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