Abstract
Non muscle myosin II (NMII) is a major motor protein present in all cell types. The three known vertebrate NMII isoforms share high sequence homology but play different cellular roles. The main difference in sequence resides in the C-terminal non-helical tailpiece (tailpiece). In this study we demonstrate that the tailpiece is crucial for proper filament size, overcoming the intrinsic properties of the coiled-coil rod. Furthermore, we show that the tailpiece by itself determines the NMII filament structure in an isoform-specific manner, thus providing a possible mechanism by which each NMII isoform carries out its unique cellular functions. We further show that the tailpiece determines the cellular localization of NMII-A and NMII-B and is important for NMII-C role in focal adhesion complexes. We mapped NMII-C sites phosphorylated by protein kinase C and casein kinase II and showed that these phosphorylations affect its solubility properties and cellular localization. Thus phosphorylation fine-tunes the tailpiece effects on the coiled-coil rod, enabling dynamic regulation of NMII-C assembly. We thus show that the small tailpiece of NMII is a distinct domain playing a role in isoform-specific filament assembly and cellular functions.
Highlights
It was shown that swapping this region between Non muscle myosin II (NMII)-A and NMII-B resulted in chimeric proteins, which adopted cellular localization according to the C-terminal part [26]
These results indicate that NMII-A and NMII-B tailpieces weakened assembly and NMII-C tailpiece strengthened it
Together these results reveal that the effects the tailextent (18 of 30 observed), indicating that some of the cellular piece exerts on NMII can account for part of the cellular differlocalization properties of NMII-B reside in the head and ences observed between the isoforms
Summary
NMII isoforms used for this study were NMII-A, accession number NP_002464, NMII-B, accession number A59252, and NMII-C accession number AY363100. Tailless NMII constructs were created by converting the coiled-coil ending proline codon on the NMII rod fragments in pET21 (described above) to a terminator codon using site-directed mutagenesis with the following primers: NMII-A, 5Ј-GGC GCG GGG ACC TGT AAT TTG TCG TGC CCC GCC G-3Ј; NMII-B, 5Ј-CGG CTG GAA GAG AAG CTG ATT TAG CCA CCC CGC CTC AGC CGG-3Ј; and NMII-C, 5Ј-GGC TCC GGC GTG GCT AAT GAG ACA TTCA CCA CAC GGA C-3Ј. NMII-Aspecific siRNA sequence 5Ј-CGA TTT CCA AAA GCG GAG GCT TCA GAA GGA CTC TCT TGA AGT CCT TCT GAA GCC TCC GCG GGG A-3Ј was synthesized and inserted into pLVHTM (Lentiweb) according to instructions This plasmid has an independent GFP protein transcribed as an infection marker. Focal adhesion area and number were automatically analyzed using Image-Pro plus software (Media Cybernetics) with calibrated background-reduced final image and subjected to two-tailed, two-sampled un-equal variance Student’s t test using at least 16 cells
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