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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) Reminder Super Worm Club begins early March Everyone must attend or else there will be dire consequences This is the biggest event to hit the lower mainland in years so don’t miss it! Remember to bring Beta blockers for Nick 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 Muscle Structure (C. elegans II) 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 unc-95 Mutants Zengal and Epstein (1980) found 2 mutants Very slow to paralyzed Lack of striations and disorganized thick and thin filaments Mutant Phenotype Disorganized thin filaments Disorganized thick filaments Disorganized dense bodies Disorganized dense bodies Long arrows indicate cell/cell boundaries, short arrows indicate dense bodies 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 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 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 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 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 DEB1/vinculin staining is diffuse in all mutant stages 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 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 Role of RNF-5 Colocalizes with UNC-95 in dense bodies Regulates levels of UNC-95 Regulates UNC-95 subcellular location 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 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 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) 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 Guess the criminal Bill Gates