Role of Negative Charge on C2 Insert of Nonmuscle Myosin II‐C: Intermittent Structural Oscillation and Aggregation in Neuritogenesis

Abstract

Previous studies reported that inclusion of 41 aa (33 aa in human) insert in loop 2 of mouse nonmuscle myosin (NM) II‐C heavy chain makes the molecule to become independent of its regulatory light chain (RLC) phosphorylation in MgATPase activity and in vitro motility, and to localize in the neurites as puncta form. However, the molecular mechanism behind these unique properties among myosin II family is not known. Here, we show that deletion of the N‐terminal 12 aa, but not the C‐terminal 8 aa or the middle 21 aa of C2 insert, diminishes the fluorescence oscillation property of GFP tagged‐NM II‐C2 molecule in the neurites of neuro‐2a cells as determined by fluorescence lifetime imaging microscopy analysis. Also, we demonstrate that inclusion of 41 aa insert increases the population of extended monomers whereas deletion of N‐terminal 12 aa, middle 21 aa or C‐terminal 8 aa of 41 aa insert increases the folded autoinhibited NM II‐C2 monomers as revealed by fluorescence correlation spectroscopy (FCS) analysis in neuronal cells. Furthermore, FCS analysis reveals that NM II‐Δ12C2‐GFP significantly diffuses very slowly compared with NM II‐Δ8C2‐ or NM II‐Δ21C2‐GFP in the neurites, suggesting that radius (rH) of NM II‐Δ12 C2‐GFP is higher than that of wildtype or mutants Δ21 and Δ8C2‐GFP. These studies suggest that N‐terminal 12 aa (contributor of net charge of C2) plays a significant role in determining the oscillation and aggregation properties of the molecule.Research Support: Department of Science and Technology, India