Myosin II Tailpiece Determines Its Paracrystal Structure, Filament Assembly Properties, and Cellular Localization

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 nonhelical 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. Non muscle myosin II (NMII)2 is a major motor protein present in all cell types participating in crucial processes, including cytokinesis, surface attachment, and cell movement (1-3). NMII units are hexamers of two long heavy chains with two pairs of light chains attached. NMII heavy chain is composed of a globular head containing the actin binding and force generating ATPase domains, followed by a large coiled-coil rod that terminates with a short non-helical tailpiece (tailpiece). To carry out its cellular functions, NMII assembles into dimers and higher order filaments by interactions of the coiled-coil rod (4). The assembly process is governed by electrostatic interactions between adjacent coiled-coil rods containing alternating charged regions with specific periodicity (5-9) and is enhanced by activation of the motor domain through regulatory light chain phosphorylation (10 -12). The charge periodicity also determines the register and orientation of each NMII hexamer in the filament. Additionally the C-terminal region of the coiled-coil rod contains a distinctive positively charged region and the assembly-competence domains that are crucial for proper filament assembly (5-9, 13).Three isoforms of NMII (termed NMII-A, NMII-B, and NMII-C) have been identified in mammals (14 -16). Although NMII isoforms share somewhat overlapping roles, each isoform has distinctive tissue distribution and specific functions. NMII-A is important for neural growth cone retraction (17,18) and is distributed to the front of migrating endothelial cells (19). While NMII-B participates in growth cone advancement (20) and was detected in the retracting tails of migrating endothelial cells (19). Furthermore NMII-A and NMII-B have an opposing effect on motility, since depletion of NMII-A leads to increased mot...

The Positively Charged Region of the Myosin IIC Non-helical Tailpiece Promotes Filament Assembly

“…7A). Thus in contrast to the complete tailpiece containing both the positively and negatively charged regions, the negatively charged The tailpiece has been shown to be important for determining the isoform specific morphology of NMII paracrystals (28). As the negatively charged region does not promote IICRod 1296 -1854 filament assembly it may be important in determining NMII paracrystal morphology.…”

“…NMII isoforms create different paracrystal morphology: NMII-A and NMII-B form large wide filaments (width measuring 1.21 Ϯ 0.4 nm and 1.22 Ϯ 0.38 nm, respectively) while NMII-C forms delicate thin filaments (0.32 Ϯ 0.07 nm) (28). Swapping the negatively charged region of either NMII-A or NMII-B with the negatively charged region of NMII-C resulted in thin filaments similar to NMII-C (0.269 nm Ϯ0.06 and 0.239 nm Ϯ0.04, respectively) ( Fig.…”

“…Furthermore, smooth muscle myosin II has a splice variant lacking the tailpiece, which exhibited significantly different properties of filament assembly that were reflected by different molecular packing inside the filament (26). Swapping the tailpieces among the different NMII isoforms showed that the tailpiece affects NMII function in vivo and determines the specific morphology of NMII filaments (28). Additionally, phosphorylating the tailpiece by protein kinase C and casein kinase II resulted in decreased NMII assembly as determined by sedimentation and critical concentration experiments (29 -34).…”

“…This tailpiece has been shown to be an important regulatory domain, affecting NMII filament assembly and cellular functions (24 -28). Removing the tailpiece from chicken smooth muscle myosin II and NMII changed their solubility and impaired the ability of NMII to form large filamentous structures (24,28). Furthermore, smooth muscle myosin II has a splice variant lacking the tailpiece, which exhibited significantly different properties of filament assembly that were reflected by different molecular packing inside the filament (26).…”

Regulation of the filament structure and assembly ofAcanthamoebamyosin II by phosphorylation of serines in the heavy-chain nonhelical tailpiece

“…Most of the reports on the role of the nonhelical tailpieces of vertebrate myosin IIs are based on in vitro studies of tail fragments of the mammalian nonmuscle myosin IIs NMIIA, NMIIB, and, to a lesser extent, NMIIC (22)(23)(24)(25)(26)(27). Phosphorylation by protein kinase C or casein kinase II of one or more serines in the nonhelical tailpieces of NMIIA and NMIIB was reported to inhibit assembly of NMIIB tail fragments but not of NMIIA tail fragments (23,24), as assayed by sedimentation, but in a later study was reported to inhibit the assembly of tail fragments of NMIIA (25).…”