with a phosphatase classified as B0379.4b.
The interest now lies in the search for other contributors to this interaction, possibly a substrate(s) for either UNC-89 or the B0379.4b phosphatase. Ultimately,
the goal of this project was to gain an increased understanding of muscle structure at its simplest level.
Yeast 2-hybrid experiments using bait clones containing a portion of UNC-96 (cloned into vector, pGBDU) and full-length B0379.4b. These baits were screened
against the RB2 library-a pool of randomly primed cDNAs from C. elegans (prey). An extensive series of selective plating was implemented to isolate cDNAs
producing translational products that interact with our proteins.
The plasmid containing cDNAs that resulted in a positive interaction were then isolated from yeast, transformed into bacteria, miniprepped, sequenced, and
compared to a nucleotide databases by a BLAST search (blastn) to determine the protein involved in the interaction. By searching WormBase (www.wormbase.org)
we determined what is already known about the interacting protein. Specifically, SAGE data is used to determine whether or not a protein is expressed in muscle.
Proteins that are likely to reside in muscle are strong candidates for interacting, directly, with UNC-96 and B0379.4b.
Considering dilution factors and the theoretical number of cDNAs represented in the library pool, millions of C. elegans cDNAs were screened. Both the A
and B bait constructs from UNC-96 have provided positives that have been functionally identified on WormBase. In fact DEB-1, which interacted with the C
terminal half of UNC-96 (construct B), is present in muscle cells at dense bodies. (Barstead et al.) UNC-15, which encodes for paramyosin, is also in muscle
cells at the thick filaments. (Waterston et al.) The C construct from UNC-96 was recently cloned into PGBDU vector. At this point the new bait has been
transformed into yeast and is ready to undergo a yeast 2-hybrid screen. Unfortunately, difficulties with contamination delayed the screen of phosphatase B.
Thus, 16 preliminary positives have been obtained from this screen and must be re-screened one-to-one with the phosphatase bait. Eventually, positives from
this screen can be sequenced and analyzed for functionality just as positives from bait A and B have been investigated.
First the screenings of the UNC-96 bait “C” and the phosphatase B bait will be completed and resulting positives will be sequenced and the corresponding
genes/proteins will be identified through WormBase. Also, these interactors will be expressed as GST or MBP fusion proteins and tested for in vitro binding
to bacterially expressed UNC-96 and B0379.4b.
Funding for this work provided by the Howard Hughes Medical Institute Grant and NIH Grants AR051466 and AR052133 to GMB.
1 Barstead R.J., R.H. Waterston. (1989). The basal component of the nematode dense-body is vinculin. Journal of Biol Chem. 264:10177-10185.
2 Chien C.T., P.L. Bartel, R. Sternglanz, and S. Fields. (1991). The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein
of interest. PNAS. 88(21): 9578-9582.
3 Ferrara, T.M., D.B. Flaherty, and G.M. Benian. (2005). Titin/connectin related proteins in C. elegans: a review and new findings. Journal of Muscle Research
and Cell Motility 26: 435-447.
4 Mercer, K.B., D.B. Flaherty, R.K. Miller, H. Qadota, T.L. Tinley, D.G. Moerman, and G.M. Benian. (2003). C. elegans UNC-98, a C2H2 Zn finger protein, is a
novel partner of UNC-97/PINCH in muscle adhesion complexes. Mol. Biol. Cell 14: 2492-2507.
5 Mercer, K.B., R.K. Miller, T.L. Tinley, S. Sheth, H. Qadota, and G.M. Benian. (2006). C. elegans UNC-96 is a new component of M-lines that interacts with
UNC-98 and paramyosin and is required in adult muscle for assembly and/or maintenance of thick filaments. Mol Biol Cell 17 (Sept.)
6 Waterston R.H., R.M. Fishpool, S. Brenner. (1977). Mutants affecting paramyosin in C. elegans. Journal Mol Biol. 117: 679-697.
Worm Base.
This project investigated muscle cells at their simplest level. C. elegans was used as a model to observe changes in muscle structure and development because
of its transparent body. Two specific muscle proteins were put through an extensive yeast screening process in order to assess whether or not proteins produced
by other genes interacted with them. Several interacting genes were identified, some of which are known to function in muscle cells.
PCR, mini-preps, agarose running gels, DNA digestion
Muscle proteins, yeast 2-hybrid, C. elegans, UNC-96, UNC-89, phosphatase
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