Regulation of Non-Muscle Myosin II Filament Assembly
Hallie Kuhn
August 15, 2008
Non-Muscle Myosin II
• Cell motility
• Spindle alignment during mitosis
• Morphogenesis
• Cytokinesis
Liu et al. 2008
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
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’ C’
RLC phosphorylation promotes filament assembly
RLC
P P
Importance of RLC Phosphorylation
O’Farrell et al. 2006
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’ C’
1744-2011 +FA
-FA1744-2011
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
1744-2011
+FA
-FA
Are IQ1 and IQ2 sufficient to suppress filament assembly?
US IQ1 IQ2
1744-2011IQ1USYFP
1744-2011
1744-2011IQ1
IQ2
IQ2
YFP
YFP
IQ2
IQ2
IQ1IQ2
Neck Region
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+NaCl Centrifuge Collect
soluble and insoluble
fractions
Soluble monomer
Insoluble filament
Results
• All three chimeras, IQ2, IQ2, and IQ1IQ2 do not form filaments
1744-2011
IQ2S S SS S SP P P P PP 0 25 50 125 300 500mM NaCl
IQ2 Truncations
1744-2011
1744-2011
1744-2011
IQ854-864
IQ854-874
IQ2
IQ864-884
IQ874-884
1744-2011
1744-2011
Results: •IQ854-864 does not form filaments
S S SS S SP P P P PP 0 25 50 125 300 500mM NaCl
Conclusions
1744-2011IQ2YFP IQ2 -No Filaments
1744-2011IQ1USYFP IQ2 -No Filaments
1744-2011IQ1 IQ2YFP IQ1IQ2 -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
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, 1987Burgess et al., 2007
Folded “shut down” state
Future Work
• IQ2 truncations
• RLC reconstitution
-FAIQRLC
IQRLC
P P
+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
Acknowledgements
• Ken Prehoda
• Derek Ricketson
• Prehoda Lab
• Peter O’day and the SPUR program