1. Regulation of Non-Muscle Myosin II Filament Assembly Hallie Kuhn August 15, 2008

2. Non-Muscle Myosin II Cell motility Spindle alignment during mitosis Morphogenesis Cytokinesis Liu et al. 2008

3. Non-Muscle Myosin II (Zipper) Filament Assembly Required for contractile force generation Dynamic assembly-disassembly is thought to allow for spatiotemporal organization in response to certain signals How is this dynamic filament assembly regulated? Inactive Active RLC phosphorylation

4. Myosin II Structure Myosin head Essential Light Chain Regulatory Light Chain (Squash) Coiled coil tail Non-helical tail piece Zipper tail domain IQ1 and IQ2 binding motifs N’N’ C’ RLC phosphorylation promotes filament assembly RLC PP

5. Importance of RLC Phosphorylation O’Farrell et al. 2006

6. Experimental Approach When a portion of the neck region is attached to 1744-2011 filament assembly is suppressed Assembly domain (AD) 1744-2011 Zipper tail domain IQ1 and IQ2 binding motifs N’N’ C’ 1744-2011 +FA -FA 1744-2011

7. Filament Assembly Regulation What are the minimal elements of the attached neck piece that allow for filament assembly? Is it possible to reconstitute filament assembly through RLC regulation? ? 1744-2011 +FA -FA

8. Are IQ1 and IQ2 sufficient to suppress filament assembly? USIQ1IQ2 1744-2011IQ1 US YFP 1744-2011 IQ1 IQ2 YFP  IQ 2 IQ 2 IQ 1 IQ 2 Neck Region

9. Filament Assembly Assay Express and purify each protein Electrostatic filaments form in a salt-dependent manner, thus at a certain concentration of NaCl the protein will form insoluble filaments and can be collected by ultra-centrifugation pBH +NaClCentrifugeCollect soluble and insoluble fractions Soluble monomer Insoluble filament

10. Results All three chimeras,  IQ2, IQ2, and IQ1IQ2 do not form filaments 1744-2011 IQ2 SSSSSSPPPPPP 0 25 50 125 300 500 mM NaCl

11. IQ2 Truncations 1744-2011 IQ854-864 IQ854-874 IQ2 IQ864-884 IQ874-884 1744-2011 Results: IQ854-864 does not form filaments SSSSSSPPPPPP 0 25 50 125 300 500 mM NaCl

12. Conclusions 1744-2011IQ2 YFP IQ 2 -No Filaments 1744-2011IQ1 US YFP  IQ 2 -No Filaments 1744-2011IQ1IQ2 YFP IQ 1 IQ 2 -No Filaments 1744-2011 IQ854-864 -No Filaments Either of the IQ pieces are sufficient to inhibit filament assembly IQ854-864 which contains the IQ binding motif for RLC inhibits filament assembly

13. Interpretations It was thought that RLC was negatively affecting the assembly domain, but in fact RLC may modulate the inhibitory effects of the IQ binding region on filament formation Trybus and Lowey, 1987 Burgess et al., 2007 Folded “shut down” state

14. Future Work IQ2 truncations RLC reconstitution -FA IQ RLC IQ RLC PP +FA Plan: Express the RLC (squash) and a phosphomimetic version, TS21EE Combine IQ chimeras with RLCs Run filament assembly assays to determine whether RLC binding can reconstitute filament formation

15. Acknowledgements Ken Prehoda Derek Ricketson Prehoda Lab Peter O’day and the SPUR program