Structural Oscillations of Non-muscle Myosin II-C2: Time Resolved Confocal Microscopy

Size and structural oscillation of the non-muscle myosin (NM) II-C2 protein are investigated using time resolved confocal microscopy. It is observed that the high enzymatic activity of GFP tagged NM II-C2 is invariably accompanied by frequent fluctuations in the fluorescence intensity (fluorescence oscillation) of GFP in Neuro-2a cells. We demonstrate that the deletion of the polar rich N-terminal 12 amino acid (aa) of the C2 insert diminishes the fluorescence oscillation of NM II-C2. On deletion of either the middle 21 aa or the C-terminal 8 aa, frequency of observing periodic fluctuation in fluorescence intensity decreases. Fluorescence correlation spectroscopy (FCS) was used to monitor the size of the GFP tagged NM II-C2. NM II-C2-GFP molecule remains in the rod-like extended conformation (6S) with a length of 120 +/- 5 nm while deletion of any region of the C2 insert causes the molecule to adopt the 10S conformation with a length of 50-65 nm. These findings suggest that enzymatic activity and assembly property of NM II-C2 are controlled by the polar amino acids present in the C2 insert.