2. The Lim domain protein UNC-95 is required for the assembly of muscle attachment structures and is regulated by the RING finger protein RNF-5 in C. elegans Broday L. et al. June issue of JCB Ruttenberg Cancer Center (NY)

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4. Back to the paper… Brief overview of C. elegans muscle Role of UNC-95 in sarcomere assembly Role of RNF-5 in localization of UNC-95

5. Muscle Structure (C. elegans II)

6. Why UNC-95? LIM domain proteins have recently been shown to aid in assembly of these structures UNC-97 is a LIM domain protein and has a central role UNC-95 is a LIM domain protein that was previously uncharacterized

7. unc-95 Mutants Zengal and Epstein (1980) found 2 mutants  Very slow to paralyzed  Lack of striations and disorganized thick and thin filaments

8. Mutant Phenotype Long arrows indicate cell/cell boundaries, short arrows indicate dense bodies Disorganized thin filaments Disorganized thick filaments Disorganized dense bodies

9. EM of unc-95 animals Irregular dense bodies Random dense body spacing Barely recognizable M-line Disorganized thin filaments Disorganized thick filaments No recognizable I-line

10. Characterization of unc-95 (su33) Y105E8.6 found associated with RNF-5 in a Y2H screen Hypothesized to be unc-95 Y105E8.6 was sequenced in su33 mutant and found to have a CAG TAG mutation causing a truncated protein without LIM domain

11. Rescue with functional fusion GFP translational fusion with standard 2.5kb upstream (promoter) created Worms were injected with construct and assayed RT-PCR showed that the mutant gene was in fact transcribed

12. Rescue with functional fusion A,B,C: Rescue with translational fusion D,E: Protein structure F: RT-PCR of mutant and wild type show comparable transcription G,H,I: RNAi with Y105E8A.6 construct

13. Role of UNC-95 during embryogenesis A-F: anti-UNC-52/perlecan staining showing wild type phenotype in mutant G-N: anti-PAT-3/  integrin staining shows wild type phenotype until post-hatching O-T: anti DEB- 1/vinculin staining is diffuse in all mutant stages

14. Conclusions from this data UNC-95 not required for localization of UNC-52 perlecan in basement membrane Recruitment of  -integrin to basal sarcolemma not dependant on UNC-95 UNC-95 is required for recruitment of vinculin

15. Analysis of unc-95 localization A-I: Expression is seen throughout muscle cells but especially in cellular attachment sites as indicated by the various arrows J-L: Expression of truncated unc-95 translational fusion shows low overall expression and no expression at cellular attachment sites

16. Role of RNF-5 Colocalizes with UNC-95 in dense bodies Regulates levels of UNC-95 Regulates UNC-95 subcellular location

17. Colocalization of RNF-5 and UNC-95 A,B: anti-RNF-5 and anti-DEB-1-vinculin C,D: same as A,B but with RNAi E: rnf-5 mutant stained with anti-DEB-1-vinculin F: unc-95 anti RNF-5 G: localization of RNF-5 in dense bodies H: colocalization of RNF-5 and UNC-95 in yellow

18. RNF regulation of UNC-95 A: UNC-95::GFP A: UNC-95::GFP with RNF-5 overexpressed with a heat shock promoter A: UNC-95::GFP with overexpression of a truncated form of RNF-5 (no RING finger domain) Conclusion: an intact RING finger domain is required for proper regulation of UNC-95

19. RNF-5 RNAi RNAi was used to deplete the levels of RNF-5 and an increase in GFP expression from UNC-95::GFP is seen in B and C In heterozygous rnf-5 mutants, a similar increase in GFP expression is seen in (D and E)

20. Summary Y105E8.6 is unc-95 UNC-95 is required for proper recruitment of vinculin for initial assembly of muscle attachment sites UNC-95 is localized primarily to muscle attachment sites RNF-5 colocalizes with UNC-95 and regulates its location and levels

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